RNA-based control of Botrytis

Abstract

Agents, compositions and methods are disclosed for treating or controlling Botrytis infections, particularly in plants. Particular agents include polynucleotide molecules that induce RNAi when administered to Botrytis cinerea fungal cells. In particular embodiments, the compositions include a fungicidally effective amount of a polynucleotide that is essentially complementary to or has at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a fragment of Botrytis cinerea DNA or target gene, or RNA transcribed from the DNA or target gene, and includes at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 225,758, filed July 26, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] The necrotrophic fungus Botrytis cinerea has been reported to invade over 1,000 species of plants (Williamson et al., 2007; Elad et al., 2016), many of which are economically very important. Agricultural crops, including vegetables (e.g., cucumber, tomato, zucchini) and fruit-bearing plants (e.g., strawberries, grapes, blueberries, raspberries), are some of the most severely affected by this pathogen (Jarvis et al., 1962; Elad et al., 2016). Botrytis cinerea is estimated to cause $10 billion to $100 billion in agricultural losses worldwide. For example, Botrytis fruit rot (BFR) contributed to a 36% decline in strawberry yields from 2007 to 2016, resulting in a net production value loss of $250 million per year (Qushim et al., 2018). Botrytis cinerea is highly destructive and therefore ranks second on the list of scientifically and economically important fungal pathogens (Dean et al., 2012). Summary of the Invention [Problem to be solved by the invention]

[0003] Botrytis cinerea is classified as a necrotrophic pathogen, meaning that it tends to invade and grow in damaged or senescent tissues, eventually causing cell and tissue death. Disease symptoms appear as greyish, soft, mushy spots on leaves, stems, flowers, and produce. Thus, diseases of various plants and plant parts caused by Botrytis cinerea are often called "gray mold" or "Botrytis rot". The spots can become covered with a coating of grey fungal spores, especially when humidity is high. Fruits and plants shrivel, rot, and often form black, stony sclerotia (highly melanized fungal overwintering structures). Fungal inoculum (e.g., asexual conidia and spores) is very abundant and ubiquitous, usually originating from infected plant tissues (Jarvis, 1962). Control or management of diseases caused by Botrytis cinerea relies heavily on chemical fungicides. Furthermore, Botrytis cinerea is considered a high-risk pathogen with regard to fungicide resistance development according to the Fungicide Resistance Action Committee (FRAC; www.frac.info). The discovery of Botrytis cinerea isolates resistant to all registered site-specific chemical fungicides for gray mold control represents an unprecedented example of resistance development in plant pathogenic fungi (Leroch et al., 2013; Fernandez-Ortuno et al., 2015). Furthermore, this finding indicates that site-specific, reduced-risk fungicides may eventually become useless for disease control and integrated pest management if these resistant fungal genotypes become dominant in field populations. Furthermore, chemical fungicides may be harmful to the environment, may lack specificity or selectivity, and may ultimately result in non-target effects (e.g., affecting non-target beneficial organisms). Therefore, there is a long-standing need for more environmentally friendly and novel disease control strategies to control Botrytis cinerea infection. [Means for solving the problem]

[0004] To address these problems, the present invention is directed to locally applied double-stranded (ds)RNA that, when sprayed on leaves and other above-ground parts of plants, such as fruit-bearing, vegetable, and ornamental plants, selectively reduces or eliminates the growth of Botrytis cinerea through the process of RNA interference (RNAi).

[0005] RNA interference (RNAi) technology has been shown to be a highly selective biological treatment that silences gene expression in pests and pathogens through internal biological processes. Exogenous application of double-stranded RNA (dsRNA) to initiate RNAi has been used to effectively control certain plant pest species. The present disclosure is directed to an approach to control the fungal pathogen Botrytis cinerea using RNAi compositions. In certain embodiments, methods and compositions are described that provide Botrytis cinerea control by using the application of exogenous dsRNA administered to plants. Such dsRNA contains specific trigger sequences that are designed to regulate the expression of specific Botrytis cinerea target genes. To identify potential target genes for RNAi regulation, the entire genome information of Botrytis cinerea is analyzed to identify gene sequences for potential targets by the RNAi trigger. Triggers are designed through proprietary computational algorithms that combine with publicly available RNAi design tools to create trigger sequences that meet specific design criteria. Several such triggers for RNAi-mediated control of Botrytis cinerea are described and claimed herein. Following treatment of Botrytis cinerea with the specific triggers claimed herein and disclosed in Table 1A, a reduction in fungal growth in vitro was observed, as described in Table 1B and explained in Example 1. The specific triggers then demonstrated the ability to reduce gray mold disease and symptoms caused by Botrytis cinerea following application to plants or plant parts in laboratory and greenhouse assays, as disclosed in Tables 2-10 and Figures 1-5 and explained in Examples 2-3. Importantly, the specific triggers and compositions described and claimed herein have been evaluated and have already shown efficacy in reducing disease caused by Botrytis cinerea in large-scale outdoor field trials on economically important plants, as explained in Example 4 and Figures 6-10.

[0006] Compositions and methods described herein include recombinant polynucleotide molecules, such as single-stranded or double-stranded DNA or RNA molecules, herein referred to as "triggers," useful for controlling or preventing Botrytis cinerea infection, or recombinant DNA constructs for generating transgenic plants resistant to Botrytis cinerea infection. In some embodiments, the polynucleotide trigger is provided as a topically applied agent for controlling or preventing plant infection with Botrytis cinerea. In some embodiments, plants with enhanced resistance to infection with Botrytis cinerea, such as transgenic plants (including seeds or propagable parts) expressing the polynucleotide trigger, are provided. In some embodiments, plants (including seeds or propagable parts) that have been topically treated with a composition comprising the polynucleotide trigger (e.g., a plant that has been sprayed with a solution of dsRNA molecules) are provided. Polynucleotide-containing compositions that are topically applied to Botrytis cinerea, or to plants, plant parts, or seeds to be protected from infection with Botrytis cinerea, are also provided. Plants that may particularly benefit from the embodiments described herein include, but are not limited to, grapes, tomatoes, strawberries, snap peas, eggplants, chili peppers, bell peppers, tomatillos, ground cherries, cape gooseberries, tobacco, apples, pears, quince, peaches, plums, cherries, almonds, apricots, blackberries, blueberries, raspberries, carnations, petunias, and roses.

[0007] Some embodiments relate to the suppression of target genes in Botrytis cinerea by polynucleotide triggers. Provided herein are nucleotide sequences of target genes, referred to herein as "target gene sequence group" or "target gene sequence", consisting of SEQ ID NOs: 1-12. Certain embodiments of the invention relate to polynucleotides designed to hybridize to RNA transcripts of these target genes resulting in RNAi. Also provided are nucleotide sequences of triggers that target target genes, referred to herein as "trigger sequence group" or "trigger sequence", consisting of SEQ ID NOs: 13-24, 49-52. Additionally provided herein are the RNA sequences of these triggers, "RNA trigger sequence group" or "RNA trigger sequence", consisting of SEQ ID NOs: 25-36, 53-56. The RNA trigger sequence group is the same as the trigger sequence group, except for the replacement of thymine with uracil. Also provided herein are reverse complements to the RNA trigger sequence group, referred to herein as "RNA trigger sequence reverse complement group" or "RNA trigger sequence reverse complement", consisting of SEQ ID NOs: 37-48, 57-60. The RNA trigger sequence reverse complement is the perfect complement to the sequence in the RNA trigger sequence read from 5' to 3'. The trigger sequence and the RNA trigger sequence are designed to affect RNAi on the corresponding mRNA transcript of the target gene sequence, preventing or reducing the translation of the associated protein, resulting in fungal control. Table 1A provided herein matches various gene target sequences with their corresponding trigger sequence, RNA trigger sequence, and RNA trigger reverse complement sequence. The sequence number refers to the sequence provided in the sequence number list submitted herewith.

[0008] In one aspect, a method for controlling Botrytis cinerea infection in a plant includes contacting Botrytis cinerea with a polynucleotide that includes at least one fragment of 18 or more contiguous nucleotides having a sequence that has about 95% to about 100% identity to a corresponding fragment of a target gene or its DNA complement having a nucleotide sequence selected from the group consisting of target gene sequences (e.g., including a fragment of 21 contiguous nucleotides having a sequence of 100% identity). In one embodiment, a method for controlling Botrytis cinerea infection in a plant includes contacting Botrytis cinerea with a polynucleotide that includes a nucleotide sequence selected from the group consisting of target gene sequences, or a target gene having a sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, 12, or a nucleotide sequence that is complementary to at least 18 contiguous nucleotides of an RNA transcribed from the target gene. In some embodiments, the polynucleotide comprises a sequence that is complementary to or about 95% to about 100% identical to at least 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 23-36. In some embodiments, the polynucleotide is designed to have complementarity to an mRNA encoded by the target gene. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a group of trigger sequences, RNA trigger sequences, or RNA trigger sequence reverse complements, or more specifically selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, or 48. In some embodiments, topical application of the polynucleotide or a composition or solution comprising the polynucleotide is accomplished by spraying the polynucleotide or a composition or solution comprising the polynucleotide onto the above-ground parts (e.g., flowers, stems, and / or leaves) of fruit-bearing plants, vegetables, or ornamental plants that are infected or potentially infected with Botrytis cinerea.In some embodiments, the plant is a grape, strawberry, tomato, bean, eggplant, chili pepper, bell pepper, tomatillo, ground cherry, cape gooseberry, tobacco, apple, pear, quince, peach, plum, cherry, almond, apricot, blackberry, blueberry, raspberry, carnation, petunia, or rose, or a variant thereof. In some embodiments, contact with the polynucleotide is achieved by topically applying (e.g., by spraying or dusting or dipping) the polynucleotide or a composition or solution comprising the polynucleotide directly to the Botrytis cinerea or to a surface or matrix (e.g., plant or soil) that will come into contact with the Botrytis cinerea. In some embodiments, contact with the polynucleotide is achieved by providing a transgenic plant that expresses a sequence that controls Botrytis cinerea infection.

[0009] Some embodiments relate to a method for controlling Botrytis cinerea infection in a plant by exposing the plant to an agent comprising a polynucleotide having at least one fragment of 18 or more contiguous nucleotides (e.g., a fragment of 21 contiguous nucleotides having a sequence of 100% identity) to a corresponding fragment of a target gene or its DNA complement having a sequence selected from the group consisting of target gene sequences, the agent functions following contact or ingestion (e.g., internal absorption / transfection) of Botrytis cinerea to inhibit a biological function in Botrytis cinerea, thereby controlling the Botrytis cinerea infection. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a group of trigger sequences, a group of RNA trigger sequences, or a group of reverse complements of RNA trigger sequences, or the polynucleotide comprises one or more nucleotide sequences that are about 95% to about 100% identical to one or more nucleotide sequences selected from a group of trigger sequences, a group of RNA trigger sequences, or a group of reverse complements of RNA trigger sequences. In some embodiments, the polynucleotide is double-stranded RNA.In some embodiments, the polynucleotide is double-stranded RNA produced through any one of the processes of cell-free production of RNA described in U.S. Patent No. 10,858,385 or No. 10,954,541, both of which are incorporated herein by reference.

[0010] In some embodiments, the agent is (a) a bactericidal amount of a polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 contiguous nucleotides that are essentially complementary to or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a DNA or target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or a fragment of RNA transcribed from said DNA or target gene; or (b) a bactericidal effective amount of at least one polynucleotide comprising at least one silencing element that is essentially complementary to, or has at least about 85%, at least about 90%, at least about 95% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides of a DNA or a target gene or an RNA transcribed from said DNA or target gene, wherein said DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12; or (c) a bactericidal effective amount of at least one RNA comprising at least one fragment that is essentially complementary to, or has at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides of, a DNA or target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or a fragment of an RNA transcribed from the DNA or target gene; or (d) an RNA molecule that, when transfected into or contacted with said Botrytis cinerea, causes death, growth inhibition, reduced virulence or pathogenicity, or reduced proliferation / fertility of said Botrytis cinerea, wherein said RNA molecule is essentially complementary to or has at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a DNA or a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides; (e) a double-stranded RNA molecule that, when transfected into or in contact with the Botrytis cinerea, causes death, growth inhibition, reduced virulence or pathogenicity, or reduced proliferation / fertility of the Botrytis cinerea, wherein at least one strand of the double-stranded RNA molecule is essentially complementary to or contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides having at least 85%, 90%, 95%, 98%, or 100% sequence identity to a DNA or a target gene or a fragment of RNA transcribed from the DNA or target gene, and the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12; or (f) a bactericidal amount of at least one double-stranded RNA comprising at least one strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to the nucleotide sequence; or (g) a fungicidally effective amount of a polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to the nucleotide sequence; or (h) a bactericidal effective amount of at least one RNA comprising at least one fragment that is essentially complementary to, or has at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60; or (i) an RNA molecule that, when transfected into or contacted with said Botrytis cinerea, causes death, reduced growth, reduced virulence or pathogenicity, or reduced reproductive / proliferative potential of Botrytis cinerea on a plant, wherein said RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 contiguous nucleotides that are essentially complementary to or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60; or (j) a double-stranded RNA molecule that, when transfected into or contacted with the Botrytis cinerea, causes death, reduced growth, reduced virulence or pathogenicity, or reduced reproductive / proliferative potential of Botrytis cinerea on Vitis vinifera, wherein at least one strand of the fungicidal double-stranded RNA molecule is essentially complementary to, or contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60; or (k) a double-stranded RNA molecule that, when transfected into or in contact with the Botrytis cinerea, causes death, reduced growth, reduced virulence or pathogenicity, or reduced reproductive / proliferative ability of the Botrytis cinerea on the plant, wherein at least one strand of the fungicidal double-stranded RNA molecule has at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60.

[0011] In certain embodiments, the agent comprising the polynucleotide is formulated, for example, as a sprayable solution or emulsion, tank mix, or powder for application to the plant field. In some embodiments, the agent is produced biologically, for example, in the form of a microbial fermentation product, or expressed in a transgenic plant cell.

[0012] Any suitable DNA encoding the RNAi molecule that targets the target gene described herein can be used in the compositions and methods described herein.The DNA can be single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA).In some embodiments, the DNA comprises one or more DNA expression cassette(s) that when transcribed produces a single-stranded RNA (ssRNA) molecule (e.g., remains single-stranded or folds into an RNA hairpin), or a complementary ssRNA molecule that anneals to produce a double-stranded RNA (dsRNA) molecule.

[0013] Some embodiments relate to a method of providing a plant with enhanced resistance to Botrytis cinerea infection, comprising topically applying to the plant a composition comprising at least one polynucleotide having at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a corresponding fragment of the DNA or its DNA complement having a sequence selected from the group consisting of target gene sequences (e.g., a fragment of 21 contiguous nucleotides having a sequence of 100% identity). In one embodiment, the method of providing a plant with enhanced resistance to Botrytis cinerea infection comprises topically applying to the plant a composition comprising at least one polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or in some embodiments, a nucleotide sequence complementary to at least 18 contiguous nucleotides of a target gene having a sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, 12, or an RNA transcribed from the target gene. In some embodiments, the at least one polynucleotide is selected from the group consisting of an RNA trigger sequence or an RNA trigger sequence reverse complement, or comprises a nucleotide sequence that is at least about 75% or at least about 80% or at least about 85% or at least about 90% or at least about 95% or at least about 98% or about 100% or 100% identical to the RNA trigger sequence or the RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA comprising one or more sequences selected from the RNA trigger sequence and a corresponding sequence selected from the RNA trigger sequence reverse complement. In one embodiment, a method for providing a plant with enhanced resistance to Botrytis cinerea infection comprises topically applying to the plant a composition comprising at least one polynucleotide such that an effective amount of the polynucleotide is transfected or contacted with Botrytis cinerea, the polynucleotide comprising at least 18 contiguous nucleotides complementary to a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or a portion of an RNA transcribed from the target gene.In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a group of trigger sequences, a group of RNA trigger sequences, or a group of reverse complements of RNA trigger sequences, or comprises a nucleotide sequence that is at least about 75% identical to an RNA trigger sequence or a reverse complement of an RNA trigger sequence. In some embodiments, the polynucleotide is a dsRNA. In some embodiments, the polynucleotide is a dsRNA that comprises one or more sequences selected from an RNA trigger sequence and one or more corresponding sequences selected from a reverse complement of an RNA trigger sequence. Some embodiments relate to compositions that comprise the polynucleotide that are formulated, for example, as a sprayable solution or emulsion, tank mix, or powder for application to a field of plants. In some embodiments, the plant is a fruit-bearing plant, a vegetable, or an ornamental plant. In some embodiments, the plant is a grape, tomato, strawberry, bean, eggplant, chili pepper, bell pepper, tomatillo, ground cherry, cape gooseberry, tobacco, apple, pear, quince, peach, plum, cherry, almond, apricot, blackberry, blueberry, raspberry, carnation, petunia, or rose.

[0014] Some embodiments relate to fungicidal compositions for controlling Botrytis cinerea comprising a fungicidally effective amount of at least one polynucleotide molecule comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a corresponding fragment of DNA or its DNA complement having a sequence selected from the group consisting of target gene sequences. In some embodiments, the polynucleotide molecule comprises at least 18 contiguous nucleotides that are complementary to a portion of a target gene, or RNA transcribed from said target gene, having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or in some embodiments, a sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, 12. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a group of trigger sequences, a group of RNA trigger sequences, or a reverse complement of an RNA trigger sequence, or comprises a nucleotide sequence that is complementary to or at least about 75% identical to a nucleotide sequence selected from a group of trigger sequences, an RNA trigger sequence, or an RNA trigger sequence reverse complement. In some embodiments, the polynucleotide molecule is RNA. In some embodiments, the polynucleotide is dsRNA comprising one or more sequences selected from an RNA trigger sequence and one or more corresponding sequences selected from an RNA trigger sequence reverse complement. In some embodiments, the polynucleotide molecule is a recombinant polynucleotide. In some embodiments, the polynucleotide molecule is double-stranded RNA.Related embodiments include fungicidal compositions comprising polynucleotide molecules formulated, for example, as a sprayable solution or emulsion, tank mix, or powder for application to a field of plants, and optionally including one or more additional components, such as a carrier agent, a surfactant, an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide fungicide, a polynucleotide insecticide, a non-polynucleotide insecticide, a polynucleotide insecticide, a non-polynucleotide insecticide, a polynucleotide fungicide, a safener, and a pathogen growth regulator.

[0015] Some embodiments relate to a method of providing a plant with enhanced resistance to Botrytis cinerea infection, comprising expressing in the plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a corresponding fragment of a DNA or its DNA complement (e.g., a fragment of 21 contiguous nucleotides with a sequence of 100% identity or complementarity) having a sequence selected from the group consisting of target gene sequences. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a group of trigger sequences, a group of RNA trigger sequences, or a group of RNA trigger sequence reverse complements, or comprises a nucleotide sequence that is at least about 75% or at least about 80% or at least about 85% or at least about 90% or at least about 95% or at least about 98% identical to a sequence selected from a RNA trigger sequence or a RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA comprising one or more sequences selected from a RNA trigger sequence and a corresponding sequence selected from a RNA trigger sequence reverse complement.

[0016] Some embodiments relate to a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element comprising at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a corresponding fragment of DNA or its DNA complement having a sequence selected from the group consisting of target gene sequences. In some embodiments, the DNA element encodes a dsRNA. In some embodiments, the dsRNA comprises one or more nucleotide sequences selected from a group of trigger sequences, an RNA trigger sequence, or an RNA trigger sequence reverse complement. Related embodiments include a plant chromosome or plastid or a recombinant plant virus vector or a recombinant baculovirus vector comprising the DNA element with or without a heterologous promoter comprising the recombinant DNA construct.

[0017] Some embodiments relate to transgenic plant cells having recombinant DNA in their genomes encoding RNA that suppresses expression of a target gene in Botrytis cinerea that is contacted with or transfected with RNA, the RNA comprising at least one silencing element having at least one fragment of 18 or more contiguous nucleotides that is complementary to a fragment of the target gene. In some embodiments, the target gene is selected from a group of target gene sequences. Particular embodiments are transgenic plant cells having recombinant DNA in their genomes encoding RNA for silencing one or more target genes selected from a group of target gene sequences. In some embodiments, the RNA comprises one or more nucleotide sequences selected from a group of trigger sequences, a group of RNA trigger sequences, or a group of RNA trigger sequence reverse complements, or comprises a nucleotide sequence that is at least about 75% or at least about 80% or at least 85% or at least about 90% or at least about 95% or at least about 98% or about 100% or 100% identical to a sequence selected from a group of RNA trigger sequences or RNA trigger sequence reverse complements.

[0018] Some embodiments relate to an isolated recombinant RNA molecule that, when transfected or contacted with Botrytis cinerea on a plant, causes death, reduced growth, reduced virulence or pathogenicity, or reduced proliferation / fertility (sporulation) of Botrytis cinerea, the recombinant RNA molecule comprising at least one fragment of 18 or more contiguous nucleotides that is essentially complementary to a corresponding fragment of DNA or its DNA complement having a sequence selected from the group consisting of target gene sequences (e.g., a fragment of 21 contiguous nucleotides having a sequence of 100% complementarity). In some embodiments, the recombinant RNA molecule is double-stranded RNA. Particular embodiments include isolated recombinant double-stranded RNA molecules having a strand with a sequence selected from the group consisting of SEQ ID NOs: 13-60, or combinations thereof. Another embodiment relates to isolated recombinant double-stranded RNA molecules having a strand with a sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 35, SEQ ID NO: 36. Another embodiment relates to an isolated recombinant dsRNA molecule having a strand with a sequence selected from the group consisting of SEQ ID NOs: 25-48.

[0019] Some embodiments relate to a method of providing a plant with enhanced resistance to Botrytis cinerea infection, comprising providing to the plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a corresponding fragment of a target gene selected from a group of target gene sequences. In one embodiment, the method of providing a plant with enhanced resistance to Botrytis cinerea infection comprises providing to the plant at least one polynucleotide comprising at least one fragment that is identical or complementary to at least 18 contiguous nucleotides of a target gene or an RNA transcribed from the target gene, wherein the target gene is selected from the group of genes identified in the group of target gene sequences. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a group of trigger sequences, a group of RNA trigger sequences, or a group of RNA trigger sequence reverse complements, or comprises a nucleotide sequence that is at least about 75% identical to an RNA trigger sequence or an RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA. In some embodiments, the dsRNA comprises one or more sequences selected from an RNA trigger sequence and one or more corresponding sequences selected from an RNA trigger sequence reverse complement.

[0020] Some embodiments relate to a method for controlling Botrytis cinerea infection in a plant, comprising contacting Botrytis cinerea with a polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a corresponding fragment of equivalent length of a portion of a DNA sequence of a target gene selected from a group of target gene sequences (e.g., a fragment of 21 contiguous nucleotides having a sequence of 100% identity or complementarity). In some embodiments, the polynucleotide is double-stranded RNA.

[0021] Some embodiments relate to artificial compositions comprising at least one polynucleotide described herein. In some embodiments, formulations useful for topical application to plants in need of protection from Botrytis cinerea infection are provided. In some embodiments, recombinant constructs and vectors useful for making transgenic plant cells and transgenic plants are provided. In some embodiments, formulations and coatings useful for treating plants, plant seeds, or propagable parts are provided. In some embodiments, commercial products and foodstuffs produced from such plants, seeds, or propagable parts treated with or containing the polynucleotides described herein, particularly commercial products and foodstuffs having detectable amounts of the polynucleotides described herein, are provided. Some embodiments relate to polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the target gene sequence group. Another aspect relates to polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the trigger sequence group or their complements. Such antibodies are made by routine methods known to those skilled in the art.

[0022] Other aspects and specific embodiments of the present invention are disclosed in the following detailed description. [Brief description of the drawings]

[0023] [Figure 1] 1 is a graph showing percent disease severity of Botrytis cinerea on whole inoculated plants treated with Trigger GS349 compared to untreated. [Diagram 2] 1 is a graph showing percent disease severity of Botrytis cinerea across inoculated plants treated with GS413 compared to untreated. [Diagram 3]1 is a graph showing percent disease severity of Botrytis cinerea on whole inoculated plants treated with GS686 compared to untreated. [Figure 4] 1 is a graph showing percent disease severity of Botrytis cinerea across inoculated plants treated with GS728 compared to untreated. [Diagram 5] 1 is a graph showing percent disease severity of Botrytis cinerea across inoculated plants treated with GS730 compared to untreated. [Figure 6] FIG. 1 is a graph comparing disease severity, measured as area under the symptom development curve, of Botrytis cinerea infection in strawberry seedlings treated with GS349, GS730, the chemical standard program, the biological standard program, and untreated controls in an outdoor field trial. [Figure 7] FIG. 1 is a graph comparing disease severity, measured as the area under the symptom development curve, of Botrytis cinerea infection in grapevine seedlings treated with GS730, the chemical standard program, the biological standard program, and untreated controls in an outdoor field trial. [Figure 8] FIG. 1 is a graph comparing disease severity, measured as the area under the symptom development curve, of Botrytis cinerea infection in grapevine seedlings treated with GS730, the chemical standard program, the biological standard program, and untreated controls in an outdoor field trial. [Figure 9] FIG. 1 is a graph comparing disease severity, measured as the area under the symptom development curve, of Botrytis cinerea infection between green bean seedlings treated with GS349, GS2280, GS2303, and GS2297, the chemical standard program, the biological standard program, and untreated controls. [Figure 10] FIG. 1 is a graph comparing disease severity, measured as the area under the symptom development curve, of Botrytis cinerea infection in strawberry seedlings treated with GS349, the chemical control program, the biological control program, and untreated controls in an outdoor field trial. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The sequence file 16206-013PC0 is submitted herein as a 96 kB xml file, and Appendix A containing the sequences set forth in the xml file is provided herein and is incorporated herein in its entirety.

[0025] I. Definition Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by those skilled in the art to which this invention belongs. When a term is described in the singular, the inventors also contemplate the embodiment of the invention described by the plural of that term. In the event of a discrepancy in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their usual meaning in the art in which they are used, as exemplified by various dictionaries specialized in the art, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to any particular mechanism or mode of action, and references thereto are provided for illustrative purposes only.

[0026] Unless otherwise stated, nucleic acid sequences in the text of this specification are given in the 5' to 3' direction when read from left to right. Those skilled in the art will recognize that a given DNA sequence is understood to define a corresponding RNA sequence that is identical to the DNA sequence except for the replacement of thymine (T) nucleotides in the DNA with uracil (U) nucleotides. Thus, providing a particular DNA sequence is understood to define the exact RNA equivalent. A given first polynucleotide sequence, whether DNA or RNA, further defines its exact complementary sequence (which may be DNA or RNA), i.e., the sequence of a second polynucleotide that hybridizes perfectly to the first polynucleotide by forming Watson-Crick base pairs. In the case of a DNA:DNA duplex (hybridized strand), the base pairs are adenine:thymine or guanine:cytosine. In the case of a DNA:RNA duplex, the base pairs are adenine:uracil or guanine:cytosine. Thus, the nucleotide sequence of a blunt-ended double-stranded polynucleotide that is fully hybridized (there is "100% complementarity" between the strands, or the strands are "complementary") is clearly defined by providing the nucleotide sequence of one strand, regardless of whether it is provided as DNA or RNA. "Essentially identical" or "essentially complementary" to a target gene or a fragment of a target gene means that the polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize to the target gene or a fragment of the target gene, or to a transcript of the target gene or a fragment of the target gene (under physiological conditions, such as those generally found in plants or fungal cells). Those skilled in the art will understand that such hybridization does not necessarily require 100% sequence identity or complementarity. In some embodiments, the trigger may be designed to be not 100% identical to the sequence of the target gene, but remain complementary to the sequence of the target gene or the RNA transcribed therefrom. A first nucleic acid sequence is "operably connected" or "linked" with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence.For example, a promoter sequence is "operably linked" to DNA if the promoter provides for transcription or expression of that DNA. Generally, operably linked DNA sequences are contiguous.

[0027] The term "polynucleotide" generally refers to a DNA or RNA molecule that contains multiple nucleotides, and generally refers to both "oligonucleotides" (polynucleotide molecules of 18-25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Polynucleotides also include molecules that contain multiple nucleotides, including non-standard or chemically modified nucleotides, as is commonly practiced in the art. See, for example, the chemical modifications disclosed in the technical manual "RNA interference (RNAi) and DsiRNAs", 2011 (Integrated DNA Technologies Coralville, Iowa). Generally, the polynucleotides described herein, whether DNA, RNA, or both, and whether single-stranded or double-stranded, contain at least one fragment of 18 or more contiguous nucleotides (or in the case of double-stranded polynucleotides, at least 18 contiguous base pairs) that are essentially identical to or complementary to a comparable-sized fragment of the DNA of the target gene or the RNA transcript of the target gene. Throughout this disclosure, "at least 18 contiguous" means "from about 18 to about 10,000, including all integer points in between."Thus, embodiments of the invention include oligonucleotides having a length of 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or intermediate length polynucleotides having a length of 26 or more nucleotides (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 1 41 pieces, 42 pieces, 43 pieces, 44 pieces, 45 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, about 65 pieces, about 70 pieces, about 75 pieces, about 80 pieces, about 85 pieces, about 9 0 pieces, about 95 pieces, about 100 pieces, about 110 pieces, about 120 pieces, about 130 pieces, about 140 pieces, about 150 pieces, about 160 pieces, about 170 pieces, about 180 pieces, about 190 pieces, about 200 pieces, about 210 pieces, about 220 pieces, about 230 pieces, about 240 pieces, about 250 pieces, about 26 0, about 270, about 280, about 290 or about 300 nucleotides), or a long polynucleotide having a length of more than about 300 nucleotides (e.g., about 500 to about 400 nucleotides, about 400 to about 600 nucleotides, about 500 to about 600 nucleotides, about 600 to about 700 nucleotides, about 700 to about 800 nucleotides, about 800 to about 900 nucleotides, about 900 to about 1,000 nucleotides, A polynucleotide may be a polynucleotide of about 300 to about 500 nucleotides, about 300 to about 600 nucleotides, about 300 to about 700 nucleotides, about 300 to about 800 nucleotides, about 300 to about 900 nucleotides, or about 1,000 nucleotides in length, or greater than about 1,000 nucleotides in length (e.g., up to the full length of the target gene, including the coding or non-coding portions of the target gene, or both). When the polynucleotide is double-stranded, its length can similarly be described in terms of base pairs.

[0028] The polynucleotides described herein can be single-stranded (ss) or double-stranded (ds). "Double-stranded" generally refers to base pairing that occurs between sufficiently complementary antiparallel nucleic acid strands to form a double-stranded nucleic acid structure under physiologically relevant conditions. Embodiments include those in which the polynucleotide is selected from the group consisting of sense single-stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), double-stranded DNA / RNA hybrid, antisense ssDNA, or antisense ssRNA, and mixtures of any of these types of polynucleotides. In some embodiments, the polynucleotide is a double-stranded RNA that is longer than the length typical of naturally occurring small regulatory RNAs (such as endogenously generated siRNAs and mature miRNAs). In some embodiments, the polynucleotide is a double-stranded RNA that is at least about 30 contiguous base pairs in length. In some embodiments, the polynucleotide is a double-stranded RNA that has a length of about 50 to about 600 base pairs. In some embodiments, a polynucleotide can include components other than standard ribonucleotides, for example, one embodiment is RNA that includes terminal deoxyribonucleotides.

[0029] In various embodiments, the polynucleotides described herein comprise naturally occurring nucleotides, such as those present in DNA and RNA. In certain embodiments, the polynucleotide is a combination of ribonucleotides and deoxyribonucleotides (e.g., a synthetic polynucleotide that is primarily composed of ribonucleotides but has one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides, or a synthetic polynucleotide that is primarily composed of deoxyribonucleotides but has one or more terminal dideoxyribonucleotides). In certain embodiments, the polynucleotide comprises non-standard nucleotides, such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide comprises chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art. See, e.g., U.S. Patent Publication Nos. 2011 / 0171287, 2011 / 0171176, 2011 / 0152353, 2011 / 0152346, and 2011 / 0160082, which are incorporated herein by reference. Illustrative examples include, but are not limited to, the phosphodiester backbones of naturally occurring oligonucleotides or polynucleotides, which can be partially or fully modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with fluorescent moieties (e.g., fluorescein or rhodamine) or other labels (e.g., biotin).

[0030] Some embodiments relate to a target gene selected from the group consisting of genes identified in the target gene sequence group (Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1), a DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript of any of them, or a polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of the DNA or RNA complement of any one of the foregoing. In some embodiments, the number of consecutive nucleotides is at least 18, e.g., 18-24, or 18-28, or 20-30, or 20-50, or 20-100, or 50-100, or 50-500, or 100-250, or 100-500, or 200-1,000, or 500-2,000, or more. In some embodiments, the number of consecutive nucleotides is more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, or more than 500 contiguous nucleotides.In some embodiments, the polynucleotide comprises at least one fragment of at least 18, 19, 20, or 21 (references to at least 18, 19, 20, or 21 as used throughout shall mean that any of these lower limits of the group may be individualized) contiguous nucleotides having a sequence of 100% identity to a target gene selected from the group consisting of genes identified in a target gene sequence group, a DNA or target gene having a sequence selected from a target gene sequence group or a trigger sequence group, or an RNA transcript of any of them, or a fragment of equivalent length of the DNA or RNA complement of any of the foregoing. In some embodiments, the polynucleotide is a double-stranded nucleic acid (e.g., dsRNA) having one strand containing at least one fragment of at least 18, 19, 20, or 21 consecutive nucleotides with 100% identity to a target gene selected from the group consisting of genes identified in the target gene sequence group, a DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript of any one of them, or a fragment of equivalent length of the DNA or RNA complement of any one of the foregoing. Such double-stranded nucleic acid expressed as base pairs contains at least one fragment of at least 18 consecutive, perfectly matched base pairs corresponding to a fragment of equivalent length of a target gene selected from the group consisting of genes identified in the target gene sequence group, a DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript of any one of them, or a fragment of equivalent length of the DNA or RNA complement of any of the foregoing. In some embodiments, each fragment contained in the polynucleotide is longer than the length typical of naturally occurring small regulatory RNAs, e.g., each fragment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the polynucleotide, or the length of each fragment contained in the polynucleotide, is less than the total length of the DNA or target gene.In some embodiments, the total length of the polynucleotide is about 50 to about 600 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of about 100 to about 600 base pairs, such as a dsRNA of any of the lengths of the dsRNA triggers disclosed in the Figures and Table 1A.

[0031] Some embodiments relate to polynucleotides designed to regulate expression by inducing regulation or repression of Botrytis cinerea target genes. In some embodiments, the Botrytis cinerea target genes are selected from the group consisting of genes identified in the target gene sequence group (Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1). In some embodiments, the polynucleotides are designed to have a nucleotide sequence that is essentially identical or essentially complementary to the nucleotide sequence of a Botrytis cinerea target gene or a fragment of a cDNA (e.g., a target gene sequence group), or to the sequence of an RNA transcribed from a Botrytis cinerea target gene, which may be a coding or non-coding sequence. These effective polynucleotide molecules that regulate expression may be referred to herein as "polynucleotides," "polynucleotide triggers," "triggers," or "triggers."

[0032] Any size of effective polynucleotide can be used alone or in combination in the various methods and compositions described herein. In some embodiments, a single polynucleotide trigger is used to generate a composition (e.g., a composition for topical application, or a recombinant DNA construct useful for generating transgenic plants). In other embodiments, a mixture or pool of different polynucleotide triggers is used. In such cases, the polynucleotide trigger can be against a single target gene or against multiple target genes.

[0033] The term "isolated" as used herein refers to the separation of a molecule from other molecules with which it is normally associated in its native or natural state. Thus, the term "isolated" may refer to a DNA molecule that is separated from other DNA molecule(s) with which it is normally associated in its native or natural state. Such DNA molecules may be present in a recombinant state, such as recombinant DNA molecules. Thus, a DNA molecule that is fused to regulatory or coding sequences with which it is not normally associated, for example as a result of recombinant techniques, is considered to be isolated even if it is integrated into the chromosome of a cell as a transgene or is present together with other DNA molecules.

[0034] As used herein, the term "target gene sequence group" or "target gene sequence" refers to a group of sequences including SEQ ID NOs: 1-12. As used herein, the term "trigger sequence group" or "trigger sequence" refers to a group of sequences including SEQ ID NOs: 13-24, 49-52. As used herein, the term "RNA trigger sequence group" or "RNA trigger sequence" refers to a group of sequences including SEQ ID NOs: 25-36, 53-56. As used herein, the term "RNA trigger sequence reverse complement group" or "RNA trigger sequence reverse complement" refers to a group of sequences including SEQ ID NOs: 37-48, 57-60.

[0035] Some embodiments relate to polynucleotides designed to suppress one or more genes ("target genes"). The term "gene" refers to any portion of a nucleic acid that provides expression of or codes for a transcript. A "gene" may include, but is not limited to, a promoter region, a 5' untranslated region, a transcribed coding region, which may include an intron region, a 3' untranslated region, or a combination of these regions. In some embodiments, the target gene(s) may include coding or non-coding sequences, or both. In other embodiments, the target gene has a sequence that is identical or complementary to a messenger RNA, for example, in some embodiments, the target gene is represented by its corresponding cDNA. In certain embodiments, the polynucleotides are designed to suppress one or more target genes, each of which is selected from the group consisting of genes identified in the target gene sequence group (Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1) or is encoded by a DNA sequence selected from the target gene sequence group. In various embodiments, the polynucleotides are designed to suppress or downregulate one or more target genes, each of which is selected from the group consisting of genes identified in the target gene sequence group or is encoded by a sequence selected from the target gene sequence group, and may be designed to suppress multiple target genes or target different regions of one or more of these target genes. In one embodiment, the polynucleotide comprises a DNA having a sequence selected from a group of target gene sequences, a group of trigger sequences, or a plurality of fragments of 21 consecutive nucleotides having 100% identity to a fragment of equivalent length of the target gene or its DNA complement. In such a case, each fragment may be identical or different in size or sequence, and may be sense or antisense to the target gene.For example, in one embodiment, the polynucleotide comprises multiple fragments in tandem or repeat sequences, each fragment comprising 21 consecutive nucleotides with a sequence of 100% identity to a DNA or target gene having a sequence selected from a gene identified in a target gene sequence group, a target gene sequence group, or a trigger sequence group, or a fragment of equivalent length of the DNA complement of any of the foregoing. In some embodiments, the fragments can be derived from different regions of the target gene, e.g., the fragments can correspond to different exon regions of the target gene. In some embodiments, "spacer" nucleotides that do not correspond to the target gene can be optionally used between or adjacent to the fragments.

[0036] The term "plant" as used herein refers to plants susceptible to Botrytis infection, i.e. Botrytis cinerea infection, unless the context of the text indicates otherwise. Examples of plants susceptible to Botrytis infection include grapes (Vitis vinifera), tomatoes (Solanum lycopersicum), strawberries (Fragaria ananassa), green beans, eggplants, chili peppers, bell peppers, beans, tomatillos, ground cherries, Cape gooseberries, tobacco, apples, pears, quince, peaches, plums, cherries, almonds, apricots, blackberries, blueberries, raspberries, and flowering ornamentals (e.g., roses, petunias, etc.).

[0037] Further definitions are provided in the sections below.

[0038] II. Polynucleotides for control of Botrytis cinerea The polynucleotides of the present disclosure are useful for controlling or preventing Botrytis cinerea infection in plants via RNAi. According to some embodiments of the present disclosure, the polynucleotides are effective in interfering with mRNAs encoded by one or more Botrytis cinerea target genes selected from the group consisting of Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1.

[0039] In some embodiments, the polynucleotide is selected from the group consisting of target gene sequences, or in certain embodiments, DNA having a sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12, or a target gene or its DNA complement, or a corresponding fragment of RNA transcribed therefrom, with about 75% to about 100% identity, about 80% to about 100% identity, about 85% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, about 98% to about 100% identity, about 10 ... In some embodiments, the nucleic acid sequence includes at least one fragment of 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 30 or more, 50 or more, 75 or more, 100 or more, 125 or more, 150 or more, 200 or more, 250 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more contiguous nucleotides having a sequence with 0.00% identity, about 100% identity, or exactly 100% identity. In one embodiment, the polynucleotide comprises a nucleotide sequence essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000 consecutive nucleotides of a DNA or target gene or its DNA complement, or RNA transcribed therefrom, having a sequence selected from the group consisting of target gene sequences, or in certain embodiments, a sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12.

[0040] In some embodiments, the polynucleotide comprises at least 21 contiguous nucleotides that are essentially complementary to a DNA or target gene having a nucleotide sequence selected from the group consisting of target gene sequences, or in certain embodiments, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12, or a corresponding fragment of RNA transcribed therefrom. In some embodiments, the polynucleotide comprises at least 400 contiguous nucleotides that are essentially complementary to a DNA or target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12, or a corresponding fragment of RNA transcribed therefrom. In some embodiments, the polynucleotide is designed to have complementarity to an mRNA encoded by a target gene of Botrytis cinerea. In some embodiments, the target gene of Botrytis cinerea is selected from the group consisting of Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1. In some embodiments, the polynucleotide is a double-stranded RNA. Also, in some embodiments, the double-stranded RNA comprises one strand comprising a sequence selected from the group consisting of SEQ ID NOs: 25-36, 53-56, or in some embodiments, the group consisting of SEQ ID NOs: 26, 31, 33, 35, and 36, and a second strand that is complementary thereto.

[0041] In some embodiments, the polynucleotide comprises a sequence of contiguous nucleotides that is essentially complementary or exactly (100%) identical to a DNA or target gene or its DNA complement, or a fragment of equivalent length of RNA transcribed therefrom, having a sequence selected from a group of target gene sequences, or in certain embodiments, selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12. In some embodiments, the polynucleotide has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a DNA or target gene or its DNA complement, or a fragment of equivalent length of RNA transcribed therefrom, having a sequence selected from a group of target gene sequences, or in certain embodiments, selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12. In some embodiments, the number of consecutive nucleotides is more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, or more than 500 contiguous nucleotides. In some embodiments, the polynucleotide comprises at least one fragment of DNA selected from a group of target gene sequences, or in certain embodiments, at least 18, 19, 20, or 21 (references to at least 18, 19, 20, 21, etc. as used throughout shall mean that any of these lower limits of the group may be individualized) having a sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12, or a sequence of 100% identity to a fragment of equivalent length of the target gene or its DNA complement.

[0042] In one embodiment, the polynucleotide comprises at least one fragment of 21 contiguous nucleotides that is essentially complementary or 100% identical to a corresponding fragment of a DNA or target gene or its DNA complement, or RNA transcribed therefrom, having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12. In some embodiments, the polynucleotide comprises one or more fragments of 21 contiguous nucleotides that have 100% identity to a corresponding fragment of the DNA or target gene, as well as one or more "neutral" sequences (sequences that have no sequence identity or complementarity to the target gene), and thus the polynucleotide as a whole has less overall complementarity or sequence identity to the DNA or target gene.

[0043] In one embodiment, the polynucleotide comprises a combination of multiple fragments of 21 or more contiguous nucleotides that are complementary or 100% identical to one or more DNA or target genes having a nucleotide sequence selected from a group of target gene sequences, or in certain embodiments, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12, or a corresponding fragment of the DNA complement or RNA transcribed therefrom. In some embodiments, the polynucleotide comprises one or more "neutral" sequences (sequences that have no sequence identity or complementarity to the target gene) in addition to one or more fragments of 21 contiguous nucleotides that are complementary or have 100% sequence identity to a corresponding fragment of the target gene, and thus the polynucleotide as a whole has less overall complementarity or sequence identity to a given target gene. In one embodiment, the polynucleotide comprises a combination of multiple fragments of 21 or more contiguous nucleotides or longer that are complementary or have 100% identity to a target gene or its DNA complement, or corresponding fragments distributed positionally throughout the length of the RNA transcribed therefrom, selected from a group of target gene sequences, or in certain embodiments, selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12. In some embodiments, the polynucleotide comprises one or more fragments of 21 contiguous nucleotides that have 100% identity to corresponding fragments distributed positionally throughout the length of the target gene, in addition to one or more fragments of 21 contiguous nucleotides that have 100% identity to corresponding fragments distributed positionally throughout the length of the target gene, and thus the polynucleotide as a whole has less overall complementarity or sequence identity to a given target gene.

[0044] In some embodiments, the polynucleotide is selected from the group consisting of RNA trigger sequences or RNA trigger sequence reverse complements, or in certain embodiments, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 28, at least 30, at least 32, at least 34, at least 36, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 101, at least 102, at least 1 The sequence may be about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 98% to about 100%, about 100%, or 100% identical to at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 contiguous nucleotides.

[0045] In some embodiments, the polynucleotide comprises a sequence selected from the group consisting of a trigger sequence, an RNA trigger sequence, or an RNA trigger sequence reverse complement, or in certain embodiments, a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60. In some embodiments, the polynucleotide comprises a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 26 and 31.

[0046] Some embodiments relate to polynucleotides comprising a sequence that is about 95% to about 100% identical to a sequence selected from the group consisting of trigger sequences, RNA trigger sequences, or reverse complements of RNA trigger sequences, or in certain embodiments, selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. Some embodiments relate to polynucleotides comprising at least a fragment of 18 or more contiguous nucleotides having a sequence that is about 95% to about 100% identical to a portion of a sequence selected from the group consisting of RNA trigger sequences, or reverse complements of RNA trigger sequences, or in certain embodiments, selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the 18 or more contiguous nucleotides are 21 or more contiguous nucleotides. In some embodiments, the number of consecutive nucleotides is at least 18, e.g., 18-24, or 18-28, or 20-30, or 20-50, or 20-100, or 50-100, or 50-500, or 100-250, or 100-500, or 200-1,000, or 500-2,000, or more. In some embodiments, the number of consecutive nucleotides is more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about The sequence may be 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 600, or more than 600 contiguous nucleotides.In some embodiments, the polynucleotide comprises at least one fragment of at least 18, 19, 20, or 21 (references to at least 18, 19, 20, 21, etc. as used throughout shall mean that any of these lower limits of the group may be individualized) contiguous nucleotides having a sequence that is 100% identical to a fragment of equivalent length found within a sequence selected from the group consisting of trigger sequences, RNA trigger sequences, and RNA trigger sequence reverse complements, or in certain embodiments selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the polynucleotide comprises at least one fragment of at least 200, 300, 400, 500, 550, 575, or 600 contiguous nucleotides having a sequence at least 85% identical to a fragment of equivalent length found in a sequence selected from the group consisting of trigger sequences, RNA trigger sequences, and RNA trigger sequence reverse complements, or in certain embodiments selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the polynucleotide comprises at least one fragment of at least 200, 300, 400, 500, 550, 575, or 600 contiguous nucleotides that is at least 85% identical to a fragment of equivalent length found in SEQ ID NO:26.

[0047] In some embodiments, the polynucleotide is a double-stranded nucleic acid (e.g., dsRNA) having one strand containing at least one fragment of at least 18, 19, 20, 21, 22, 23, 24, 50, 75, 100, 150, 200, 250, 300, 400, 500, 550, 575, or 600 contiguous nucleotides having about 95%-100% identity to a DNA having a sequence selected from a group of target gene sequences or a sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12, in particular a fragment of the target gene or its DNA complement. Such double-stranded nucleic acids, expressed as base pairs, include at least one fragment of at least 18, 19, 20, 21, 22, 23, 24, 50, 75, 100, 150, 200, 250, 300, 400, 500, 550, 575, or 600 contiguous, perfectly matched base pairs corresponding to a fragment of equivalent length of a DNA or target gene or its DNA complement, selected from the group of target gene sequences or having a sequence specifically selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12. In some embodiments, each fragment included in the polynucleotide is longer than a length typical of naturally occurring small regulatory RNAs, e.g., each fragment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the polynucleotide, or the length of each fragment contained in the polynucleotide, is less than the total length of a DNA or target gene having a sequence selected from a group of target gene sequences, or specifically selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12. In some embodiments, the total length of the polynucleotide is about 50 to about 600 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides).

[0048] In some embodiments, the polynucleotide is a dsRNA of about 100 to about 600 base pairs, such as a dsRNA of any of the lengths of the RNA trigger sequences disclosed in the figures and tables. In some embodiments, the dsRNA comprises one strand comprising a sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the dsRNA comprises a strand that includes at least one fragment of at least 200, 300, 400, 500, 550, 575, or 600 contiguous nucleotides having a sequence at least 85%, or at least 90%, or at least 95%, or at least 98%, or 100% identical to a fragment of equivalent length found in a sequence selected from the group consisting of trigger sequences, RNA trigger sequences, and RNA trigger sequence reverse complements, or in certain embodiments selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the dsRNA comprises a strand comprising at least one fragment of at least 200, 300, 400, 500, 550, 575, or 600 contiguous nucleotides that is at least 85% identical to a fragment of equivalent length found in SEQ ID NO:26.

[0049] In some embodiments, the polynucleotide is designed to have complementarity to the mRNA encoded by the target gene. In some embodiments, the target gene is selected from the group consisting of genes identified in the target gene sequence group (Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5 and Bcded1). In some embodiments, the polynucleotide is a dsRNA. In some embodiments, the dsRNA comprises a first strand that binds to (e.g., is essentially complementary to) the mRNA encoded by the target gene and a second strand that is complementary to the first strand. The dsRNA may comprise RNA strands of the same length or different lengths. In some embodiments, the dsRNA comprises a first strand (e.g., an antisense strand) that is the same length as the second strand (e.g., a sense strand). In some embodiments, the dsRNA comprises a first strand (e.g., antisense strand) of a different length than the second strand (e.g., sense strand). The first strand may be about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or more than 20% longer than the second strand. The first strand may be 1-5, 2-5, 2-10, 5-10, 5-15, 10-20, 15-20, or more than 20 nucleotides longer than the second strand. dsRNA molecules can also be assembled from a single oligonucleotide of stem-loop structure, and circular single-stranded RNA with two or more loop structures and a stem that includes self-complementary sense and antisense strands, the self-complementary sense and antisense regions of the RNA molecule are linked by a nucleic acid-based or non-nucleic acid-based linker(s), and the circular RNA can be processed in vivo or in vitro to generate an active RNAi molecule that can mediate RNAi. The RNAi molecule can include a 3' overhang at one end of the molecule, and the other end can be blunt-ended or have an overhang (5' or 3'). When the RNAi molecule includes an overhang at both ends of the molecule, the length of the overhang can be the same or different.

[0050] In some embodiments, the polynucleotide is designed to regulate expression of a protein product of a target gene in Botrytis cinerea selected from the group consisting of genes identified in the target gene sequence group (Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1).

[0051] In some embodiments, the dsRNA comprises one strand that includes one or more nucleotide sequences that are at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to a sequence selected from an RNA trigger sequence or an RNA trigger sequence reverse complement. In some embodiments, the dsRNA comprises at least a fragment of 18, 19, 20, 21, 22, 23, 24, 50, 75, 100, 150, 200, 250, 300, 400, 500, 550, 575, or 600 or more contiguous nucleotides having about 95% to about 100% identity to a portion of a sequence selected from a group of RNA trigger sequences or RNA trigger sequence reverse complements, or in certain embodiments, selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the dsRNA comprises a sequence of 21 consecutive nucleotides with 95% sequence identity to a portion of a sequence selected from the group consisting of SEQ ID NO: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. Such dsRNA may further comprise a second strand that is complementary to the first strand. In some embodiments, the dsRNA comprises a first strand that comprises a nucleotide sequence selected from an RNA trigger sequence, and a second strand that is selected from the corresponding RNA trigger sequence reverse complement or another sequence that is complementary to the sequence of the first strand. Particular embodiments include those in which the polynucleotide is a dsRNA that comprises a first strand that comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 26, 31, 33, 35, and 36.

[0052] III. Polynucleotide Length The RNAi molecules targeted to the target genes provided herein may vary in length. In some embodiments, long RNA (e.g., dsRNA or ssRNA) molecules are applied (e.g., to plants) as fungicides, but it should be understood that after entering the cell, the dsRNA is cleaved by the Dicer enzyme into shorter double-stranded RNA fragments, e.g., having a length of 15-25 nucleotides. Thus, the RNAi molecules of the present disclosure may be delivered as, e.g., 15-25 nucleotide fragments, or as longer double-stranded nucleic acids (e.g., at least 100 nucleotides).

[0053] The total length of the polynucleotide of the invention may be greater than or equal to 18 contiguous nucleotides and may include nucleotides in addition to the contiguous nucleotides having a sequence of about 75% to about 100% identity to a DNA having a sequence selected from the group consisting of target gene sequences or to a fragment of comparable length of the target gene or its DNA complement or RNA transcribed therefrom. Similarly, the polynucleotide of the invention may include one or more sequences that are about 75% to about 100% identical to 18 or more contiguous nucleotides of a sequence selected from the group consisting of trigger sequences, RNA trigger sequences, or RNA trigger sequence reverse complements, and may include additional unrelated sequences. In other words, the total length of the polynucleotide may be greater than the length of the section or fragment of the polynucleotide that is designed to suppress one or more target genes.

[0054] For example, the polynucleotide can have nucleotides adjacent to the "active" fragment that suppresses the target gene (e.g., the "active" fragment can be a sequence that is essentially complementary to a fragment of the target gene or the mRNA transcribed therefrom, and can be a sequence selected from a group of trigger sequences, a group of RNA trigger sequences, or a group of reverse complements of RNA trigger sequences), or can include "spacer" nucleotides between the active fragments, or can have additional nucleotides at the 5' or 3' or both the 5' and 3' ends. In one embodiment, the polynucleotide can include additional nucleotides that are not specifically related (have a sequence that is not complementary or identical) to the sequences disclosed herein for the control of Botrytis cinerea. For example, such polynucleotides can include nucleotides that provide a stabilizing secondary structure or can include nucleotides for convenience of cloning or manufacturing. In one embodiment, the polynucleotide can include additional nucleotides located immediately adjacent to the active fragment. In one embodiment, the polynucleotide includes one such fragment and has an additional 5'G or an additional 3'C or both adjacent to the fragment. In another embodiment, the polynucleotide is a double stranded RNA that includes additional nucleotides to form one or more overhangs, e.g., a dsRNA that includes two deoxyribonucleotides to form a 3' overhang. In other embodiments, the polynucleotide may include one or more of the active fragments listed herein, as well as additional fragments active against other target genes of Botrytis cinerea, or additional fragments active against another fungus or pest.

[0055] Thus, in various embodiments, the nucleotide sequence of the entire polynucleotide is not 100% identical or complementary to the trigger sequence, RNA trigger sequence, or RNA trigger sequence reverse complement, and is not 100% identical or complementary to the sequence of consecutive nucleotides in the target gene. For example, in some embodiments, the polynucleotide comprises at least two fragments of 21 consecutive nucleotides having a sequence of 100% identity to a fragment of DNA or its DNA complement having a sequence selected from the group consisting of target gene sequences, where (1) the at least two fragments are separated by one or more spacer nucleotides, or (2) the at least two fragments are arranged in an order different from the order in which the corresponding fragments are present in DNA or its DNA complement having a sequence selected from the group consisting of target gene sequences.

[0056] Some embodiments relate to polynucleotides designed to regulate expression by inducing downregulation or repression of a target gene of Botrytis cinerea. In some embodiments, the polynucleotides are designed to have a nucleotide sequence that is essentially identical to or essentially complementary to a nucleotide sequence of a target gene or cDNA of Botrytis cinerea (e.g., a group of target gene sequences), or a sequence of an RNA transcribed from a target gene of Botrytis cinerea, which may be a coding or non-coding sequence. These effective polynucleotide molecules that regulate expression may be referred to herein as "polynucleotides," "polynucleotide triggers," "triggers," or "triggers." Examples of such embodiments include polynucleotides that include one or more sequences selected from the group of RNA trigger sequences, the group of RNA trigger sequence reverse complements. Further examples include polynucleotides that include at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a portion of a sequence selected from the group of RNA trigger sequences or the group of RNA trigger sequence reverse complements.

[0057] Any size of effective polynucleotide can be used alone or in combination in the various methods and compositions described herein. In some embodiments, a single polynucleotide trigger is used to generate a composition (e.g., a composition for topical application, or a recombinant DNA construct useful for generating transgenic plants). In other embodiments, a mixture or pool of different polynucleotide triggers is used. In such cases, the polynucleotide trigger can be for a single target gene or multiple target genes.

[0058] IV. Allowed Mismatches As used herein, "essentially identical" or "essentially complementary" means that a polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to a target gene or an RNA transcribed from a target gene (e.g., a transcript) to inhibit expression of the target gene (e.g., affect a reduction in the level or activity of a target gene transcript and / or encoded protein). The polynucleotides described herein do not need to have 100% identity or complementarity to a target gene or an RNA transcribed from a target gene to inhibit expression of a target gene (e.g., affect a reduction in the level or activity of a target gene transcript or encoded protein or provide control of Botrytis cinerea). In some embodiments, a polynucleotide or a portion thereof is designed to be essentially identical to or essentially complementary to a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In some embodiments, the polynucleotide or a portion thereof is designed to be 100% identical or 100% complementary to one or more sequences of 21 consecutive nucleotides in the target gene or RNA transcribed from the target gene. In certain embodiments, an "essentially identical" polynucleotide has 100% sequence identity, or at least about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when compared to the sequence of 18 or more consecutive nucleotides of the endogenous target gene or RNA transcribed from the target gene. In certain embodiments, an "essentially complementary" polynucleotide has 100% sequence complementarity, or at least about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity when compared to a sequence of 18 or more contiguous nucleotides of a target gene or RNA transcribed from a target gene.

[0059] Sequence identity: As used herein, the term "sequence identity" or "identity" in the context of two polynucleotides or polypeptides refers to the residues in the sequences of the two molecules that are the same when aligned for maximum correspondence over a specified comparison window.

[0060] Percent identity is calculated by determining the number of positions where identical nucleotides or amino acid residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. A sequence that is identical in all positions compared to a reference sequence is said to be 100% identical to the reference sequence, and vice versa. The percent identity of two nucleotide sequences can be determined by comparing two optimally aligned sequences of molecules (e.g., nucleic acid sequences or polypeptide sequences) over a comparison window, where the portion of the sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Optimal alignment for comparing two or more sequences can be performed using local or global alignment by various available computer programs. The algorithm of Smith TF and Waterman MS (1981), Identification of common molecular subsequences J. Mol. Biol. 147(1):195-, PubMed:7265238 DOI:10.1016 / 0022-2836(81)90087-5 is a suitable local alignment strategy and is used in tools such as EMBOSS Water (https: / / www.ebi.ac.uk / Tools / psa / emboss_water / ).The algorithm of Needleman SB and Wunsch CD (1970), A general method applicable to the search for similarities in the amino acid sequence of two proteins, J. Mol. Biol. 48 (3): 443-53, PubMed: 5420325, DOI: 10.1016 / 0022-2836 (70) 90057-4 is a suitable global alignment strategy and is utilized in tools such as EMBOSS Needle (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). Depending on the parameters associated with the sequences being compared, either local or global alignment strategies are likely to find the optimal alignment, although both strategies can be utilized to ascertain the optimal alignment that gives the most accurate percent identity.

[0061] The term "about" with respect to numerical values ​​of sequence length means the stated value with a + / - variance of up to 1-5%. For example, about 30 contiguous nucleotides means a range of 27-33 contiguous nucleotides, or any range therebetween. The term "about" with respect to numerical values ​​of percent sequence identity means the stated percentage value with a + / - variance of up to 1-3% rounded to the nearest integer. For example, about 90% sequence identity means a range of 87%-93%. However, the percent sequence identity cannot exceed 100%. Thus, about 98% sequence identity means a range of 95%-100%.

[0062] Polynucleotides containing mismatches to target genes or transcripts can be used in certain embodiments of the compositions and methods described herein. In some embodiments, the variants provided herein contain randomly placed mutations with four nucleotides (A, U, G, C) selected with approximately equal probability for a given mutation. In some embodiments, these mutations can be distributed over a small region of the sequence or distributed broadly throughout the length of the sequence. In some embodiments, the polynucleotide contains at least 18 or at least 19 or at least 21 contiguous nucleotides that are essentially identical or essentially complementary to a fragment of equivalent length in the target gene or transcript of the target gene. In certain embodiments, polynucleotides of 18, 19, 20, or 21 or more contiguous nucleotides that are essentially identical or essentially complementary to a fragment of equivalent length in the target gene or transcript of the target gene can have one or two mismatches to the target gene or transcript (i.e., one or two mismatches between the 21 contiguous nucleotides of the polynucleotide and the fragment of equivalent length in the target gene or transcript of the target gene). In certain embodiments, a polynucleotide of about 50, 100, 150, 200, 250, 300, 350 or more nucleotides that contains a contiguous 18, 19, 20, or 21 or more nucleotide span of identity or complementarity to a fragment of comparable length in a target gene or a transcript of a target gene may have one or more mismatches to the target gene or transcript.

[0063] When designing a polynucleotide with mismatches to an endogenous target gene or to an RNA transcribed from a target gene, mismatches of a particular type and at a particular position that are likely to be tolerated can be used. In a particular embodiment, mismatches formed between adenine and cytosine or guanosine and uracil residues are used, as described by Du et al. (2005) Nucleic Acids Res., 33:1671-1677. (2005) Nucleic Acids Res., 33:1671-1677. In some embodiments, mismatches in the 19 base pair overlap region are at low tolerance positions 5, 7, 8 or 11 (from the 5' end of the 19-nucleotide target), at moderate tolerance positions 3, 4, and 12-17 (from the 5' end of the 19-nucleotide target), and / or at high tolerance positions at either end of the region of complementarity, i.e. positions 1, 2, 18, and 19 (from the 5' end of the 19-nucleotide target), as described by Du et al. (2005) Nucleic Acids Res., 33:1671-1677. Permitted mismatches can be determined empirically by routine assays.

[0064] V. Embedding silencing elements in neutral sequences In some embodiments, the silencing element that contains the sequence corresponding to the target gene and is responsible for the observed suppression of the target gene is embedded in a "neutral" sequence, i.e., inserted with additional nucleotides that have no sequence identity or complementarity to the target gene. Neutral sequences may be desirable, for example, to increase the overall length of the polynucleotide or to confer desirable properties, such as increased binding to the silencing complex. For example, it may be desirable for the polynucleotide to be of a certain size for reasons of stability in production, cost-effectiveness, or effective biological activity, such as silencing efficiency. In some embodiments, neutral sequences are also useful for forming preferred secondary structures, such as loops in hairpin triggers, or as spacers between trigger regions.

[0065] Thus, in one embodiment, a 21 base pair dsRNA silencing element that corresponds to an equivalent length fragment of a target gene in the target gene sequence group and that is found to provide control of Botrytis cinerea infection is embedded in an additional 39 base pair neutral sequence, thereby forming a polynucleotide of about 60 base pairs. In some embodiments, the dsRNA trigger comprises a neutral sequence of about 60 to about 600 base pairs, or 100 to about 500 base pairs, within which is embedded at least one fragment of 21 contiguous nucleotides having a sequence of 100% identity or 100% complementarity to an equivalent length fragment of a target gene having a sequence selected from the target gene sequence group. In another embodiment, a single 21 base pair silencing element with 100% identity or 100% complementarity to a comparable length fragment of a target gene is found to be effective when embedded in a larger section of neutral sequence (e.g., when the total polynucleotide length is about 60 base pairs to about 300 base pairs). In an embodiment where a polynucleotide comprises a region of neutral sequence, the polynucleotide has a relatively low overall sequence identity compared to the target gene. For example, a dsRNA with a total length of 210 base pairs, including a single 21 base pair trigger (100% identity or complementarity to a 21-nucleotide fragment of a target gene) embedded in an additional 189 base pairs of neutral sequence, has an overall sequence identity of about 10% to the target gene.

[0066] VI. Related Technologies The polynucleotide and nucleic acid molecule embodiments described herein may include additional elements, such as promoters, early transcription sequences, transcription initiation elements, transcription elongation elements, transcription termination elements, small RNA recognition sites, aptamers or ribozymes, additional expression cassettes for expressing coding sequences (e.g., to express a transgene such as a bactericidal protein or a selectable marker) or non-coding sequences (e.g., to express additional inhibitory elements). For example, one aspect of the invention provides a recombinant DNA construct comprising a heterologous promoter having a transcription initiation sequence operably linked to DNA comprising at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the complement of a target gene sequence group. Another aspect of the invention provides a recombinant DNA construct comprising a heterologous promoter having a transcription initiation sequence operably linked to DNA encoding an RNA hairpin having an antisense region having a sequence or fragment of a sequence selected from the group selected from a trigger sequence group, an RNA trigger sequence group, or an RNA trigger sequence reverse complement group. In another embodiment, a recombinant DNA construct comprising (a) an RNA silencing element for suppressing a target gene selected from a group of target gene sequences, and (b) a promoter operably linked to DNA encoding an aptamer is stably integrated into the genome of a plant, from where the RNA transcript comprising the RNA aptamer and the RNA silencing element are expressed in the plant's cells, and the aptamer serves to guide the RNA silencing element to a desired location in the cell.In another embodiment, the inclusion of one or more recognition sites for binding and cleavage by small RNA (e.g., by miRNA or siRNA that are expressed only in specific cells or tissues) allows for a more precise expression pattern in the plant, and the expression of the recombinant DNA construct is suppressed at the location where the small RNA is expressed. Such additional elements are described below.

[0067] VII. Control of Botrytis cinerea infection by contact with polynucleotides Provided herein are methods for controlling Botrytis cinerea infection in plants. Such methods include contacting Botrytis cinerea with any of the polynucleotides and other compositions described herein. Some embodiments relate to methods for controlling Botrytis cinerea infection in plants by contacting the plant with any of the polynucleotides described in Section II above or elsewhere herein. Some embodiments relate to contacting Botrytis cinerea with a polynucleotide that suppresses expression of a target gene of Botrytis cinerea selected from the group consisting of Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1. Some embodiments relate to a method of controlling Botrytis cinerea infection in a plant by contacting the plant with a DNA selected from the group consisting of a target gene sequence or a polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides having about 95% to about 100% identity or complementarity to a corresponding fragment of the target gene or its DNA complement. In one embodiment, the method of controlling Botrytis cinerea infection in a plant comprises contacting Botrytis cinerea with a DNA having a DNA sequence selected from the group consisting of SEQ ID NOs: 1-12 or a polynucleotide comprising at least 18 contiguous nucleotides having 100% identity to a corresponding fragment of the target gene or its DNA complement. In another embodiment, the method of controlling Botrytis cinerea infection in a plant comprises contacting the plant with a DNA having a DNA sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12 or a polynucleotide comprising at least 18 contiguous nucleotides having 100% identity to a corresponding fragment of the target gene or its DNA complement. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized, or produced by expression in a microbial organism or expression in a plant cell.Embodiments include those in which the polynucleotide is a dsRNA comprising a strand having a sequence selected from the group consisting of trigger sequences, RNA trigger sequences, or reverse complements of RNA trigger sequences. Embodiments further include those in which the polynucleotide comprises at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a portion of a sequence selected from the group consisting of RNA trigger sequences or reverse complements of RNA trigger sequences. Polynucleotides for use in the method can be designed for multiple target genes. Related aspects of the invention include isolated polynucleotides for use in the method, and plants with improved Botrytis cinerea resistance provided by the method. Particular embodiments include those in which the polynucleotide is a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 26, 31, 33, 35, 36, or a complement thereof.

[0068] In some embodiments, the contiguous nucleotides have a sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a fragment of equivalent length of DNA or a target gene or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA or a target gene or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the polynucleotide has an overall sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a fragment of equivalent length of DNA or a target gene or its DNA complement having a sequence selected from the target gene sequence group.

[0069] In one embodiment, the polynucleotide comprises at least one fragment of 21 contiguous nucleotides that has 100% identity to a corresponding fragment of DNA or the target gene or its DNA complement having a DNA sequence selected from the group consisting of SEQ ID NOs: 1-12. In some embodiments, the polynucleotide comprises "neutral" sequences (sequences that have no sequence identity or complementarity to the target gene) in addition to one or more fragments of 21 contiguous nucleotides that have 100% identity to a corresponding fragment of DNA or the target gene, and thus the polynucleotide as a whole has less overall sequence identity to the target gene.

[0070] In some embodiments, the polynucleotides used in the method are provided as isolated DNA or RNA fragments. In some embodiments, the polynucleotides used in the method are not part of an expression construct and lack additional elements such as promoter or terminator sequences). Such polynucleotides may be relatively short, such as single- or double-stranded polynucleotides of about 18 to about 500 or about 50 to about 600 nucleotides (for single-stranded polynucleotides) or about 18 to about 500 or about 50 to about 600 base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of about 100 to about 600 base pairs, such as a dsRNA of any of the lengths of the dsRNA triggers of SEQ ID NOs: 13-60. Alternatively, the polynucleotides can be provided in more complex constructs, for example, provided as part of a recombinant expression construct, or included in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression constructs or vectors are designed to include additional elements, such as an expression cassette for expressing a gene of interest (eg, a bactericidal protein).

[0071] Some embodiments relate to a method of controlling Botrytis cinerea infection in a plant comprising contacting Botrytis cinerea with a polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of DNA of a target gene selected from the group consisting of genes identified in a group of target gene sequences. In some embodiments, the polynucleotide comprises a dsRNA having a strand with a sequence selected from the group consisting of trigger sequences. In some embodiments, the present invention provides a method of controlling Botrytis cinerea infection in a plant comprising contacting Botrytis cinerea with an effective amount of a solution comprising double-stranded RNA having a strand comprising a sequence selected from the group consisting of RNA trigger sequences, the solution further comprising an organosilicone surfactant and / or other agricultural formulation ingredients known in the art.

[0072] In various embodiments of this method, the contacting comprises application of a suitable composition comprising any of the polynucleotides described herein (e.g., a polynucleotide described in Section II, a dsRNA described in Section VIII, or a composition described in Section IX) to the surface of a plant that is infected or potentially infected with Botrytis cinerea. Such compositions can be provided, for example, as a solid, a liquid (including homogeneous mixtures such as soluble liquid concentrates, and heterogeneous mixtures such as suspensions, colloids, micelles, and emulsions), a powder, a suspension, an emulsion, a spray, an encapsulated or microencapsulated formulation, in or on microbeads or other carrier particles, in a film or coating, or on or in a matrix, or as a leaf, seed, root, or stem treatment. In one embodiment, the surface is a plant leaf, flower, or fruit. In such an embodiment, application can be accomplished by spraying the plant leaf, flower, or fruit. The contacting can be in the form of a seed treatment. As known to those skilled in the art of pesticide formulation and seed treatment, suitable binders, inert carriers, surfactants, and the like can optionally be included in the composition. In some embodiments, the contacting comprises providing the polynucleotide within a composition that further comprises one or more carrier agents and / or one or more surfactants (e.g., organosilicones, organosilicon surfactants), non-polynucleotide fungicides, polynucleotide herbicide molecules, polynucleotide insecticides, non-polynucleotide insecticides, non-polynucleotide herbicide molecules, non-polynucleotide insecticides, polynucleotide insecticides, safeners, and pathogen growth regulators. In one embodiment, the contacting comprises providing the polynucleotide within a composition that can be transfected into or otherwise internally absorbed by Botrytis cinerea on a plant.

[0073] VIII. Bactericidal double-stranded RNA molecules Another aspect of the invention provides a bactericidal double-stranded RNA molecule that, when transfected into or contacted with Botrytis cinerea, causes death, reduced growth, reduced virulence or pathogenicity, or reduced growth / fertility (sporulation) of Botrytis cinerea, the bactericidal double-stranded RNA comprising the nucleotide sequence of any of the polynucleotides described in Section II above or elsewhere herein. Certain embodiments of the invention provide a bactericidal double-stranded RNA molecule that, when transfected into or contacted with Botrytis cinerea, causes death, reduced growth, reduced virulence or pathogenicity, or reduced growth / fertility (sporulation) of Botrytis cinerea on a plant, the bactericidal double-stranded RNA molecule comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical to or essentially complementary to a fragment of equivalent length of a target gene or DNA having a sequence selected from a group of target gene sequences. In some embodiments, the bactericidal dsRNA comprises a first strand that includes one or more sequences selected from a group of trigger sequences, a group of RNA trigger sequences, or a group of reverse complements of RNA trigger sequences. In some embodiments, the bactericidal dsRNA comprises a first strand that includes one or more sequences selected from the group consisting of SEQ ID NOs: 13-60, or at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 50, at least 75, at least 10, at least 12, at least 14, at least 16, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 50, at least 75, at least 10, at least 12, at least 16, at least 18 ... the first strand comprises a sequence that is essentially complementary to, or is about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, 95% to about 100%, about 98% to about 100%, about 100%, or 100% identical to, 0, at least 150, at least 200, at least 300, at least 400, at least 500, at least 550, at least 575, or at least 600 contiguous nucleotides.In some embodiments, the bactericidal dsRNA comprises at least one fragment of 18 or more contiguous nucleotides having about 95% to about 100% identity to a portion of a sequence selected from the group consisting of RNA trigger sequences or RNA trigger sequence reverse complements, or selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the 18 or more contiguous nucleotides is 21 contiguous nucleotides. In some embodiments, the bactericidal dsRNA comprises a first strand comprising a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to a sequence selected from the group consisting of RNA trigger sequences or RNA trigger sequence reverse complements. In some embodiments, the bactericidal dsRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60, or selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the bactericidal dsRNA further comprises a second strand complementary to the first strand. In some embodiments, the bactericidal dsRNA comprises a first strand comprising a nucleotide sequence selected from an RNA trigger sequence, and further comprises a second strand comprising a sequence selected from a corresponding RNA trigger sequence reverse complement. In some embodiments, the bactericidal dsRNA comprises a first strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26, 31, 33, 35, and 36, and further comprises a second strand comprising a sequence selected from a corresponding complementary sequence selected from the group consisting of SEQ ID NOs: 38, 43, 45, 47, and 48.

[0074] The total length of one strand of the bactericidal dsRNA may be greater than or equal to 18 contiguous nucleotides and may include nucleotides in addition to the contiguous nucleotides having about 95% to about 100% sequence to a portion of a sequence selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement, or selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. A bactericidal dsRNA comprising a nucleotide sequence selected from the RNA trigger sequence group and the RNA trigger sequence reverse complement may include nucleotides in addition to the nucleotides of a sequence selected from the RNA trigger sequence group and the RNA trigger sequence reverse complement. In other words, the total length of the dsRNA strand may be greater than the length of a sequence or a portion of a sequence selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement. For example, the dsRNA may have nucleotides adjacent to the "active" fragments that suppress the target gene, or may include "spacer" nucleotides between the active fragments, or may have additional nucleotides at the 5' end or at the 3' end or at both the 5' and 3' ends. In one embodiment, the dsRNA may include additional nucleotides that are not specifically related (have a sequence that is not complementary or identical) to the target gene targeted by a given trigger, such as nucleotides that provide a stabilizing secondary structure or provide convenience in cloning or manufacturing. In one embodiment, the dsRNA may include additional nucleotides located immediately adjacent to a sequence or a portion of a sequence selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement group. In one embodiment, the dsRNA includes one such fragment with an additional 5'G or an additional 3'C or both adjacent to the fragment. In another embodiment, the dsRNA further includes additional nucleotides to form an overhang, for example, the dsRNA includes two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire dsRNA is not 100% identical or complementary to the RNA trigger sequence or the RNA trigger sequence reverse complement.For example, in some embodiments, the dsRNA comprises at least two fragments of 21 contiguous nucleotides having 100% sequence identity to a portion of a sequence selected from the RNA trigger sequence or the RNA trigger sequence reverse complement, and (1) the at least two fragments are separated by one or more spacer nucleotides, or (2) the at least two fragments are arranged in an order different from the order in which the corresponding fragments are present in the target gene.

[0075] In some embodiments, the bactericidal dsRNA molecule is about 50 to about 600 base pairs in length. In some embodiments, the bactericidal dsRNA molecule comprises a plurality of fragments of 18 or more contiguous nucleotides that are essentially identical to or essentially complementary to equivalent length fragments of a target gene or DNA having a sequence selected from a group of target gene sequences, the fragments being derived from different regions of the target gene (e.g., the fragments can correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene can optionally be used between or adjacent to the fragments) or from different target genes. In some embodiments, the bactericidal dsRNA molecule comprises a plurality of fragments of 18 or more contiguous nucleotides that are essentially identical to or essentially complementary to equivalent length fragments of a target gene or DNA having a sequence selected from a group of target gene sequences, the fragments being derived from different regions of the target gene, and arranged in the bactericidal dsRNA molecule in an order that is different from the order in which the fragments naturally occur in the target gene. In some embodiments, the bactericidal dsRNA molecule comprises multiple fragments of 18 contiguous nucleotides having sequences that are 100% identical or 100% complementary to fragments of equivalent length of a target gene or DNA having a sequence selected from a group of target gene sequences, said fragments being derived from different regions of the target gene and arranged in the bactericidal double-stranded RNA molecule in an order that is different from the order in which the fragments naturally occur in the target gene. In some embodiments, the sRNA molecule comprises one strand that comprises a sequence selected from the group consisting of a group of trigger sequences, or a complement thereof.

[0076] The fungicidal dsRNA molecule can be applied topically to plants to control or prevent infection by Botrytis cinerea. The fungicidal dsRNA molecule can be provided in a form suitable for transfection or direct contact by Botrytis cinerea, for example in the form of a spray or powder. Other methods and suitable compositions for providing the fungicidal dsRNA molecule are similar to those described herein for other aspects of the invention.

[0077] Some embodiments relate to tank mixes comprising one or more fungicidal polynucleotides, water or other solvent, and optionally an organosilicone surfactant. An embodiment comprises a tank mix formulation of polynucleotides and optionally at least one insecticide. Such composition embodiments include those in which the one or more fungicidal polynucleotides are provided within a living or dead microorganism, such as a bacterial or fungal or yeast cell, or provided as a microbial fermentation product, or provided within a living or dead plant cell, or provided as a synthetic recombinant polynucleotide. In one embodiment, the composition comprises a non-pathogenic strain of a microorganism comprising a polynucleotide, as described herein. Ingestion of the microorganism causes inhibition of growth, reduced virulence or pathogenicity, reduced growth / fertility (sporulation), or death of Botrytis cinerea. Non-limiting examples of suitable microorganisms include E. coli, B. thuringiensis, Pseudomonas spp., Photorhabdus spp., Xenorhabdus spp., Serratia entomophila and related Serratia spp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae, Clostridium bifermentans and other Clostridium species, or other sporulating Gram-positive bacteria. In one embodiment, the composition comprises a plant virus vector comprising a polynucleotide as described herein. Treatment of plants infected with Botrytis cinerea with a plant virus vector causes reduced growth, death, or reduced growth / fertility (sporulation) of Botrytis cinerea. In one embodiment, the composition comprises a baculovirus vector comprising a polynucleotide as described herein. Ingestion of the vector causes reduced growth, death, or reduced virulence or pathogenicity, or reduced growth / fertility (sporulation) of Botrytis cinerea. In one embodiment, the polynucleotides described herein are encapsulated in a synthetic matrix, such as a polymer, or attached to a microparticle and applied topically to the surface of the plant.Topical treatment of plants infected with Botrytis cinerea causes suppressed growth, death, or reduced toxicity or pathogenicity, or reduced growth / reproductive ability (sporulation) of Botrytis cinerea. In one embodiment, the polynucleotide described herein is provided in the form of a plant cell (e.g., a transgenic plant cell of the invention) expressing the polynucleotide. The plant cell or the contents of the plant cell against Botrytis cinerea infection causes suppression, death, or reduced toxicity or pathogenicity, or reduced growth / reproductive ability (sporulation) of Botrytis cinerea on the plant.

[0078] In some embodiments, one or more polynucleotides described herein are provided with suitable adhesives and wetting agents necessary for efficient leaf coverage, and UV protectants to protect polynucleotides such as dsRNA from UV damage.In some embodiments, one or more polynucleotides described herein are further provided with carrier agents, surfactants, organosilicones, organosilicon surfactants, non-polynucleotide fungicides, polynucleotide herbicide molecules, non-polynucleotide herbicide molecules, non-polynucleotide insecticides, polynucleotide insecticides, polynucleotide insecticides, non-polynucleotide insecticides, toxic mitigators, and pathogen growth regulators.In some embodiments, the composition further comprises at least one insecticide or fungicide.

[0079] Such compositions are applied by any convenient method, for example, by spraying or dusting directly onto Botrytis cinerea, or by spraying or dusting onto plants (e.g., including the leaves, stems, and / or flowers of the plants) or the environment in which it is desired to prevent or control infection with Botrytis cinerea, or by applying a coating to the surface of a plant, or by applying a coating to seeds in preparation for planting the seeds, or by applying a soil drench around the roots of plants in which it is desired to prevent or control infection with Botrytis cinerea.

[0080] An effective amount of the polynucleotide described herein is an amount sufficient to provide control of Botrytis cinerea (e.g., by causing death, growth inhibition, toxicity or virulence, growth or reproduction inhibition or reduction of Botrytis cinerea) or prevent infection by Botrytis cinerea. Determination of the effective amount of polynucleotide is performed by routine assays. There is no upper limit to the concentration and dose of fungicidal polynucleotide that may be useful in the methods and compositions provided herein, although lower effective concentrations and doses are generally desired for efficiency and economy. Non-limiting embodiments of effective amounts of polynucleotide include about 10 nanograms to about 100 micrograms of polynucleotide per milliliter in liquid form sprayed on a single plant, or about 10 milligrams to about 100 grams of polynucleotide per acre applied to a field of plants. When the polynucleotides described herein are applied topically to plants, the concentration can be adjusted to account for the amount of spray or treatment applied to the surface of the plant leaves or other plant parts such as petals, stems, fruits, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using the 25-mer polynucleotides described herein is about 1 nanomole (nmol) of polynucleotide per plant, e.g., about 0.05 to 1 nmol of polynucleotide per plant. Other embodiments of herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotide per plant. In certain embodiments, about 40 to about 50 nmol of ssDNA polynucleotide is applied. In certain embodiments, about 0.5 nmol to about 2 nmol of dsRNA is applied. In certain embodiments, compositions containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of dsRNA or ssDNA (21-mer) are applied. In certain embodiments, compositions of about 0.5 to about 1.5 milligrams per milliliter of dsRNA polynucleotides of the present invention of about 50 to about 200 or more nucleotides are applied.In certain embodiments, about 1 nmol to about 5 nmol of the dsRNA of the present invention is applied to the plant. In certain embodiments, the polynucleotide composition topically applied to the plant contains at least one polynucleotide of the present invention at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. In some embodiments, a concentration of about 5 g to about 100 g of polynucleotide active ingredient per hectare is applied. Very large plants, trees, or vines may require correspondingly larger amounts of polynucleotide. Lower concentrations can be used when using long dsRNA molecules of the present invention that can be processed into multiple oligonucleotides (e.g., multiple triggers encoded by a single recombinant DNA molecule of the present invention). Non-limiting examples of effective polynucleotide treatment regimens include treatment with about 0.1 to about 1 nmol of polynucleotide molecule per plant, or about 1 nmol to about 10 nmol of polynucleotide molecule per plant, or about 10 nmol to about 100 nmol of polynucleotide molecule per plant.

[0081] In some embodiments, one or more polynucleotides are provided with a "transfer agent," which is an agent that allows the topically applied polynucleotide to enter the cells of the organism. Such a transfer agent can be incorporated as part of a composition comprising the polynucleotide described herein, or can be applied before, after, or simultaneously with the application of the polynucleotide. In some embodiments, the transfer agent is an agent that improves uptake of the polynucleotide of the invention by Botrytis cinerea. In some embodiments, the transfer agent is an agent that conditions the surface of the plant tissue, e.g., seeds, leaves, stems, roots, flowers, or fruits, for penetration into the plant cells by the polynucleotide. In some embodiments, the transfer agent allows a pathway for the polynucleotide to pass through the epidermal wax barrier, stomata, and / or cell wall or membrane barriers to enter the plant cells.

[0082] Suitable transfer agents include agents that increase the permeability of the outside of the organism or increase the permeability of the cells of the organism to the polynucleotide. Suitable transfer agents include chemical agents, physical agents, or combinations thereof. Chemical agents for conditioning or transferring include (a) surfactants, (b) organic solvents or aqueous solutions or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or any combination thereof. In some embodiments, application of the polynucleotide and transfer agent optionally includes an incubation step, a neutralization step (e.g., to neutralize the acid, base, or oxidizing agent or to inactivate the enzyme), a water washing step, or a combination thereof. Suitable transfer agents may be in the form of an emulsion, a reverse emulsion, a liposome, or other micelle-like composition, or may cause the polynucleotide to take the form of an emulsion, a reverse emulsion, a liposome, or other micelle-like composition. The transfer agent embodiment includes counterions or other molecules known to bind to nucleic acid molecules, such as inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine or putrescine, and other cations. The transfer agent embodiment includes organic solvents such as DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or other solvents that are miscible with water or dissolve phosphonucleotides in non-aqueous systems (e.g., those used in synthesis reactions). The transfer agent embodiment includes naturally derived or synthetic oils with or without surfactants or emulsifiers, such as oils of plant origin, crop oils (e.g., those listed in the 9th Compendium of Herbicide Adjuvants, publicly available online at herbicide.adjuvants.com), paraffin oils, polyol fatty acid esters, or oils with short chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.

[0083] The embodiment of the introduction agent includes an organosilicone formulation. For example, a suitable introduction agent is an organosilicone formulation, commercially available as SILWET L-77® brand surfactant with CAS number 27306-78-1 and EPA number: CAL.REG.NO.5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY. BREAK-THRU S 240 brand polyether modified polysiloxane (CASRN proprietary) surfactant, currently available from Goldschmidt Chemical Corporation, Hopewell, VA. BREAK-THRU S 279 end-capped polyether trisiloxane surfactant, whose components are listed in the following existing chemicals list: EINECS, TSCA, ENCS, AICS, ECL, PICCS CHINA, NDSL. INDUCE brand adjuvant NMFC item 42652, class 60, currently available from Helena Chemical Company, Collierville, TN. FRANCHISE® with LECI-TECH® brand surfactants, having CA REG No. 34704-50065, currently available from Loveland Products, Inc. Greely, Colo. One embodiment includes a composition comprising a polynucleotide and BREAK-thru 301. In one embodiment, the polynucleotide and Silwet L-77, break-thru, in the range of about 0.015 to about 2 weight percent (wt%) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt%). These include compositions that include an introducer, such as S240, Break-thru S279, Induce or Franchise, which include an organosilicone formulation.One embodiment includes a composition comprising a polynucleotide of the invention and an induction agent comprising SILWET L-77®, BREAK-THRU S240, BREAK-THRU S279, Induce, or Franchise brand surfactant in the range of about 0.3 to about 1 weight percent (wt%) or about 0.5 to about 1 weight percent (wt%).

[0084] Organosilicone compounds useful as introduction agents for use in the present invention include, but are not limited to, compounds that include (a) a covalently linked trisiloxane head group, (b) a covalently linked alkyl linker, including, but not limited to, an n-propyl linker, (c) a covalently linked polyglycol chain, and (d) a terminal group. The trisiloxane head group of such organosilicone compounds includes, but is not limited to, heptamethyltrisiloxane. The alkyl linker can include, but is not limited to, an n-propyl linker. The polyglycol chain includes, but is not limited to, polyethylene glycol or polypropylene glycol. The polyglycol chain can include a mixture that provides an average chain length "n" of about "7.5". In certain embodiments, the average chain length "n" can vary from about 5 to about 14. The terminal group can include, but is not limited to, an alkyl group, such as a methyl group. Organosilicone compounds useful as introduction agents include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyltrisiloxane. An example of a transfer agent for use in the present invention is Compound I [ka] (Compound I: polyalkylene oxide heptamethyltrisiloxane, average n=7.5).

[0085] Organosilicon compounds useful as introduction agents are used in freshly made concentrations ranging from, for example, about 0.015 to about 2 weight percent (wt%) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt%).

[0086] An embodiment of the introduction agent includes one or more salts, such as ammonium chloride, tetrabutylphosphonium bromide, and ammonium sulfate, provided in or used with a composition comprising a polynucleotide. In some embodiments, the ammonium chloride, tetrabutylphosphonium bromide, and / or ammonium sulfate are used at a concentration of about 0.5% to about 5% (w / v), or about 1% to about 3% (w / v), or about 2% (w / v). In certain embodiments, the composition comprising a polynucleotide includes an ammonium salt at a concentration of 300 millimolar or greater. In certain embodiments, the composition comprising a polynucleotide includes an organosilicone introduction agent at a concentration of about 0.015 to about 2 weight percent (wt%), and ammonium sulfate at a concentration of about 80 to about 1200 mM or about 150 mM to about 600 mM.

[0087] An embodiment of the introduction agent comprises a phosphate salt. Phosphates useful in compositions comprising polynucleotides include, but are not limited to, calcium, magnesium, potassium, or sodium phosphate salts. In certain embodiments, compositions comprising polynucleotides comprise a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, compositions comprising polynucleotides comprise a phosphate salt in the range of about 1 mM to about 25 mM or in the range of about 5 mM to about 25 mM. In certain embodiments, compositions comprising polynucleotides comprise sodium phosphate at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, compositions comprising polynucleotides comprise sodium phosphate at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, compositions comprising polynucleotides comprise a sodium phosphate salt in the range of about 1 mM to about 25 mM or in the range of about 5 mM to about 25 mM. In certain embodiments, the composition comprising the polynucleotide comprises a sodium phosphate salt in the range of about 10 mM to about 160 mM or in the range of about 20 mM to about 40 mM. In certain embodiments, the composition comprising the polynucleotide comprises a sodium phosphate buffer at a pH of about 6.8.

[0088] The embodiment of the transfer agent includes a surfactant and / or an effective molecule contained therein. The surfactant and / or the effective molecule contained therein includes, but is not limited to, sodium or lithium salts of fatty acids (such as tallow or tallow amines or phospholipids) and organosilicone surfactants. In certain embodiments, the composition containing the polynucleotide is formulated with counterions or other molecules known to bind to nucleic acid molecules. Non-limiting examples include tetraalkylammonium ions, trialkylammonium ions, sulfonium ions, lithium ions, and polyamines such as polyethyleneimine, spermine, spermidine, or putrescine. In certain embodiments, compositions comprising a polynucleotide are formulated with a non-polynucleotide herbicide, such as auxin-like benzoic acid herbicides including glyphosate, dicamba, chloramben, and TBA, auxin-like herbicides including glufosinate, phenoxycarboxylic acid herbicides, pyridine carboxylic acid herbicides, quinoline carboxylic acid herbicides, pyrimidine carboxylic acid herbicides, and benazolin ethyl herbicides, sulfonylureas, imidazolinones, bromoxynil, dalapon, cyclohexandione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibitor herbicides.

[0089] IX. Fungicidal compositions for controlling Botrytis cinerea infections Another aspect of the invention provides a fungicidal composition for controlling Botrytis cinerea comprising a fungicidally effective amount of at least one RNA. Such RNA may be any of the RNAs described in Section II or elsewhere herein. In one embodiment, the RNA has about 100% identity, about 75% to about 100% identity, about 80% to about 100% identity, about 85% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, about 98% to about 100% identity, about 100% identity, or a similar sequence to a DNA having a sequence selected from the group consisting of target gene sequences or a corresponding fragment of a target gene or its DNA complement or RNA transcribed therefrom. comprises at least one fragment of 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 30 or more, 50 or more, 75 or more, 100 or more, 125 or more, 150 or more, 200 or more, 250 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1,000 or more contiguous nucleotides of exactly 100% identity. In one embodiment, the RNA comprises a nucleotide sequence that is essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000 consecutive nucleotides of a DNA having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12 or a target gene or its DNA complement or an RNA transcribed from such a target gene.

[0090] In some embodiments, the RNA comprises at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 50, at least 75, at least 1 of a sequence selected from the group consisting of SEQ ID NOs: 13-60, or selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. 00, at least 150, at least 200, at least 300, at least 400, at least 500, at least 550, at least 575, or at least 600 contiguous nucleotides, or a sequence that is essentially complementary to or is about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, 95% to about 100%, about 98% to about 100%, about 100%, or 100% identical to the sequence. In some embodiments, the polynucleotide comprises a sequence that is essentially complementary to, or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to, a sequence selected from an RNA trigger sequence or a reverse complement of an RNA trigger sequence, or a sequence selected from the group consisting of SEQ ID NOs: 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48. In some embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60, or a sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48.

[0091] In this context, "control" includes, but is not limited to, inducing a biological change in Botrytis cinerea, such as increased mortality, reduced growth, reduced toxicity and / or pathogenicity, or reduced growth / fertility (sporulation). "Fungicidal effective amount" or "fungicidal" refers to an amount of an agent effective to induce a biological change in Botrytis cinerea, including, but not limited to, increased mortality, reduced growth, reduced toxicity and / or pathogenicity, and reduced growth / fertility. In some embodiments, application of a fungicidally effective amount of RNA to a plant improves the plant's resistance to Botrytis cinerea infection. The RNA may be longer than one or more of the fragments contained therein (i.e., the RNA may include additional nucleotides 3' and / or 5' of the fragments), but the fragments corresponding to each fragment of the target gene are of comparable length. RNA for use in this method can be designed for multiple target genes. In an embodiment, the fungicidal composition comprises a fungicidally effective amount of a polynucleotide comprising at least 18, 19, 20, or 21 contiguous nucleotides that are complementary to a portion of a DNA or target gene or RNA transcribed therefrom having a nucleotide sequence selected from a group of target gene sequences, or a fungicidally effective amount of at least one polynucleotide comprising at least one silencing element that is essentially complementary or essentially identical to at least 21 contiguous nucleotides of the DNA or target gene or RNA transcribed therefrom, wherein the DNA or target gene has a nucleotide sequence selected from a group of target gene sequences, or a fungicidally effective amount of at least one RNA comprising at least one fragment that is identical or complementary to at least 18, 19, 20, or 21 contiguous nucleotides of the DNA or target gene or RNA transcribed therefrom having a nucleotide sequence of SEQ ID NOs: 1-12, or a fungicidally effective amount of at least one RNA that, when transfected into or contacted with Botrytis cinerea, causes death, inhibits growth, reduces toxicity or pathogenicity of Botrytis cinerea,or a bactericidal double-stranded RNA molecule that, when transfected into or in contact with Botrytis cinerea, causes death, reduced growth, reduced virulence or pathogenicity, or reduced growth / fertility (sporulation) of Botrytis cinerea, wherein at least one strand of the bactericidal double-stranded RNA molecule comprises 21 consecutive nucleotides that are complementary to a fragment or equivalent length of the DNA or target gene or RNA transcribed therefrom, and wherein the DNA or target gene comprises a bactericidal double-stranded RNA molecule having a sequence selected from the group consisting of SEQ ID NOs: 1-12, or a bactericidal amount of at least one double-stranded RNA that comprises a sequence selected from the group of RNA trigger sequences or the group of RNA trigger sequence reverse complements. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide is chemically or enzymatically synthesized, or produced by expression in a microorganism or expression in a plant cell. The embodiments include a fungicidal composition comprising a dsRNA or its complement comprising a sequence selected from the group consisting of trigger sequences, RNA trigger sequences, RNA trigger sequence reverse complements, or selected from the group consisting of SEQ ID NO: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, or 48, or in a more specific embodiment, a sequence identical to 21 or more consecutive nucleotides of the sequence of SEQ ID NO: 19.

[0092] In various embodiments, the fungicidal composition for controlling Botrytis cinerea is in at least one form selected from the group consisting of a solid, a liquid (including homogeneous mixtures such as soluble liquid concentrates, and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions), a powder, a suspension, an emulsion, an aerosol, an encapsulated or microencapsulated formulation, in or on a microbead or other carrier particle, in a film or coating, or on or in a matrix, or as a leaf, seed, root, or stem treatment. Suitable binders, inert carriers, surfactants, etc., can be optionally included in the polynucleotide-containing composition, as known to those skilled in the art for formulating fungicides and treating seeds, stems, fruits, or leaves. The Botrytis cinerea to be controlled is a pathogen that generally invades plants. In some embodiments, the plant is grape, tomato, strawberry, snap bean, eggplant, chili pepper, bell pepper, tomatillo, ground cherry, cape gooseberry, tobacco, apple, pear, quince, peach, plum, cherry, almond, apricot, blackberry, blueberry, raspberry, carnation, petunia, or rose. In some embodiments, the fungicidal composition is at least one embedded formulation selected from the group consisting of particles, pellets, or capsules that are embedded in the plant. In such embodiments, the method includes embedding the embedded formulation in the plant. In one embodiment, the fungicidal composition can be transfected into or otherwise internally absorbed by Botrytis cinerea. In some embodiments, the fungicidal composition further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, an organosilicone surfactant, a non-polynucleotide fungicide, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide insecticide, a polynucleotide insecticide, a polynucleotide insecticide, a non-polynucleotide insecticide, and a safener, a pathogen growth regulator.In one embodiment, the germicidal composition further comprises a nonionic organosilicone surfactant, such as a SILWET® brand surfactant, e.g., SILWET L-77® brand surfactant, having CAS No. 27306-78-1 and EPA No.: CAL.REG.NO.5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY. One embodiment includes a germicidal composition further comprising BREAK-thru 301. Other surfactants include, for example, BREAK-THRU S 240 brand polyether modified polysiloxane (CASRN proprietary) surfactant currently available from Goldschmidt Chemical Corporation, Hopewell, VA, BREAK-THRU S 279 end-capped polyether trisiloxane surfactant whose ingredients are listed on the EINECS, TSCA, ENCS, AICS, ECL, PICCS CHINA, NDSL Existing Chemical Substance List, and INDUCE brand adjuvant NMFC Item 42652, Class 60 currently available from Helena Chemical Company, Collierville, TN. FRANCHISE® with LECI-TECH® brand surfactant, having CA REG No. 34704-50065, is currently available from Loveland Products, Inc. Greely, CO. Alternatively, plants are topically treated with the fungicidal composition with a separate (preceding, subsequent, or simultaneous) application of a substance that improves the efficacy of the fungicidal composition. For example, plants can be sprayed with a first topical application of a solution containing a nonionic organosilicone surfactant, such as a SILWET® brand surfactant, for example SILWET L-77®, BREAK-THRU S24, BREAK-THRU S279, INDUCE or FRANCAHISE brand surfactant, followed by a second topical application of the fungicidal composition, or vice versa.

[0093] The combination of a particular RNA (e.g., a dsRNA trigger as described in the Examples) used in this method with one or more non-polynucleotide fungicides is expected to provide enhanced prevention or control of Botrytis cinerea infections compared to the effects achieved with the RNA alone or the non-polynucleotide fungicide alone.

[0094] In various embodiments, the bactericidal composition comprises a microbial cell or is produced in a microorganism. For example, the bactericidal composition can comprise a bacterial or yeast cell or be produced in a bacterial or yeast cell. In similar embodiments, the bactericidal composition comprises a transgenic plant cell or is produced in a plant cell (e.g., a plant cell that transiently expresses a polynucleotide). Such a plant cell can be a cell in a plant or a cell grown in tissue culture or cell suspension.

[0095] In one embodiment, the fungicidal composition is provided in the form of any plant susceptible to infection by Botrytis cinerea, comprising an RNA in or on said plant. Such plants may be stable transgenic plants expressing the RNA, or non-transgenic plants that transiently express the RNA or that have been treated with the RNA, for example by spraying or coating. Stable transgenic plants generally comprise a recombinant construct encoding the RNA integrated into their genome.

[0096] RNA useful in the bactericidal composition can be single-stranded (ss) or double-stranded (ds). Embodiments include those in which the RNA is at least one selected from the group consisting of sense single-stranded RNA (ssRNA), antisense single-stranded (ssRNA), or double-stranded RNA (dsRNA), and mixtures of any of these types of RNA can be used. In one embodiment, a double-stranded DNA / RNA hybrid is used. The RNA can include components other than standard ribonucleotides, for example, one embodiment is an RNA that includes terminal deoxyribonucleotides.

[0097] The RNA in the bactericidal composition has at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of a target gene or DNA or its DNA complement having a sequence selected from the target gene sequence group. In one embodiment, the RNA comprises at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group or trigger sequence group. In some embodiments, the contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA or its DNA complement having a sequence selected from the target gene sequence group or trigger sequence group. In some embodiments, the contiguous nucleotides are 100% identical to a fragment of equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group or trigger sequence group. In some embodiments, the RNA has an overall sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a fragment of DNA or its DNA complement having a sequence selected from a group of target gene sequences or a group of trigger sequences.

[0098] The RNA in the bactericidal composition comprises at least one fragment of 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group or the trigger sequence group. In some embodiments, the RNA comprises at least one fragment of 18 or more, e.g., 18 to 24, or 18 to 28, or 20 to 30, or 20 to 50, or 20 to 100, or 50 to 100, or 50 to 500, or 100 to 250, or 100 to 600, or 200 to 1000, or 500 to 2000 or more consecutive nucleotides. In some embodiments, the fragments comprise more than 18 consecutive nucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, About 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 575, about 600, or more than 600 consecutive nucleotides. In certain embodiments, the RNA comprises at least one fragment of at least 18, 19, 20, or 21 contiguous nucleotides having a sequence that is 100% identical to a DNA having a sequence selected from a group of target gene sequences or a group of trigger sequences or to an RNA transcript of either of the target gene or any of the target gene or to a fragment of equivalent length of the DNA or RNA complement of any of the foregoing.In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) with one strand comprising at least one fragment of at least 18, 19, 20, or 21 consecutive nucleotides with a sequence of 100% identity to a fragment of equivalent length of DNA or a target gene or an RNA transcript of any of the foregoing, or a DNA or RNA complement of any of the foregoing, having a sequence selected from a group of target gene sequences or a group of trigger sequences. Such double-stranded nucleic acid, expressed as base pairs, comprises at least one fragment of at least 18, 19, 20, or 21 consecutive, perfectly matched base pairs corresponding to a fragment of equivalent length of DNA or a target gene or an RNA transcript of any of the foregoing, having a sequence selected from a group of gene sequences or a group of trigger sequences, or a DNA or RNA complement of any of the foregoing. In certain embodiments, each fragment contained in the RNA is longer than the length typical of naturally occurring small regulatory RNAs, for example, each fragment is at least about 30 consecutive nucleotides (or base pairs) in length. In some embodiments, the total length of the RNA, or the length of each fragment contained in the RNA, is less than the total length of the sequence of interest (a DNA or a target gene having a sequence selected from the group consisting of a group of target gene sequences or a group of trigger sequences). In some embodiments, the total length of the RNA is about 50 to about 600 nucleotides (for single-stranded RNA) or base pairs (for double-stranded RNA). In some embodiments, the RNA comprises at least one RNA strand about 50 to about 600 nucleotides in length.

[0099] The RNA in the fungicidal composition is generally designed to inhibit one or more target genes. Such target genes may include coding or non-coding sequences or both. In certain embodiments, the RNA is designed to inhibit one or more Botrytis cinerea target genes selected from the group consisting of Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, Bcded1, and may be designed to target one or more different regions of these genes. In various embodiments, the RNA is designed to inhibit one or more genes, each gene having a sequence selected from the group consisting of target gene sequences, and may be designed to inhibit multiple genes from this group or to target one or more different regions of these genes. In one embodiment, the RNA comprises multiple sections or fragments, each of said multiple sections or fragments comprising at least one fragment of 21 consecutive nucleotides with a sequence of 100% identity to a DNA having a sequence selected from a target gene sequence group or a trigger sequence group, or an RNA transcript of any of the above, or a fragment of equivalent length of the DNA or RNA complement of any of the above. In such a case, each section may be identical or different in size or sequence, and may be sense or antisense to the target gene. For example, in one embodiment, the RNA comprises multiple sections in tandem or repeat sequences, each of which comprises at least one fragment of 21 consecutive nucleotides with a sequence of 100% identity to a DNA having a sequence selected from a target gene sequence group or a trigger sequence group, or an RNA transcript of any of the above, or a fragment of equivalent length of the DNA or RNA complement of any of the above. The fragments can be derived from different regions of the target gene, for example, the fragments can correspond to different exon regions of the target gene, and "spacer" nucleotides not corresponding to the target gene can be optionally used between or adjacent to the fragments.

[0100] The total length of the RNA in the bactericidal composition can be more than 18 contiguous nucleotides and can include nucleotides in addition to contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of comparable length of DNA or its DNA or RNA complement having a sequence selected from the group of target gene sequences or trigger sequences. In other words, the total length of the RNA can be more than the length of a section or fragment of the RNA designed to inhibit one or more target genes, each target gene having a DNA sequence or its complement selected from the group consisting of the group of target gene sequences. For example, the RNA can have nucleotides adjacent to the "active" fragment of at least one fragment of 18 or more contiguous nucleotides that inhibits the target gene, or can include "spacer" nucleotides between the active fragments, or can have additional nucleotides at the 5' end or at the 3' end or at both the 5' and 3' ends. In one embodiment, the RNA includes additional nucleotides that are not specifically related (having a sequence that is not complementary or identical) to the DNA or target gene or its DNA complement having a sequence selected from the group of target gene sequences, e.g., nucleotides that provide a stabilizing secondary structure or provide convenience in cloning or production. In one embodiment, the RNA comprises additional nucleotides located immediately adjacent to one or more fragments of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity or complementarity to a DNA having a sequence selected from a group of target gene sequences or a group of trigger sequences or a fragment of comparable length of a target gene or a DNA or RNA complement of any of the foregoing. In one embodiment, the RNA comprises one such fragment and has an additional 5'G or an additional 3'C or both adjacent to the fragment. In another embodiment, the RNA is a double stranded RNA containing additional nucleotides to form an overhang, e.g., a dsRNA containing two deoxyribonucleotides to form a 3' overhang.Thus, in various embodiments, the nucleotide sequence of the entire RNA is not 100% identical or complementary to a segment of consecutive nucleotides in a DNA or target gene having a sequence selected from the group consisting of a target gene sequence group or a trigger sequence group. For example, in some embodiments, the RNA comprises at least two segments of 21 consecutive nucleotides having a sequence of 100% identity to a segment of a DNA or its DNA complement having a sequence selected from the target gene sequence group, and (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two fragments are arranged in an order different from the order in which the corresponding fragments are present in a DNA or its DNA complement having a sequence selected from the target gene sequence group.

[0101] In various embodiments, the RNA in the fungicidal composition is composed of naturally occurring ribonucleotides. Embodiments include, for example, synthetic RNA composed entirely of ribonucleotides, or synthetic RNA composed primarily of ribonucleotides but with one or more terminal deoxyribonucleotides or one or more terminal deoxyribonucleotides. In certain embodiments, the RNA comprises non-standard nucleotides, such as inosine, thiuridine, or pseudouridine. In certain embodiments, the RNA comprises chemically modified nucleotides. The RNA in the fungicidal composition is provided by suitable means known to those skilled in the art. Embodiments include those in which the RNA is chemically or enzymatically synthesized (e.g., by in vitro transcription, such as transcription using T7 polymerase or other polymerases), or is produced by expression in a microorganism or cell culture (e.g., plant or fungal cells grown in culture), or is produced by microbial fermentation.

[0102] In some embodiments, the RNA is provided as an isolated RNA that is not part of an expression construct. In some embodiments, the RNA is provided as an isolated RNA that lacks additional elements such as promoter or terminator sequences. Such RNA may be relatively short, e.g., single- or double-stranded RNA of about 18 to about 300 or about 50 to about 600 nucleotides (for single-stranded RNA) or about 18 to about 300 or about 50 to about 600 base pairs (for double-stranded RNA). Alternatively, the RNA can be provided in a more complex construct, e.g., as part of a recombinant expression construct or included in a recombinant vector, e.g., a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression constructs or vectors are designed to include additional elements, such as additional RNA encoding an aptamer or ribozyme, or an expression cassette for expressing a gene of interest (e.g., a bactericidal protein).

[0103] X. Methods for Providing Plants Having Enhanced Resistance to Botrytis cinerea Infection, and Plants, Plant Parts, and Seeds So Provided Some embodiments relate to a method of providing a plant with enhanced resistance to Botrytis cinerea infection / colonization, comprising providing to the plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a target gene selected from the group consisting of genes identified in a target gene sequence group or a fragment of RNA transcribed from the target gene. These target gene embodiments include genes identified by name in Table 1A and having a sequence selected from the group consisting of the target gene sequence group, and related genes including orthologs from related plant pathogens. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized or produced by expression / production in a microorganism or expression in a plant cell. In some embodiments, the polynucleotide comprises at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a sequence selected from the group consisting of the target gene sequence group. In some embodiments, the polynucleotide is a dsRNA having a strand with a sequence selected from a trigger sequence group or its complement. In some embodiments, the polynucleotide comprises a dsRNA having a strand with a sequence selected from a trigger sequence group.

[0104] In a related aspect, the invention is directed to plants having enhanced resistance to Botrytis cinerea infection, said resistance being provided by expressing in the plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of a target gene selected from the group consisting of genes identified in the target gene sequence group, whereby the resulting plant has greater resistance to Botrytis cinerea infection compared to a control plant in which the polynucleotide is not expressed. In a related aspect, the invention is directed to plants having enhanced resistance to Botrytis cinerea infection, said resistance being provided by expressing in the plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that has a sequence of about 95% to about 100% identity or complementarity to a fragment of a target gene selected from the group consisting of genes identified in the target gene sequence group, whereby the resulting plant has improved resistance to Botrytis cinerea infection compared to a control plant in which the polynucleotide is not expressed.

[0105] In yet another aspect, the invention is directed to seeds or propagable parts (particularly transgenic progeny seeds or propagable parts) produced by the plants having enhanced resistance to Botrytis cinerea infection provided by this method. Products produced by the plants having enhanced resistance to Botrytis cinerea infection provided by this method, and products produced from transgenic progeny seeds or propagable parts of such plants, are also contemplated.

[0106] Another aspect of the invention provides a method for providing a plant with enhanced resistance to Botrytis cinerea infection comprising topically applying to a plant a composition comprising at least one polynucleotide having at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of a target gene or its DNA complement having a sequence selected from a group of target gene sequences, such that plants treated with the polynucleotide-containing composition exhibit greater resistance to Botrytis cinerea infection compared to untreated plants. In one embodiment, the at least one polynucleotide comprises at least one fragment of 18 or more contiguous nucleotides that is essentially identical to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from a group of target gene sequences. A polynucleotide may be longer than one or more of the fragments contained therein, but each fragment and the corresponding fragment of the target gene are of equivalent length. In one embodiment, the present invention provides a method for providing a plant with enhanced resistance to Botrytis cinerea infection comprising topically applying to the plant a composition comprising at least one polynucleotide comprising a nucleotide sequence that is complementary to at least 18 contiguous nucleotides of a target gene or RNA transcribed from the target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12. In one embodiment, the present invention provides a method for providing a plant with enhanced resistance to Botrytis cinerea infection comprising topically applying to the plant a composition comprising at least one polynucleotide such that an effective amount of the polynucleotide is transfected into Botrytis cinerea in or on the plant, the polynucleotide comprising at least 18 contiguous nucleotides that are complementary to a region of a target gene or RNA transcribed from the target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12.

[0107] The polynucleotides used in this method can be designed for multiple target genes.Embodiments include those in which the composition comprises a dsRNA having a strand with a sequence selected from the group consisting of trigger sequences.Related aspects of the invention include compositions for topical application, and isolated polynucleotides for use in this method, and plants with enhanced Botrytis cinerea resistance provided by this method.

[0108] As used throughout the specification, "topical application" refers to application to the surface or exterior of a subject, such as application to the surface of a plant part, such as, for example, leaves, stems, flowers, fruits, shoots, roots, stems, seeds, flowers, anthers, or pollen, or application to the entire plant, or to the above-ground or below-ground parts of the plant. Topical application can be on a non-living surface, such as application to the soil, or application to a surface or matrix where Botrytis cinerea can encounter the polynucleotide. In various embodiments of the method, a composition comprising at least one polynucleotide is applied locally to the plant in an appropriate form, such as, for example, a solid, liquid (including homogeneous mixtures, such as soluble liquid concentrates, and heterogeneous mixtures, such as, for example, soluble liquid concentrates), suspensions, colloids, micelles, and emulsions), powders, suspensions, emulsions, sprays, encapsulated or microencapsulated formulations, in or on microbeads or other carrier particles, in a film or coating, or on or within a matrix, or as a treatment of leaves, seeds, roots, stems. In some embodiments of this method, the polynucleotide-containing composition is applied topically to the above-ground parts of the plant, for example, sprayed or dusted onto the leaves, stems, and flowering parts of the plant.

[0109] An embodiment of the method includes topical application of a foliar spray (e.g., spraying a liquid polynucleotide-containing composition onto the leaves of a plant) or foliar dusting (e.g., dusting a plant with a polynucleotide-containing composition comprising a composition in the form of a powder or on carrier particles). In other embodiments, the polynucleotide-containing composition is applied locally to underground portions of the plant, such as the roots, for example, by soil drench. In other embodiments, the polynucleotide-containing composition is applied locally to the seeds growing into the plant. Topical application can be in the form of topical treatment of the fruit of the plant or the seeds from the fruit of the plant. Suitable binders, inert carriers, surfactants, and the like can be optionally included in the polynucleotide-containing composition, as known to those skilled in the art of fungicide formulation and seed or stem treatment.

[0110] In some embodiments, the polynucleotide-containing composition is at least one locally embedded formulation selected from the group consisting of microparticles, pellets, or encapsulation that are locally embedded in the plant. In such embodiments, the method includes locally embedding the locally embedded formulation in the plant. In one embodiment, the polynucleotide-containing composition can be transfected or otherwise internally absorbed by Botrytis cinerea. In some embodiments, the polynucleotide-containing composition further includes a carrier agent and / or a surfactant (e.g., a non-ionic surfactant). Examples of non-ionic organosilicone surfactants include SILWET® brand surfactants, BREAK THRU S240, BREAK THRU S279, BREAK THRU 301, Induce and Franchise, e.g., SILWET L-77® brand surfactants. A first topical application of the surfactant may be followed by a second topical application of the polynucleotide-containing composition, or vice versa. In some embodiments, the plant is treated by applying a substance that enhances the efficacy of the polynucleotide-containing composition before, after, or simultaneously with the topical treatment with the polynucleotide-containing composition. For example, the plants can be sprayed with an initial topical application of a solution containing a nonionic organosilicone surfactant, such as SILWET® brand surfactants BREAK THRU S240, BREAK THRU S279, BREAK THRU 301, Induce and Franchise, e.g., SILWET L-77®, followed by a second topical application of the polynucleotide-containing composition, or vice versa.

[0111] Combinations of certain polynucleotides useful in polynucleotide-containing compositions (e.g., the polynucleotide triggers described in the Examples) with one or more non-polynucleotide insecticides are expected to provide improved efficacy in preventing or controlling Botrytis cinerea infections compared to the efficacy obtained with the polynucleotide alone or the non-polynucleotide insecticide alone.

[0112] Polynucleotides useful in the polynucleotide-containing compositions are provided by suitable means known to those of skill in the art. Embodiments include those in which the polynucleotides are chemically or enzymatically synthesized (e.g., by in vitro transcription, such as transcription using T7 polymerase or other polymerases), produced by expression in a microorganism or cell culture (e.g., a plant cell, a microbial cell, or a pathogen cell (grown in culture), a plant cell, or produced by microbial fermentation.

[0113] In many embodiments, polynucleotides useful in polynucleotide-containing compositions are provided as isolated DNA or RNA fragments. In some embodiments, polynucleotides useful in polynucleotide-containing compositions are not part of an expression construct and lack additional elements such as promoter or terminator sequences. Such polynucleotides can be relatively short, such as single-stranded or double-stranded polynucleotides of about 18 to about 500 or about 50 to about 600 nucleotides (for single-stranded polynucleotides) or about 18 to about 500 or about 50 to about 600 base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of about 100 to about 600 base pairs, such as a dsRNA of any of the lengths of the dsRNA triggers disclosed in Figure and Table 1A. Alternatively, the polynucleotide can be provided in a more complex construct, for example, provided as part of a recombinant expression construct, or included in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. Such recombinant expression constructs or vectors can be designed to contain additional elements, such as an expression cassette for expressing a gene of interest (eg, a bactericidal protein).

[0114] A polynucleotide useful in a polynucleotide-containing composition has at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the group consisting of target gene sequences. In one embodiment, the polynucleotide comprises at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of DNA having a sequence selected from the group consisting of target gene sequences. In some embodiments, the contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA having a sequence selected from the group consisting of target gene sequences. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the group consisting of target genes. In some embodiments, the polynucleotide has an overall sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a fragment of DNA or its DNA complement having a sequence selected from a group of target gene sequences.

[0115] Polynucleotides useful in polynucleotide-containing compositions include fragments of 18 or more (e.g., 18-24, or 18-28, or 20-30, or 20-50, or 20-100, or 50-100, or 50-500, or 100-250, or 100-600, or 200-1000, or 500-2000 or more) consecutive nucleotides. In some embodiments, the fragments are greater than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, The polynucleotide comprises about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 575, about 600, or more than 600 consecutive nucleotides. In certain embodiments, the polynucleotide comprises DNA having a sequence selected from a group of target gene sequences or at least one fragment of at least 18, 19, 20, or 21 consecutive nucleotides having a sequence 100% identical to a fragment of equivalent length of the target gene or its DNA complement. In certain embodiments, the polynucleotide is a double-stranded nucleic acid (e.g., dsRNA) in which one strand comprises at least one fragment of at least 18, 19, 20, or 21 contiguous nucleotides having a sequence of 100% identity to a DNA having a sequence selected from a group of target gene sequences or a fragment of equivalent length of the target gene or its DNA complement. Such double-stranded nucleic acid, expressed as base pairs, comprises at least one fragment of at least 18, 19, 20, or 21 contiguous perfectly matched base pairs corresponding to a DNA having a sequence selected from a group of target gene sequences or a fragment of equivalent length of the target gene or its DNA complement.In certain embodiments, each fragment contained in the polynucleotide is longer than the length typical of naturally occurring small regulatory RNAs, e.g., each fragment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the polynucleotide, or the length of each fragment contained in the polynucleotide, is less than the total length of the sequence of interest (a DNA having a sequence selected from a group of target gene sequences or a target gene or its DNA complement). In some embodiments, the total length of the polynucleotide is about 50 to about 600 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of about 100 to about 600 base pairs, such as a dsRNA of any of the lengths of the dsRNA triggers disclosed in Table 1A. In some embodiments, the polynucleotide is a dsRNA encoded by a sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, and 24.

[0116] The locally applied polynucleotide is generally designed to suppress one or more genes ("target genes") via RNAi. Such target genes may include coding or non-coding sequences or both. In certain embodiments, the polynucleotide is designed to suppress one or more target genes, each target gene having a DNA sequence selected from the group consisting of target gene sequences. In various embodiments, the locally applied polynucleotide is designed to suppress one or more genes, each gene having a sequence selected from the group consisting of target gene sequences, and can be designed to suppress multiple genes from this group or target one or more different regions of these genes. In one embodiment, the locally applied polynucleotide comprises multiple sections or fragments, each of which comprises at least one fragment of 21 contiguous nucleotides having a sequence 100% identical to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequences. In such cases, each section may be identical or different in size or sequence and may be sense or antisense to the target gene. For example, in one embodiment, the locally applied polynucleotide can comprise multiple sections in tandem or repeat sequences, each section comprising at least one fragment of 21 contiguous nucleotides having a sequence that is 100% identical to a fragment of equivalent length of DNA having a sequence selected from a group of target gene sequences.

[0117] The total length of the locally applied polynucleotide can be more than 18 contiguous nucleotides and can include nucleotides in addition to contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of comparable length of DNA or its DNA complement having a sequence selected from the group of target gene sequences. In other words, the total length of the locally applied polynucleotide can be longer than the length of the section or fragment of the polynucleotide designed to suppress one or more target genes, each target gene having a DNA sequence selected from the group consisting of the group of target gene sequences. For example, the locally applied polynucleotide can have nucleotides adjacent to the "active" fragment of at least one fragment of 18 or more contiguous nucleotides that suppresses the target gene, or can include "spacer" nucleotides between the active fragments, or can have additional nucleotides at the 5' end or at the 3' end or at both the 5' end and the 3' end. In one embodiment, the locally applied polynucleotide comprises DNA having a sequence selected from a group of target gene sequences or additional nucleotides not specifically related (having a sequence that is not complementary or identical) to the target gene or its DNA complement (e.g., nucleotides provided to stabilize secondary structures or for convenience of cloning or manufacturing).

[0118] In one embodiment, the locally applied polynucleotide comprises additional nucleotides located immediately adjacent to one or more fragments of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity or complementarity to a fragment of comparable length of DNA or target gene having a sequence selected from a group of target gene sequences. In one embodiment, the locally applied polynucleotide comprises one such fragment having an additional 5'G or an additional 3'C or both adjacent to the fragment. In another embodiment, the locally applied polynucleotide is a double-stranded RNA comprising additional nucleotides forming an overhang, e.g., a dsRNA comprising two deoxyribonucleotides forming a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire locally applied polynucleotide is not 100% identical or complementary to a fragment of contiguous nucleotides in DNA or target gene having a sequence selected from a group of target gene sequences. For example, in some embodiments, the locally applied polynucleotide comprises at least two fragments of 21 contiguous nucleotides having sequences that are 100% identical to fragments of DNA or DNA complements having a sequence selected from a group of target gene sequences, where (1) the at least two fragments are separated by one or more spacer nucleotides, or (2) the at least two fragments are arranged in an order that is different from the order in which the corresponding fragments are present in the DNA or DNA complements having a sequence selected from a group of target gene sequences.

[0119] In a related aspect, the present invention is directed to a plant having enhanced resistance to Botrytis cinerea infection provided by a method comprising topically applying to the plant a composition comprising at least one polynucleotide having at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of DNA of equivalent length having a sequence selected from a group of target gene sequences or its DNA complement, wherein plants treated with the polynucleotide composition by the method exhibit greater resistance to Botrytis cinerea infection compared to untreated plants.

[0120] One embodiment is a plant having enhanced resistance to Botrytis cinerea infection compared to a control plant, provided by topically applying to the plant or to a seed growing on the plant a dsRNA trigger having a sequence selected from the group of trigger sequences or its complement or a dsRNA trigger encoded by a sequence of SEQ ID NO: 13-24, 49-52. In yet another aspect, the invention is directed to seeds (particularly transgenic progeny seeds) produced by the plant having enhanced resistance to Botrytis cinerea infection provided by this method. Products produced by the plant having enhanced resistance to Botrytis cinerea infection provided by this method, and products produced from transgenic progeny seeds or stems / shoots of such plants are also contemplated.

[0121] XI. Methods for Providing Transgenic Plants with Enhanced Resistance to Botrytis cinerea Infection, and Plants and Seeds So Provided Another aspect of the invention is directed to a method for providing a plant having enhanced resistance to Botrytis cinerea infection comprising expressing in the plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of a target gene or DNA or its DNA complement (or both) having a sequence selected from a target gene sequence group or a trigger sequence group, such that the plant obtained by such method has a stronger resistance to Botrytis cinerea infection compared to a control plant in which the polynucleotide is not expressed. In one embodiment, the method comprises expressing in the plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that has a sequence of about 95% to about 100% identity to a fragment of equivalent length of a target gene or DNA or its DNA complement having a sequence selected from a target gene sequence group or a trigger sequence group. In one embodiment, the invention provides a method of providing a plant with enhanced resistance to Botrytis cinerea infection, comprising expressing in the plant at least one polynucleotide comprising at least one fragment identical to or complementary to at least 18, 19, 20, or 21 consecutive nucleotides of DNA having a sequence selected from the group consisting of SEQ ID NOs: 1-12. "Expressing a polynucleotide in a plant" generally means "expressing an RNA transcript in a plant", e.g., expressing in a plant an RNA comprising a ribonucleotide sequence that is antisense or essentially complementary to at least one fragment of a target gene or DNA having a sequence selected from a group of target gene sequences or its DNA complement. Embodiments include those in which the polynucleotide expressed in the plant is an RNA or its complement comprising at least one fragment having a sequence selected from a group of trigger sequences. However, the polynucleotide expressed in the plant may be DNA (e.g., DNA generated in the plant during genome replication), or RNA encoded by such DNA.Related aspects of the invention include isolated polynucleotides for use in the method, and plants with improved Botrytis cinerea resistance provided by the method.

[0122] The method includes expressing at least one polynucleotide in a plant, the polynucleotide comprising at least one fragment of 18 or more consecutive nucleotides that is essentially identical or complementary to a fragment of a target gene or DNA or its DNA complement having a sequence selected from the target gene sequence. In some embodiments, the first polynucleotide is provided to the plant in the form of DNA (e.g., in the form of an isolated DNA molecule, or as an expression construct, or as a transformation vector), and the polynucleotide expressed in the plant is a second polynucleotide in the plant (e.g., an RNA transcript of the first polynucleotide). In one embodiment, the polynucleotide is expressed in the plant by transgenic expression, i.e., by stably integrating the polynucleotide into the genome of the plant from where it can be expressed in one or more cells of the plant. In one embodiment, a first polynucleotide (e.g., a recombinant DNA construct comprising a promoter operably linked to DNA comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical to or complementary to a target gene or DNA selected from a group of target gene sequences or a fragment of its DNA complement) is stably integrated into the genome of the plant, from where a secondarily generated polynucleotide (e.g., an RNA transcript comprising a transcript of a fragment of 18 or more contiguous nucleotides that is essentially identical to or complementary to a target gene or DNA selected from a group of target gene sequences or a fragment of its DNA complement) is expressed in one or more cells of the plant. Methods for providing stably transformed plants are discussed in the section "Generation and Use of Transgenic Plant Cells and Transgenic Plants."

[0123] In another embodiment, the polynucleotide expressed in the plant is expressed by transient expression (i.e., expression that is not due to stable integration of the sequence into the plant genome). In such an embodiment, the method can include introducing a polynucleotide (e.g., dsRNA or dsDNA) into the plant by conventional techniques known in the art. For example, transient expression can be achieved by infiltrating the plant leaves or stems with a polynucleotide solution using a needleless syringe.

[0124] In some embodiments, where the polynucleotide expressed in the plant is expressed by transient expression, the first polynucleotide is provided to the plant in the form of RNA or DNA, or both RNA and DNA, and the second polynucleotide generated subsequently is transiently expressed in the plant. In some embodiments, the first polynucleotide is one or more selected from (a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (ssDNA), a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule comprising a modified Pol III gene transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule comprising a modified Pol III gene transcribed into an RNA molecule, and (i) a double-stranded, hybridized RNA / DNA molecule, or a combination thereof. In certain embodiments, the first polynucleotide is introduced into the plant by topical application of the polynucleotide-containing composition to the plant in a suitable form, for example, as a solid, liquid (homogeneous mixture such as soluble liquid concentrate, and non-homogeneous mixture such as suspension, colloid, micelle, and emulsion), powder, suspension, emulsion, spray, encapsulation or microencapsulation formulation, in or on microbeads or other carrier particles, in a film or coating, or on or in a matrix, or in the form of treatment of the leaves, seeds, roots, or stems of the plant.As known to those skilled in the art of pesticide formulation and seed treatment, suitable binders, inert carriers, surfactants, etc. can be optionally included in the composition. In such embodiments, the polynucleotide-containing composition comprises a carrier agent, a surfactant, an organosilicone, an organosilicone surfactant, a non-polynucleotide fungicide, a polynucleotide insecticide, a non-polynucleotide insecticide, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide insecticide, a polynucleotide insecticide, a polynucleotide insecticide, a non-polynucleotide insecticide, a safener, and a pathogen growth regulator.In one embodiment, the composition may further comprise a non-ionic organosilicone surfactant such as a SILWET® brand surfactant, for example SILWET L-77® brand surfactant, currently available from Momentive Performance Materials, Albany, NY, having CAS number 27306-78-1 and EPA number: CAL.REG.NO.5905-50073-AA. BREAK-THRU S 240 brand is a polyether modified polysiloxane (CASRN proprietary) surfactant currently available from Goldschmidt Chemical Corporation, Hopewell, VA. BREAK-THRU S 279 is an end-capped polyether trisiloxane surfactant, whose components are listed on the following existing chemicals lists: EINECS, TSCA, ENCS, AICS, ECL, PICCS CHINA, NDSL. INDUCE brand adjuvant NMFC item 42652, class 60, currently available from Helena Chemical Company, Collierville, TN. FRANCHISE® with LECI-TECH® brand surfactants, currently available from Loveland Products, Inc. Greely, CO, have CA REG No. 34704-50065. Alternatively, such additional ingredients or insecticides can be provided separately, for example, by separate topical application or by transgenic expression in the plant. Alternatively, in addition to being treated with the polynucleotide-containing composition, the plant can be topically treated with another substance that enhances the efficacy of the polynucleotide-containing composition before, after or simultaneously with the treatment. For example, the plant can be sprayed with a first topical application of a solution containing a nonionic organosilicone surfactant, such as SILWET® brand surfactants, such as SILWET L-77®, BREAK-THRU S24, BREAK-THRU S279, INDUCE or FRANCAHISE brand surfactants, followed by a second topical application of the polynucleotide-containing composition, or vice versa.One embodiment includes a composition further comprising BREAK-thru 301.

[0125] It is expected that the combination of a particular polynucleotide used in the method (e.g., a polynucleotide trigger as described in the Examples) with one or more non-polynucleotide fungicides will provide improved prevention or control against Botrytis cinerea infections compared to the efficacy achieved with the polynucleotide alone or with a non-polynucleotide fungicide alone.

[0126] In some embodiments where the polynucleotides expressed in the plant are expressed by transient expression, a first polynucleotide is provided to the plant in the form of RNA or DNA or both RNA and DNA, and a second polynucleotide that is generated secondarily is transiently expressed in the plant. The site of application of the first polynucleotide need not be the same as the site where the second polynucleotide is transiently expressed. For example, the first polynucleotide can be provided to the plant by topical application to the leaves, seed treatment, root drench, or by injection into the stem, and the second polynucleotide can be transiently expressed elsewhere in the plant, e.g., in the roots or throughout the plant. In some embodiments of this method, a composition comprising at least one polynucleotide is applied locally to the above-ground parts of the plant (e.g., sprayed or dusted on the leaves, stems, and flowering parts of the plant). In other embodiments, a composition comprising at least one polynucleotide is applied locally to the underground parts of the plant, such as the roots, e.g., by soil drench. In other embodiments, a composition comprising at least one polynucleotide is applied locally to the seeds that will grow into plants with enhanced resistance to Botrytis cinerea infection. In some embodiments, the polynucleotide expressed in the plant is RNA, which can be single-stranded (ss) RNA or double-stranded (ds) RNA or a combination of both.

[0127] In some embodiments, a first polynucleotide (DNA or RNA or both) is provided to a plant, and a second polynucleotide having a sequence corresponding to (identical or complementary to) the first polynucleotide is then expressed in the plant. In such embodiments, the polynucleotide expressed in the plant is an RNA transcript, which may be ssRNA or dsRNA or a combination of both. In some embodiments in which the polynucleotide is expressed by transient expression, the first polynucleotide is provided to the plant in the form of RNA or DNA or both RNA and DNA, and the second polynucleotide that is generated secondarily is transiently expressed in the plant. In such embodiments, the first polynucleotide is one or more selected from (a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule that includes a modified Pol III gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule that includes a modified Pol III gene that is transcribed into an RNA molecule, and (i) a double-stranded, hybridized RNA / DNA molecule, or a combination thereof. In such embodiments in which the polynucleotide is expressed by transient expression, the first polynucleotide may be composed of naturally occurring nucleotides, such as those present in DNA and RNA. In such embodiments in which the polynucleotide is expressed by transient expression, the first polynucleotide may be chemically modified or include chemically modified nucleotides. The first polynucleotide is provided by suitable means known to those skilled in the art.The first polynucleotide can be provided as an RNA or DNA fragment.Alternatively, the first polynucleotide can be provided in a more complex construct, for example as part of a recombinant expression construct, or can be provided in a recombinant vector, for example in a recombinant plant virus vector or in a recombinant baculovirus vector.Such recombinant expression constructs or vectors can be designed to contain additional elements, such as an expression cassette for expressing a gene of interest (eg, a bactericidal protein).

[0128] In some embodiments, the polynucleotide expressed in the plant is an RNA molecule, which may be relatively short, such as a single-stranded or double-stranded RNA of about 18 to about 300 or about 50 to about 600 nucleotides (for single-stranded RNA), or about 18 to about 300 or about 50 to about 600 base pairs (for double-stranded RNA). Alternatively, the polynucleotide may be provided in a more complex construct, for example, provided as part of a recombinant expression construct, or included in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression constructs or vectors are designed to include additional elements, such as an expression cassette for expressing a gene of interest (e.g., a bactericidal protein).

[0129] The polynucleotide expressed in the plant has at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group, trigger sequence group. In one embodiment, the polynucleotide expressed in the plant comprises at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA or its DNA complement having a sequence selected from the target gene sequence group, trigger sequence group. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group or trigger sequence group. In some embodiments, the polynucleotide expressed in the plant has an overall sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a fragment of DNA or its DNA complement having a sequence selected from the group consisting of target gene sequences, trigger sequences.

[0130] Polynucleotides expressed in plants are generally designed to suppress one or more genes ("target genes"). Such target genes may include coding or non-coding sequences or both. In certain embodiments, polynucleotides expressed in plants are designed to suppress one or more target genes, each target gene having a DNA sequence selected from the group consisting of target gene sequences. In various embodiments, polynucleotides expressed in plants are designed to suppress one or more genes, each gene having a sequence selected from the group consisting of target gene sequences, and can be designed to suppress multiple genes from this group or target one or more different regions of these genes. In one embodiment, polynucleotides expressed in plants include multiple sections or fragments, each of said sections or fragments comprising at least one fragment of 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the group of target gene sequences, trigger sequences. In such cases, each section may be identical or different in size or sequence and may be sense or antisense to the target gene. For example, in one embodiment, the polynucleotide expressed in the plant can include multiple sections in tandem or repeat sequences, each section including at least one fragment of 21 consecutive nucleotides with a sequence of 100% identity to a fragment of comparable length of DNA or its DNA complement with a sequence selected from the target gene sequence group or trigger sequence group. The fragments can be derived from different regions of the target gene, for example, the fragments can correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene can be used between or adjacent to the fragments as needed.

[0131] The total length of the polynucleotide expressed in the plant can be more than 18 contiguous nucleotides and can include nucleotides in addition to contiguous nucleotides having a sequence of about 95%-100% identity to a fragment of comparable length of DNA or its DNA complement having a sequence selected from the group consisting of the target gene sequence group or the trigger sequence group. In other words, the total length of the polynucleotide expressed in the plant can be more than the length of a section or fragment of the polynucleotide designed to suppress one or more target genes, each fragment having a DNA sequence selected from the group consisting of the target gene sequence group. For example, the polynucleotide expressed in the plant can have nucleotides adjacent to the "active" fragment of at least one fragment of 18 or more contiguous nucleotides that suppresses the target gene, or can include "spacer" nucleotides between the active fragments, or can have additional nucleotides at the 5' end or at the 3' end or at both the 5' end and the 3' end. In one embodiment, the polynucleotide expressed in the plant includes additional nucleotides (e.g., nucleotides that provide a stable secondary structure or nucleotides for convenience of cloning or manufacturing) that are not specifically related (i.e., have a sequence that is not complementary or identical) to the DNA or target gene or its DNA complement sequence having a sequence selected from the target gene sequence group. In one embodiment, the polynucleotide expressed in the plant includes additional nucleotides located immediately adjacent to one or more fragments of 18 or more consecutive nucleotides that have a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length of the DNA or target gene having a sequence selected from the target gene sequence group. In one embodiment, the polynucleotide expressed in the plant includes one such fragment, adjacent to which is an additional 5'G or an additional 3'C or both. In another embodiment, the polynucleotide expressed in the plant is a double-stranded RNA that includes additional nucleotides that form an overhang, e.g., a dsRNA that includes two deoxyribonucleotides that form a 3' overhang.Thus, in various embodiments, the nucleotide sequence of the entire polynucleotide expressed in the plant is not 100% identical or complementary to a segment of consecutive nucleotides in the DNA or target gene having a sequence selected from the group consisting of target gene sequences.For example, in some embodiments, the polynucleotide expressed in the plant comprises at least two segments of 21 consecutive nucleotides having a sequence of 100% identity to a segment of the DNA or its DNA complement having a sequence selected from the group of target gene sequences, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two fragments are arranged in an order different from the order in which the corresponding fragments are present in the DNA or its DNA complement having a sequence selected from the group of target gene sequences.

[0132] In a related embodiment, the present invention is directed to a plant having enhanced resistance to Botrytis cinerea infection provided by expressing in a plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group or the trigger sequence group, whereby the resulting plant has a stronger resistance to Botrytis cinerea infection compared to a control plant in which the polynucleotide is not expressed. In a related embodiment, the present invention is directed to a plant having enhanced resistance to Botrytis cinerea infection provided by expressing in a plant at least one polynucleotide comprising at least one fragment of 18 or more contiguous nucleotides that has a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group or the trigger sequence group, whereby the resulting plant has a stronger resistance to Botrytis cinerea infection compared to a control plant in which the polynucleotide is not expressed. One embodiment is a plant having greater resistance to Botrytis cinerea infection compared to a control plant, provided by expressing in the plant an RNA having a sequence selected from the group of trigger sequences or its complement. In yet another aspect, the invention is directed to a seed or stem cutting (particularly a transgenic progeny seed or cloned stem) produced by the plant having enhanced resistance to Botrytis cinerea infection provided by this method. Products produced by the plant having enhanced resistance to Botrytis cinerea infection provided by this method, and products produced from transgenic progeny seeds of such plants are also contemplated.

[0133] XII. Recombinant DNA constructs for controlling Botrytis cinerea infections Another aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element comprising at least one fragment of 18 or more contiguous nucleotides having a sequence that is about 95% to about 100% identical to a fragment of DNA or its DNA complement having a sequence selected from a group of target gene sequences or a group of trigger sequences. In some embodiments, the recombinant DNA construct is: (a) a DNA comprising a nucleotide sequence complementary to at least 18, 19, 20, or 21 consecutive nucleotides of a DNA having a sequence selected from the group consisting of SEQ ID NOs: 1-12, or a fragment of equivalent length of RNA transcribed therefrom; or (b) a DNA comprising 18, 19, 20, or 21 or more consecutive nucleotides having 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group consisting of SEQ ID NOs: 1-12, or its DNA complement; or (c) a DNA encoding at least one silencing element complementary to at least 18, 19, 20, or 21 consecutive nucleotides of a target gene having a sequence selected from the group consisting of SEQ ID NOs: 1-12, or an RNA transcribed therefrom; or (d) a target gene or target selected from the genes in the target gene sequence group. (e) a DNA encoding at least one silencing element comprising at least 18, 19, 20 or 21 consecutive nucleotides complementary to a portion of an RNA transcribed from a gene, or (f) a DNA encoding an RNA comprising at least 18, 19, 20 or 21 consecutive nucleotides complementary to a nucleotide sequence selected from the group of trigger sequences, or an orthologous nucleotide sequence from Botrytis cinerea, said orthologous nucleotide sequence having at least 95% sequence identity to a nucleotide sequence selected from the group of trigger sequences, said percent sequence identity being calculated over the same length; or (g) a DNA encoding at least one silencing element comprising at least 18, 19, 20 or 21 consecutive nucleotides complementary to a portion of an RNA transcribed from a gene, said silencing element comprising at least 18, 19, 20 or 21 consecutive nucleotides complementary to a nucleotide sequence selected from the group of trigger sequences, or an orthologous nucleotide sequence from Botrytis cinerea, said orthologous nucleotide sequence having at least 95% sequence identity to a nucleotide sequence selected from the group of trigger sequences, said percent sequence identity being calculated over the same length.(g) a DNA encoding an RNA comprising at least one double-stranded RNA region comprising 20 or 21 contiguous nucleotides, said orthologous nucleotide sequence having at least 95% sequence identity to a nucleotide sequence selected from the group consisting of a trigger sequence, said percent sequence identity being calculated over the same length; or (g) a DNA encoding an RNA comprising a nucleotide sequence selected from the group consisting of an RNA trigger sequence or an RNA trigger sequence reverse complement. Embodiments include recombinant DNA constructs comprising a heterologous promoter operably linked to a DNA element encoding an RNA having a sequence selected from the group consisting of SEQ ID NOs: 25-48, 53-60, or a combination thereof or a complement thereof.

[0134] An embodiment includes a recombinant DNA construct comprising a promoter from a heterologous or homologous plant operably linked to DNA encoding a dsRNA having a strand with a sequence selected from the group consisting of trigger sequences. The recombinant DNA construct is useful, for example, for providing a plant with enhanced resistance to Botrytis cinerea infection by expressing a transcript of such a recombinant DNA construct in the plant. The recombinant DNA construct is also useful for producing a polynucleotide useful for making a composition that can be applied to a plant, seed, propagable plant part, soil or field, or surface in need of protection from Botrytis cinerea infection. Related aspects of the invention include compositions comprising the recombinant DNA construct, a plant chromosome or plastid or a recombinant plant viral vector or a recombinant baculoviral vector comprising the recombinant DNA construct, a transgenic plant cell having the recombinant DNA construct in its genome, and a transgenic plant or fruit, seed or propagable part of a transgenic plant comprising such a transgenic plant cell, and a plant having enhanced resistance to Botrytis cinerea and pests provided by expression or treatment of the recombinant DNA construct or the RNA encoded therein.

[0135] The recombinant DNA construct comprises a heterologous promoter operably linked to DNA comprising at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the fragment of 18 or more contiguous nucleotides has a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the DNA has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA or its DNA complement having a sequence selected from the target gene sequence group.

[0136] Thus, the recombinant DNA construct comprises a heterologous promoter operably linked to DNA comprising at least one fragment of 18 or more contiguous nucleotides designed to repress expression of a target gene having a sequence selected from a group of target gene sequences or its DNA complement. In some embodiments, the Botrytis cinerea target gene is selected from the group consisting of Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1. In some embodiments, the DNA comprises at least one fragment of 18 or more, e.g., 18-24, or 18-28, or 20-30, or 20-50, or 20-100, or 50-100, or 50-500, or 100-250, or 100-600, or 200-1000, or 500-2000 or more contiguous nucleotides. In some embodiments, the fragments comprise more than 18 consecutive nucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, The DNA comprises about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 575, about 600, or more than 600 consecutive nucleotides. In certain embodiments, the DNA encodes a DNA having a sequence selected from a group of target gene sequences or their DNA complements, or an RNA comprising at least one fragment of at least 18, 19, 20, or 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of the target gene.In certain embodiments, the DNA encodes a double-stranded nucleic acid (e.g., dsRNA) in which one strand comprises at least one fragment of at least 18, 19, 20, or 21 contiguous nucleotides with a sequence of 100% identity to a fragment of equivalent length of DNA or a target gene or its DNA complement, expressed as base pairs, and such double-stranded nucleic acid comprises at least one fragment of at least 18, 19, 20, or 21 contiguous perfectly matched base pairs corresponding to a fragment of equivalent length of DNA or a target gene or its DNA complement, with a sequence selected from the group of target gene sequences. In certain embodiments, each fragment in the DNA is longer than the length typical of naturally occurring small regulatory RNA. In some embodiments, each fragment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the DNA or each fragment in the polynucleotide is shorter than the total length of the sequence of interest (DNA or a target gene with a sequence selected from the group consisting of target gene sequences). In some embodiments, the total length of the DNA is about 50 to about 600. In some embodiments, the DNA encodes RNA having a sequence selected from the group consisting of SEQ ID NOs: 13 to 24, 49 to 52, or a combination thereof, or a complement thereof.

[0137] The recombinant DNA construct generally comprises a heterologous promoter operably linked to DNA encoding an RNA designed to repress one or more target genes. Such target genes may comprise coding or non-coding sequences or both. In certain embodiments, the recombinant DNA construct is designed to repress one or more target genes of Botrytis cinerea selected from the group consisting of Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1. In various embodiments, the recombinant DNA construct is designed to repress one or more genes, each gene having a sequence selected from the group consisting of target gene sequences, and can be designed to repress multiple genes from this group or target one or more different regions of these genes. In one embodiment, the recombinant DNA construct comprises a heterologous promoter operably linked to a plurality of sections or fragments, each of which comprises at least one fragment of 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from a group of target gene sequences. In such a case, each section may be identical or different in size or sequence and may be sense or antisense to the target gene. For example, in one embodiment, the recombinant DNA construct may comprise a heterologous promoter operably linked to a plurality of sections in tandem or repeat arrangement, each of which comprises at least one fragment of 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from a group of target gene sequences. The fragments may be derived from different regions of the target gene, for example, the fragments may correspond to different exon regions of the target gene, and "spacer" nucleotides not corresponding to the target gene may be used between or adjacent to the fragments, if necessary.

[0138] The recombinant DNA construct comprises a heterologous promoter operably linked to DNA that may have a total length greater than 18 contiguous nucleotides and may contain nucleotides in addition to a fragment of at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from a group of target gene sequences. In other words, the total length of the DNA may be longer than the length of the fragment of DNA designed to repress one or more target genes, each target gene having a DNA sequence selected from a group consisting of a group of target gene sequences. For example, the DNA may have nucleotides adjacent to the "active" fragment of at least one fragment of 18 or more contiguous nucleotides that represses the target gene, or may contain "spacer" nucleotides between the active fragments, or may have additional nucleotides at the 5' end or at the 3' end or at both the 5' and 3' ends. In one embodiment, the heterologous promoter is operably linked to DNA having a sequence selected from a group of target gene sequences or DNA containing additional nucleotides (e.g., nucleotides that provide a stable secondary structure or nucleotides for convenience of cloning or manufacturing) that are not specifically related (have a sequence that is not complementary or identical) to the target gene or its DNA complement. In one embodiment, the heterologous promoter is operably linked to DNA having a sequence selected from a group of target gene sequences or DNA containing additional nucleotides located immediately adjacent to one or more fragments of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length of the target gene or its DNA complement. In one embodiment, the heterologous promoter is operably linked to DNA containing one such fragment and having an additional 5'G or an additional 3'C or both adjacent to the fragment. In another embodiment, the heterologous promoter is operably linked to DNA encoding a double-stranded RNA containing additional nucleotides that form an overhang.Thus, in various embodiments, the entire nucleotide sequence of the DNA operably linked to the heterologous promoter is not 100% identical or complementary to a segment of contiguous nucleotides in the DNA having a sequence selected from the group consisting of the target gene sequences or in the target gene. For example, in some embodiments, the heterologous promoter is operably linked to a DNA comprising at least two segments of 21 contiguous nucleotides having a sequence of 100% identity to a segment of the DNA having a sequence selected from the group of target gene sequences or its DNA complement, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding segments are present in the DNA having a sequence selected from the group of target gene sequences or its DNA complement.

[0139] In the recombinant DNA construct, a heterologous promoter is operably linked to DNA encoding a transcript that can be single-stranded (ss) or double-stranded (ds), or a combination of both. Embodiments of this method include those in which the DNA encodes a transcript that includes a sense single-stranded RNA (ssRNA), an antisense ssRNA, or a double-stranded RNA (dsRNA), or any combination thereof.

[0140] The recombinant DNA construct is provided by any suitable means known to those of skill in the art. Embodiments include those in which the recombinant DNA construct is synthesized in vitro, produced by expression in a microorganism or cell culture (such as a plant cell grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.

[0141] The heterologous promoter used in the recombinant DNA construct is selected from the group consisting of promoters that function in plants, promoters that function in prokaryotes, promoters that function in fungal cells, and baculovirus promoters. Non-limiting examples of promoters are listed in the section entitled "Promoters".

[0142] In some embodiments, the recombinant DNA construct also comprises a second promoter operably linked to the DNA. For example, the DNA comprising at least one fragment of 18 or more consecutive nucleotides can be flanked by two promoters arranged such that the promoters transcribe in opposite directions and in a convergent manner to generate opposite strand transcripts of the DNA that are complementary and can hybridize with each other to form double-stranded RNA. In one embodiment, the DNA is located between two root-specific promoters, which allows the DNA to be transcribed in opposite directions, resulting in the formation of dsRNA.

[0143] In some embodiments, the recombinant DNA construct comprises other DNA elements in addition to a heterologous promoter operably linked to DNA comprising at least one fragment of 18 or more contiguous nucleotides having a sequence of about 95%-100% identity to a fragment of comparable length of DNA or its DNA complement having a sequence selected from a target gene sequence group or a trigger sequence group. Such DNA elements are known in the art and include, but are not limited to, introns, recombinase recognition sites, aptamers or ribozymes, and additional expression cassettes for expressing coding sequences (e.g., for expressing transgenes such as bactericidal proteins or selectable markers) or for expressing non-coding sequences (e.g., for expressing additional inhibitory elements). The inclusion of one or more recognition sites for binding and cleavage by small RNAs (e.g., miRNAs or siRNAs that are only expressed in certain cells or tissues) allows for a more precise expression pattern in the plant, with expression of the recombinant DNA construct being inhibited where the small RNA is expressed.

[0144] In some embodiments, the recombinant DNA construct is provided in a recombinant vector. "Recombinant vector" refers to a recombinant polynucleotide molecule that is used to transfer genetic information from one cell to another. Suitable embodiments of the present invention include, but are not limited to, recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors, such as recombinant plant viral vectors and recombinant baculovirus vectors. Alternative embodiments include recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors, such as recombinant plant viral vectors and recombinant baculovirus vectors, that contain DNA elements that do not contain heterologous promoters.

[0145] In some embodiments, the recombinant DNA construct is provided in a plant chromosome or plastid, for example, in a transgenic plant cell or transgenic plant. Thus, the present invention also encompasses transgenic plant cells having a recombinant DNA construct in their genome, as well as transgenic or partially transgenic plants comprising such transgenic plant cells. Partially transgenic plants include, for example, non-transgenic scions grafted onto transgenic rootstocks comprising transgenic plant cells. An embodiment includes a transgenic tomato rootstock comprising a transgenic plant cell. The plant may be any plant susceptible to infection by Botrytis cinerea. An embodiment includes those in which the plant is an ungerminated plant seed, a plant in a vegetative stage, or a plant in a reproductive stage. In yet another aspect, the present invention is directed to seeds (particularly transgenic progeny seeds) produced by transgenic plants having a recombinant DNA construct described herein in their genome. Products produced by such transgenic plants and products produced from transgenic progeny seeds of such transgenic plants are also contemplated.

[0146] The recombinant DNA construct can be provided in a composition for topical application to the surface of a plant or the seeds, roots, or stems of a plant, or for topical application to any substrate requiring protection from Botrytis cinerea infection. Similarly, the recombinant DNA construct can be provided in a composition for topical application to Botrytis cinerea, or for internal absorption (e.g., transfection) by Botrytis cinerea. In various embodiments, such compositions containing the recombinant DNA construct are provided in at least one form selected from the group consisting of a solid, a liquid (including homogeneous mixtures such as solutions, and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions), a powder, a suspension, an emulsion, a spray, an encapsulated or microencapsulated formulation, in or on microbeads or other carrier particles, in a film or coating, or on or within a matrix, or a treatment of leaves, seeds, roots, or stems. The topical application can be in the form of a topical treatment of the fruit of the plant or the seeds from the fruit of the plant. As known to those skilled in the art of pesticide formulation and seed treatment, suitable binders, inert carriers, surfactants, etc. can be included in the composition containing the recombinant DNA construct. In some embodiments, the composition for topical application containing the recombinant DNA construct is at least one topically embedded formulation selected from the group consisting of microparticles, pellets, or capsules that are topically embedded in the plant. In such embodiments, the method includes topically embedding the topically embedded formulation in the plant. In one embodiment, the composition for topical application containing the recombinant DNA construct can be internalized (e.g., transfected) by Botrytis cinerea. In some embodiments, the composition containing the recombinant DNA construct further includes one or more components selected from a carrier agent, a surfactant (incorporated herein by reference), an organosilicone, an organosilicone surfactant, a non-polynucleotide fungicide, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide insecticide, a polynucleotide insecticide, a polynucleotide insecticide, a non-polynucleotide insecticide, a toxic safener, and a pathogen growth regulator.In one embodiment, the composition containing the recombinant DNA construct further comprises a non-ionic organosilicone surfactant, such as a SILWET® brand surfactant, exemplified by SILWET L-77® brand surfactant having CAS number 27306-78-1 and EPA number: CAL.REG.NO. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY. BREAK-THRU S 240 brand is a polyether modified polysiloxane (caSRN proprietary) surfactant currently available from Goldschmidt Chemical Corporation, Hopewell, VA. BREAK-THRU S 279 is an end-capped polyether trisiloxane surfactant, whose components are listed on the following existing chemicals list: EINECS, TSCA, ENCS, AICS, ECL, PICCS CHINA, NDSL. INDUCE brand adjuvant NMFC Item 42652, Class 60, is currently available from Helena Chemical Company, Collierville, TN. FRANCHISE® with LECI-TECH® brand surfactant, having CA REG No. 34704-50065, is currently available from Loveland Products, Inc. Greely, CO. One embodiment includes a germicidal composition further comprising BREAK-thru 301.

[0147] It is expected that the combination of certain recombinant DNA constructs described herein (e.g., recombinant DNA constructs containing a polynucleotide trigger as described in the Examples) with one or more non-polynucleotide insecticides, whether transgenically expressed or topically applied, will provide improved prevention and control against Botrytis cinerea infection and pest infestation compared to the recombinant DNA construct alone or the non-polynucleotide insecticide alone. In one embodiment, a recombinant DNA construct for expressing one or more polynucleotides and one or more genes encoding a non-polynucleotide insecticide is found to provide enhanced resistance against Botrytis cinerea infection and pest infestation in plants expressing the recombinant DNA construct. One embodiment relates to a recombinant DNA construct for expressing an RNA comprising a fragment having a sequence selected from a group of trigger sequences and one or more genes encoding a non-polynucleotide insecticide.

[0148] In various embodiments, the composition comprising the recombinant DNA construct comprises a microbial cell or is produced in a microorganism.For example, the composition for containing the recombinant DNA construct can comprise a bacterial or yeast cell or be produced in a bacterial or yeast cell.In a similar embodiment, the composition comprising the recombinant DNA construct comprises a transgenic plant cell or is produced in a plant cell (e.g., a plant cell that transiently expresses the recombinant DNA construct).Such a plant cell can be a cell in a plant or a cell grown in tissue culture or cell suspension.

[0149] XIII. Transgenic Plant Cells Some embodiments relate to transgenic plant cells expressing polynucleotides useful in the methods described herein for suppressing expression of a target gene in Botrytis cinerea or for controlling Botrytis cinerea infection. In one aspect, the invention provides a transgenic plant cell having in its genome a recombinant DNA encoding an RNA comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of DNA having a sequence selected from a group of target gene sequences, a group of trigger sequences, or a DNA complement thereof. In one aspect, the invention provides a transgenic plant cell having in its genome a recombinant DNA encoding an RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of Botrytis cinerea, wherein the target gene sequence is selected from a group of target gene sequences or a DNA complement thereof. In one aspect, the invention provides a transgenic plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in Botrytis cinerea that is in contact with or internalized by the RNA, wherein the RNA is a silencing element having at least one fragment of 18 or more contiguous nucleotides that is complementary to a fragment of the target gene, the target gene being selected from the group consisting of genes in a target gene sequence group. A particular embodiment is a transgenic plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in Botrytis cinerea that is in contact with or internalized by the RNA, the transgenic plant cell comprising at least one silencing element having at least one fragment of 18 or more contiguous nucleotides that is complementary to a fragment of one or more target gene sequences. In one aspect, the invention provides a transgenic plant cell having in its genome a recombinant DNA encoding an RNA having a sequence selected from a trigger sequence group.Such transgenic plant cells are useful for providing transgenic plants having enhanced resistance to Botrytis cinerea infection compared to control plants lacking such plant cells. The transgenic plant cells can be isolated transgenic plant cells, transgenic plant cells grown in culture, or transgenic cells of any transgenic plant that is subject to infection by Botrytis cinerea.

[0150] In one embodiment, the recombinant DNA is stably integrated into the genome of the transgenic plant, from which it can be expressed in one or more cells of the transgenic plant. Methods for providing stably transformed plants are discussed in the section "Generation and Uses of Transgenic Plant Cells and Transgenic Plants."

[0151] Some embodiments relate to a transgenic plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in Botrytis cinerea that contacts or internalizes the RNA, the RNA comprising at least one silencing element that is complementary to the target gene, the target gene sequence being selected from a group of target gene sequences or their complements. In some embodiments, the silencing element comprises at least 18 or more contiguous nucleotides that have a sequence that is about 95% to about 100% complementary to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from a group of target gene sequences or a group of trigger sequences. In some embodiments, the silencing element comprises at least 18 or more contiguous nucleotides that can hybridize in vivo or under physiological conditions (e.g., physiological conditions normally found in cells of Botrytis cinerea) to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from a group of target gene sequences or a group of trigger sequences. The number of consecutive nucleotides is at least 18, for example, 18 to 24, or 18 to 28, or 20 to 30, or 20 to 50, or 20 to 100, or 50 to 100, or 50 to 500, or 100 to 250, or 100 to 500, or 200 to 1000, or more.In some embodiments, the number of consecutive nucleotides is more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, About 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 575, about 600, or more than 600 consecutive nucleotides. In certain embodiments, the silencing element comprises at least one fragment of at least 18, 19, 20, or 21 consecutive nucleotides having a sequence selected from a target gene sequence group, a trigger sequence group, a DNA, or a fragment of equivalent length of a target gene or a DNA complement having a sequence of 100% identity. In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) comprising at least one fragment of at least 18, 19, 20, or 21 contiguous nucleotides, one strand of which has a sequence of 100% identity to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the group of target gene sequences or the group of trigger sequences. Such double-stranded nucleic acids, expressed as base pairs, comprise at least one fragment of at least 18, 19, 20, or 21 contiguous perfectly matched base pairs corresponding to a fragment of equivalent length of DNA or the target gene or its DNA complement having a sequence selected from the group of target gene sequences or the group of trigger sequences. In certain embodiments, each silencing element contained in the RNA is longer than the length typical of naturally occurring small regulatory RNAs. In some embodiments, each fragment is at least about 30 contiguous nucleotides (or base pairs) in length. In certain embodiments, the length of the RNA is about 50 to about 600 nucleotides. In certain embodiments, the RNA has a sequence selected from the group of trigger sequences.

[0152] In some embodiments, the transgenic plant cell can further express additional heterologous DNA sequences. In certain embodiments, the transgenic plant cell stably integrates in its genome (i) a recombinant DNA encoding at least one RNA having a sequence selected from a group of trigger sequences, and (ii) a DNA encoding at least one fungicide.

[0153] In related aspects, the present invention is directed to products produced from transgenic plants comprising transgenic plant cells, transgenic plants and transgenic progeny plant seeds of transgenic plants or transgenic propagable portions of transgenic plants. Also contemplated are products produced by transgenic plants and products produced from transgenic progeny seeds of such transgenic plants.

[0154] XIV. Methods for generating polynucleotides for RNAi The polynucleotides of the claimed methods and compositions can be produced by any suitable method known in the art. Examples of methods for producing the RNA molecules of the present disclosure include, but are not limited to, in vitro transcription (IVT) (such as transcription using T7 polymerase or other polymerases), chemical synthesis, expression in an organism (such as a plant or microorganism) or in a cell culture (such as a plant cell culture), and microbial fermentation. In some embodiments, the RNA described herein is produced through any one of the cell-free production processes of RNA described in U.S. Pat. No. 10,858,385 or U.S. Pat. No. 10,954,541, both of which are incorporated herein by reference. In some embodiments, the dsRNA described herein is produced through the use of recombinant plasmids to enhance expression of dsRNA, as described in WIPO Patent Application Publication No. WO2021 / 113774, which is incorporated herein by reference.

[0155] XV. Promoter The promoters used in the present invention function in the cells in which the construct is intended to be transcribed.Generally, these promoters are heterologous promoters used in recombinant constructs, i.e., they are not naturally found operably linked to other nuclear elements used in the constructs described herein.In various embodiments, the promoter is selected from the group consisting of constitutive promoters, spatially specific promoters, temporally specific promoters, developmentally specific promoters, and inducible promoters.In many embodiments, the promoter is a promoter that functions in plants, such as a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.

[0156] Non-constitutive promoters suitable for use with the recombinant DNA constructs of the invention include spatially specific promoters, temporally specific promoters, and inducible promoters. Spatially specific promoters may include organelle-specific, cell-specific, tissue-specific, or organ-specific promoters (e.g., plastid-specific, root-specific, pollen-specific, or seed-specific promoters for expression in plastids, roots, pollen, or seeds, respectively). Often, seed-specific, embryo-specific, aleurone-specific, or endosperm-specific promoters are particularly useful. Temporally specific promoters may include promoters that tend to promote expression during a particular developmental stage in the plant's growth cycle, or at different times of the day or night, or during different seasons of the year. Inducible promoters include, but are not limited to, promoters that are induced by environmental conditions such as chemicals, or biotic or abiotic stresses (e.g., water shortage or drought, heat, cold, high or low nutrient or salinity levels, high or low light levels, or pest or pathogen infection). MicroRNA promoters are particularly useful as promoters with temporally specific, spatially specific, or inducible expression patterns. Examples of miRNA promoters, as well as methods for identifying miRNA promoters with specific expression patterns, are provided in U.S. Patent Application Publication Nos. 2006 / 0200878, 2007 / 0199095, and 2007 / 0300329, which are specifically incorporated herein by reference. Expression-specific promoters can also include promoters that are constitutively expressed but differ in the degree or "strength" of expression, including promoters that are generally considered to be "strong promoters" or "weak promoters."

[0157] Specific promoters of interest include, for example, the opaline synthase promoter isolated from the Agrobacterium T-DNA, the Cauliflower Mosaic Virus (CaMV) 35S promoter, enhanced promoter elements, or enhancer elements (Zea chimeric promoter elements such as the enhanced CaMV35S promoter linked to an intron from heat shock protein 70 of M. mays; root specific promoters such as those disclosed in U.S. Pat. Nos. 5,837,848, 6,437,217, and 6,426,446, the maize L3 oleosin promoter disclosed in U.S. Pat. No. 6,433,252, the promoter of a plant nuclear gene encoding a plastid-localized aldolase disclosed in U.S. Patent Application Publication No. 2004 / 0216189, cold inducible promoters disclosed in U.S. Pat. No. 6,084,089, salt inducible promoters disclosed in U.S. Pat. No. 6,140,078, light inducible promoters disclosed in U.S. Pat. No. 6,294,714, pathogen inducible promoters disclosed in U.S. Pat. No. 6,252,138, and water deficit inducible promoters disclosed in U.S. Patent Application Publication No. 2004 / 0123347A1. All of the above patents and patent publications disclosing promoters and their uses, particularly in recombinant DNA constructs that function in plants, are hereby incorporated by reference.

[0158] Examples of plant vascular or phloem specific promoters of interest include the rolC or rolA promoter of Agrobacterium rhizogenes, the promoter of the oncogenic Agrobacterium T-DNA gene 5, the rice sucrose synthase RSs1 gene promoter, the Commelina yellow spotted badna virus promoter, the palm leaf rot virus promoter, the rice sclerotinia virus promoter, the pea glutamine synthase GS3A gene promoter, the potato invertase gene invCD111 and invCD141 promoters, the Kertbundit et al. These include a promoter isolated from Arabidopsis shown to have phloem-specific expression in tobacco by K. et al. (1991) Proc. Natl. Acad. Sci. USA., 88:5212-5216, the VAHOX1 promoter region, the pea cell wall invertase gene promoter, the acid invertase gene promoter from carrot, the promoter of the sulfate transporter gene Sultr1, the promoter of a plant sucrose synthase gene, and the promoter of a plant sucrose transporter gene.

[0159] Promoters suitable for use in the recombinant DNA constructs or polynucleotides of the invention can include polymerase II ("pol II") and polymerase III ("pol III") promoters. RNA polymerase II transcribes structural or catalytic RNAs, which are usually shorter than 400 nucleotides in length, and recognizes a run of simple T residues as a termination signal. It is used to transcribe siRNA duplexes (see, e.g., Lu et al. (2004) Nucleic Acids Res., 32:e171). Thus, in certain embodiments in which short RNA transcripts are generated from the recombinant DNA constructs of the invention, a Pol II promoter is used. In one embodiment, the recombinant DNA construct includes a pol II promoter that expresses an RNA transcript flanked by self-cleaving ribozyme sequences (e.g., a self-cleaving hammerhead ribozyme), resulting in a processed RNA, such as a single-stranded RNA that binds to a transcript of a target gene in Botrytis cinerea with defined 5' and 3' ends and no potentially interfering flanking sequences. Another approach uses pol III promoters to generate transcripts with relatively defined 5' and 3' ends, i.e., transcribe RNA with minimal 5' and 3' flanking sequences. In some embodiments, the Pol III promoter (e.g., U6 or H1 promoter) is used to add a short AT-rich transcription termination site that results in a two-base pair overhang (UU) to the transcribed RNA. This is useful for the expression of siRNA type constructs, for example. The use of pol III promoters to drive the expression of siRNA constructs has been reported. See van de Wetering et al. (2003) EMBO Rep., 4:609-615, and Tuschl (2002) Nature Biotechnol., 20:446-448. Baculovirus promoters, such as the baculovirus polyhedrin and p10 promoters, are known in the art and are commercially available.See, e.g., Invitrogen's "Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques", 2002 (Life Technologies, Carlsbad, Calif.) and FJ Haines et al., "Baculovirus Expression Vectors", published date (Oxford Expression Technologies, Oxford, UK).

[0160] A promoter element may include a nucleic acid sequence that is not a naturally occurring promoter or promoter element or a homolog thereof, but is capable of regulating expression of a gene. Examples of such "gene-independent" regulatory sequences include naturally occurring or artificially designed nucleic acid sequences that include ligand-binding regions or aptamers (see "Aptamers" below) and regulatory regions (which may be cis-acting). See, e.g., Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005) Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal and Breaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such "riboregulators" can be selected or engineered for a particular spatial or temporal specificity, e.g., to regulate the translation of DNA encoding a silencing element that represses a target gene in Botrytis cinerea only in the presence (or absence) of a given concentration of the appropriate ligand. One example is bioregulatory in nature, responding to endogenous ligands (e.g., jasmonic acid or salicylic acid) that are produced by the plant when it is under stress (e.g., abiotic stress such as water, temperature, or nutrient stress, or biotic stress such as infestation by pests or pathogens). Under stress, the level of the endogenous ligand increases to a level sufficient for the bioregulator to initiate transcription of DNA encoding a silencing element that represses the target gene in Botrytis cinerea.

[0161] XVI. Recombinase Sites In some embodiments, the recombinant DNA construct or polynucleotide of the invention comprises DNA encoding one or more site-specific recombinase recognition sites. In one embodiment, the recombinant DNA construct comprises at least one pair of loxP sites, and site-specific recombination of DNA between the loxP sites is mediated by Cre recombinase. The position and relative orientation of the loxP sites are selected to achieve the desired recombination. For example, if the loxP sites are in the same orientation, the DNA between the loxP sites is excised into a circular shape. In another embodiment, the recombinant DNA construct comprises DNA encoding one loxP site. In the presence of Cre recombinase and another DNA with a loxP site, the two DNAs are recombined.

[0162] XVII. Transgene Transcription Unit In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises a transgene transcription unit. The transgene transcription unit comprises a DNA sequence that codes for a gene of interest, such as a native or heterologous protein. The gene of interest can be any coding or non-coding sequence from any species, including but not limited to non-eukaryotes such as bacteria, viruses, fungi, protists, plants, invertebrates, and vertebrates. The transgene transcription unit can further comprise 5' or 3' sequences or both required for transcription of the transgene.

[0163] XVIII. Intron In some embodiments, the recombinant DNA construct or polynucleotide of the invention comprises DNA encoding a splicable intron. By "intron" is generally meant a fragment of DNA (or RNA transcribed from such a fragment) located between exons (protein-coding fragments of DNA or corresponding transcribed RNA), and during maturation of messenger RNA, the intron present is enzymatically "spliced ​​out" or removed from the RNA strand by a cleavage / ligation process that occurs in the eukaryotic nucleus. The term "intron" also applies to non-coding DNA sequences that are transcribed into RNA fragments that can be spliced ​​out from the mature RNA transcript, but are not introns found between protein-coding exons. One example of these is a splicable sequence that has the ability to enhance expression of downstream coding sequences in plants (in some cases, particularly monocotyledonous plants). These spliceable sequences are naturally located in the 5' untranslated regions of some plant genes and of some viral genes (e.g., the 5' leader sequence or "omega" leader of the tobacco mosaic virus, which has been described to enhance the expression of plant genes by Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638). These spliceable sequences or "expression-enhancing introns" can be artificially inserted into the 5' untranslated region between the promoter and the protein-coding exon of a plant gene. Examples of such expression-enhancing introns include, but are not limited to, the maize alcohol dehydrogenase (Zm-Adh1), maize Bronze-1 expression-enhancing intron, rice actin 1 (Os-Act1) intron, Shrunken-1 (Sh-1) intron, maize sucrose synthase intron, heat shock protein 18 (hsp18) intron, and the heat shock protein 82 kilodalton (hsp82) intron.U.S. Pat. Nos. 5,593,874 and 5,859,347 (specifically incorporated herein by reference) describe methods of improving recombinant DNA constructs for use in plants by including an expression-enhancing intron derived from the 70 kilodalton maize heat shock protein (hsp70) in the untranslated leader located 3' from the gene promoter and 5' from the exon encoding the first protein.

[0164] XIX. Ribozymes In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding one or more ribozymes. Ribozymes of particular interest include self-cleaving ribozymes, hammerhead ribozymes, or hairpin ribozymes. In one embodiment, the recombinant DNA construct comprises DNA encoding one or more ribozymes that function to cleave transcribed RNA to provide a defined fragment of RNA, such as a silencing element for suppressing a target gene in Botrytis cinerea.

[0165] XX. Gene suppression elements In some embodiments, the recombinant DNA construct or polynucleotide of the invention comprises DNA encoding additional gene suppression elements for suppressing a target gene other than the Botrytis cinerea target gene. The target gene to be suppressed may comprise coding or non-coding sequences, or both.

[0166] Suitable gene suppression elements are described in detail in US Patent Application Publication No. 2006 / 0200878, the disclosure of which is specifically incorporated herein by reference, and include one or more of the following: (a) DNA comprising at least one antisense DNA fragment that is antisense to at least one fragment of a gene to be inhibited; (b) DNA comprising multiple copies of at least one antisense DNA fragment that is antisense to at least one fragment of the gene to be inhibited; (c) DNA comprising at least one sense DNA fragment which is at least one fragment of the gene to be suppressed; (d) DNA comprising multiple copies of at least one sense DNA fragment which is at least one fragment of a gene to be suppressed; (e) a DNA which can be transcribed into RNA for suppressing a gene to be suppressed by forming a double-stranded RNA, the DNA comprising at least one antisense DNA fragment which is antisense to at least one fragment of the gene to be suppressed, and at least one sense DNA fragment which is at least one fragment of the gene to be suppressed; (f) a DNA that can be transcribed into an RNA for suppressing a gene to be suppressed by forming a single double-stranded RNA, the DNA comprising a plurality of serial antisense DNA fragments that are antisense to at least one fragment of the gene to be suppressed, and a plurality of serial sense DNA fragments that are at least one fragment of the gene to be suppressed; (g) a DNA which can be transcribed into RNA for suppressing a gene to be suppressed by forming multiple duplexes of RNA, the DNA comprising multiple antisense DNA fragments which are antisense to at least one fragment of the gene to be suppressed, and multiple sense DNA fragments which are at least one fragment of the gene to be suppressed, the multiple antisense DNA fragments and the multiple sense DNA fragments being arranged in a series of inverted repeats; (h) DNA containing nucleotides derived from plant miRNA; (i) DNA comprising siRNA nucleotides; (j) DNA that is transcribed into an RNA aptamer capable of binding to a ligand; and (k) DNA that is transcribed into an RNA aptamer capable of binding to a ligand, and DNA that is transcribed into a regulatory RNA capable of controlling the expression of a gene to be inhibited, wherein the control depends on the three-dimensional structure of the regulatory RNA, and the three-dimensional structure of the regulatory RNA is allosterically affected by the binding state of the RNA aptamer.

[0167] In some embodiments, introns are used to deliver gene suppression elements in the absence of any protein-coding exons (coding sequences).In one example, introns, such as expression-enhancing introns, are interrupted by embedding gene suppression elements within the intron, and the gene suppression elements are excised from the intron during transcription.Therefore, protein-coding exons are not required to provide gene suppression function of the recombinant DNA construct disclosed herein.

[0168] XXI. Transcriptional Regulatory Elements In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding a transcriptional regulatory element. The transcriptional regulatory element comprises an element that regulates the expression level of the recombinant DNA construct of the present invention (relative to the expression in the absence of such regulatory element). Examples of suitable transcriptional regulatory elements include riboswitches (cis-acting or trans-acting), transcript stabilizing sequences, transcriptional initiation sites, transcriptional elongation sequences, transcriptional termination elements, and miRNA recognition sites, as described in detail in US Patent Application Publication No. 2006 / 0200878, which is incorporated herein by reference.

[0169] XXII. Production and Uses of Transgenic Plant Cells and Plants Plant transformation can include any of several well-known methods and compositions. Methods suitable for plant transformation include virtually any method that can introduce DNA into cells. One method of plant transformation is biolistic bombardment, as shown, for example, in U.S. Pat. No. 5,015,580 (soybean), U.S. Pat. No. 5,538,880 (corn), U.S. Pat. No. 5,550,318 (corn), U.S. Pat. No. 5,914,451 (soybean), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (corn), U.S. Pat. No. 6,288,312 (rice), U.S. Pat. No. 6,365,807 (rice), and U.S. Pat. No. 6,399,861 (corn), and U.S. Pat. No. 6,403,865 (corn), all of which are incorporated by reference to allow the generation of transgenic plants.

[0170] Another useful method of plant transformation is Agrobacterium-mediated transformation with Agrobacterium containing a two-component Ti plasmid system, which carries a first Ti plasmid (often released), a second chimeric plasmid containing at least one T-DNA border of the wild-type Ti plasmid, and a promoter that functions in transformed plant cells and is operably linked to a polynucleotide or recombinant DNA construct of the present invention. See the two-component system described in U.S. Pat. No. 5,159,135, which is incorporated by reference. See also De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. In such a binary system, a smaller plasmid containing one or more T-DNA borders can be conveniently constructed and engineered in a suitable alternative host such as E. coli, and then transferred to Agrobacterium.

[0171] Detailed procedures for Agrobacterium-mediated transformation of plants, particularly crop plants, are described in U.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton), 5,416,011, 5,569,834, 5,824,877, and 6,384,301 (soybean), 5,591,616, and 5,981,840 (rice), and in U.S. Pat. Nos. 5,463,174 (rapeseed, including canola), 7,026,528 (wheat), and 6,329,571 (rice), and U.S. Patent Application Publication Nos. 2004 / 0244075 (corn) and 2001 / 0042257A1 (sugar beet), all of which are specifically incorporated by reference to enable the generation of transgenic plants. U.S. Patent Application Publication No. 2011 / 0296555 discloses in Example 5 transformation vectors (including vector sequences) and detailed protocols for transforming corn, soybean, canola, cotton, and sugarcane, which are specifically incorporated by reference to enable the generation of transgenic plants.Similar methods have been reported for many plant species, both dicotyledons and monocotyledons, including peanut (Cheng et al. (1996) Plant Cell Rep., 15:653), asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA, 84:5345), barley (Wan and Lemaux (1994) Plant Physiol., 104:37), rice (Toriyama et al. (1988) Bio / Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7:379), wheat (Vasil et al. (1992) Bio / Technology, 10:667; Becker et al. (1994) Plant J., 5:299), alfalfa (Masoud et al. (1996) Plant Cell Rep., 15:653), and sorghum (Schidigera cereale, 1996). al. (1996) Transgen. Res., 5:313), and tomato (Sun et al. (2006) Plant Cell Physiol., 47:426-431). See also U.S. Patent Application Publication No. 2003 / 0167537A1, incorporated by reference, for a description of vectors, transformation methods, and production of transformed Arabidopsis thaliana plants in which transcription factors are constitutively expressed by the CaMV35S promoter. Transformation methods particularly useful for plants susceptible to Botrytis infection are well known in the art.See, for example, publicly described transformation methods for tomato (Sharma et al. (2009), J. Biosci., 34:423-433), eggplant (Arpaia et al. (1997) Theor. Appl. Genet., 95:329-334), potato (Bannerjee et al. (2006) Plant Sci., 170:732-738; Chakravarty et al. (2007) Amer. J. Potato Res., 84:301-311; S. Millam “Agrobacterium-mediated transformation of potato.” Chapter 19 (pp. 257-270), “Transgenic Crops of the World: Essential Protocols”, Ian S. Curtis (editor), Springer, 2004), and pepper (Li et al. (2003) Plant Cell Reports, 21:785-788). Stable transgenic potatoes, tomatoes, and eggplants have been commercially introduced in various regions, see, e.g., K. Redenbaugh et al. “Safety Assessment of Genetically Engineered Fruits and Vegetables: A Case Study of the FLAVR SAVR Tomato”, CRC Press, Boca Raton, 1992, and for a description of commercial transgenic crops in the extensive publicly available GM crop database, see CERA. (2012). GM Crop Database. Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington DC, available electronically at cera-gmc.org / ?action=gm_crop_database.Various methods for transformation of other plant species are known in the art and are described, for example, in the encyclopedic reference "Compendium of Transgenic Crop Plants", edited by Chittaranjan Kole and Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (available electronically at mrw.interscience.wiley.com / emrw / 9781405181099 / hpt / toc) includes cereals and forage grasses (rice, corn, wheat, barley, oats, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahia grass), oilseed crops (soybean, oilseed rape, sunflower, peanut, flax, sesame, safflower), legume grains and forages (bean, cowpea, pea, broad bean, lentil, tepary bean, Asian bean, pigeon pea, vetch, chickpea, lupin, alfalfa, and clover), temperate fruits and nuts (apple, pear, peach, plum, berry, rye ... Transformation procedures are described for tropical and subtropical fruits and nuts (citrus fruits, grapefruit, bananas and plantains, pineapple, papaya, mango, avocado, kiwifruit, passion fruit, and persimmon), vegetable crops (tomato, eggplant, bell pepper, canola, radish, carrot, melon, leek, asparagus, and leafy vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turfgrasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, and turfgrasses), and forest tree species.

[0172] The transformation method that provides transgenic plant cells and transgenic plants containing stably integrated recombinant DNA is preferably performed in tissue culture on medium and in a controlled environment. "Medium" refers to a number of nutrient mixtures used to grow cells in vitro, i.e. outside of an intact living organism. Recipient cell targets include, but are not limited to, meristematic cells, callus, immature embryos or parts of embryos, and gamete cells such as microspores, pollen, sperm, egg cells, etc. Any cell capable of regenerating a reproductive plant is intended to be a recipient cell useful in the practice of the present invention. Callus can be initiated from a variety of tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling tip meristems, microspores, etc. These cells that are capable of growing as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for producing the transgenic plants of the invention (e.g., transformation of various media and recipient target cells, immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. No. 6,194,636 and U.S. Patent Application Publication No. 2004 / 0216189, which are specifically incorporated by reference.

[0173] In typical transformation practices, DNA is only introduced into a low percentage of target cells in any one transformation experiment. Generally, marker genes are used to provide an efficient system for identifying cells that are stably transformed by receiving and integrating the transgenic DNA construct into their genome. A preferred marker gene provides a selective marker that confers resistance to a selective agent, such as an antibiotic or herbicide. Any antibiotic or herbicide to which the plant cell is resistant can be a useful drug agent for selection. Potentially transformed cells are exposed to the selective agent that corresponds to the marker. In the population of surviving cells, generally, cells are of interest in which the resistance-conferring gene (selective marker) is integrated and expressed at a sufficient level to allow the cell to survive. The cells can be further tested to confirm stable integration of the recombinant DNA. Commonly used selectable marker genes include those that confer resistance to antibiotics such as kanamycin or paromomycin (nptll), hygromycin B (aphIV), and gentamicin (aac3 and aacC4), or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of useful selectable marker genes and selectable agents are set forth in U.S. Patent Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047, all of which are specifically incorporated by reference. Screenable markers or reporters can also be used, such as markers that provide the ability to visually identify transformants. Examples of useful screenable markers include, for example, genes that produce a detectable color by acting on a chromogenic substrate (e.g., β-glucuronidase (GUS) (uidA) or luciferase (luc)) or express a protein that is itself detectable, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those of skill in the art will recognize that many other useful markers or reporters are available.

[0174] Detecting or measuring transcription of the recombinant DNA construct in the transgenic plant cells can be accomplished by any appropriate method, including protein detection methods (e.g., Western blots, ELISAs, and other immunochemical methods), enzyme activity measurements, or nucleic acid detection methods (e.g., Southern blots, Northern blots, PCR, RT-PCR, fluorescence in situ hybridization).

[0175] Other suitable methods for detecting or measuring transcription in plant cells of a recombinant polynucleotide of the invention targeting a target gene in Botrytis cinerea include measuring other traits that directly or surrogately indicate the expression level of the target gene in Botrytis cinerea, such as, for example, growth rate, mortality rate, reproduction rate, or replenishment rate of Botrytis cinerea, compared to the expression level observed in the absence of the recombinant polynucleotide, or measuring injury (e.g., root damage) or yield loss in plants or fields of plants infested with Botrytis cinerea. In general, suitable methods for detecting or measuring transcription in plant cells of a recombinant polynucleotide of interest include, for example, macroscopic or microscopic morphological traits, growth rate, yield, reproduction rate or replenishment rate, resistance to pests or pathogens, or resistance to biotic or abiotic stress (e.g., water deficit stress, salt stress, nutrient stress, heat or cold stress). Such methods can use direct measurements of phenotypic traits or surrogate assays (e.g., in plants, these assays include plant part assays such as leaf or root assays to determine tolerance to abiotic stresses). Such methods include direct measurements (e.g., damage to plant tissues) or surrogate assays (e.g., plant yield assays, or bioassays) of resistance to Botrytis cinerea.

[0176] The recombinant DNA constructs of the invention can be stacked with other recombinant DNA to confer additional traits, for example, by expressing or suppressing other genes (e.g., in the case of transformed plants, traits including herbicide resistance, pest resistance, low temperature germination resistance, water deficit tolerance, etc.). Constructs for the coordinated reduction and increase of gene expression are disclosed in U.S. Patent Application Publication No. 2004 / 0126845A1, which is specifically incorporated by reference.

[0177] The seeds of the fertile transgenic plants can be harvested and used to grow progeny generations, including hybrid generations of the transgenic plants of the present invention, containing the recombinant DNA construct in its genome. Thus, in addition to direct transformation of plants with the recombinant DNA construct of the present invention, the transgenic plants of the present invention can be prepared by crossing a first plant containing the recombinant DNA with a second plant lacking the construct. For example, the recombinant DNA can be introduced into a plant line suitable for transformation to generate a transgenic plant, which can then be crossed with a second plant line to introgress the recombinant DNA into the resulting progeny. The transgenic plants of the present invention can be crossed with plant lines carrying other recombinant DNA that confers one or more additional traits (e.g., including, but not limited to, herbicide resistance, disease or pest resistance, environmental stress resistance, modified nutrient content, and improved yield) to generate progeny plants carrying recombinant DNA that confers both a given target sequence expression behavior and the additional trait.

[0178] In such combination breeding of traits, the transgenic plant that provides the additional trait can be the male line (pollinator) and the transgenic plant that retains the base trait can be the female line. The progeny of this cross will segregate such that some plants retain the DNA for the traits of both parents and some plants retain the DNA for the trait of one parent. Such plants can be identified by markers associated with the parental recombinant DNA, and progeny plants that retain the DNA for the traits of both parents can be crossed back to the female parent line multiple times, e.g., usually for six to eight generations, to produce homozygous progeny plants that have substantially the same genotype as one original transgenic parent line and the recombinant DNA of the other transgenic parent line. o Yet another aspect of the invention is a transgenic plant grown from a transgenic seed of the invention. The invention contemplates transgenic plants grown directly from a transgenic seed containing recombinant DNA, as well as progeny generation plants including inbred or hybrid lines created by crossing a transgenic plant grown directly from a transgenic seed with a second plant that was not grown from the same transgenic seed. Crossing can, for example, comprise the steps of: (a) planting a seed or stem cutting of a first parent plant (e.g. non-transgenic or transgenic) and a second parent plant that is transgenic according to the invention; (b) growing seeds or stem cuttings of the first and second parent plants into flowering plants; (c) pollinating flowers from the first parent with pollen of the second parent; and (d) harvesting the seeds produced on the parent plant having the pollinated flowers.

[0179] It is often desirable to introgress recombinant DNA into elite varieties, for example by backcrossing, to transfer a particular desired trait from a source to an inbred or other plant lacking that trait. This can be accomplished, for example, by crossing a first superior inbred line ("A") (the recurrent parent) to a donor inbred line ("B") (the non-recurrent parent) carrying the appropriate gene for the trait of interest (the construct prepared according to the present invention). The progeny of this cross are first selected in the resulting progeny for the desired trait transferred from the non-recurrent parent "B", and the selected progeny are backcrossed to the superior recurrent parent "A". After five or more generations of backcrossing with selection for the desired trait, the progeny are essentially hemizygous for the locus controlling the trait transferred, but like the superior parent for most or almost all other genes. The final backcross generation is selfed (selfing is allowed) to provide progeny that are pure breeding for the gene(s) transferred, such as one or more transformation events.

[0180] Through a series of breeding operations, a selected DNA construct can be moved from one line to a completely different line without the need for further recombinant operations. Thus, inbred plants can be generated that are true breedings of one or more DNA constructs. By crossing different inbred plants, a large number of different hybrids can be generated that have various combinations of DNA constructs. In this way, plants can be generated that have the desirable agronomic characteristics commonly associated with hybrids ("hybrid vigor"), as well as the desirable characteristics imparted by one or more DNA constructs.

[0181] In certain transgenic plant cells and transgenic plants of the invention, it is sometimes desirable to express a gene of interest while also regulating the expression of a target gene in Botrytis cinerea. Thus, in some embodiments, the transgenic plant contains recombinant DNA that further comprises a gene expression element for expressing at least one gene of interest, and transcription of the recombinant DNA construct of the invention is effected with co-transcription of the gene expression element.

[0182] The present invention also provides products produced from the transgenic plant cells, plants, or seeds of the present invention, including, but not limited to, harvested leaves, roots, shoots, stems, fruits, seeds, or other parts of plants, oils, extracts, fermentation or digestion products, or any food or non-food product containing such products produced from the transgenic plant cells, plants, or seeds of the present invention. Detection of one or more nucleic acid sequences of a recombinant DNA construct of the present invention in one or more of the goods or products contemplated herein is factual evidence that the goods or products contain or are derived from the transgenic plant cells, plants, or seeds of the present invention.

[0183] Generally, the genome of a transgenic plant having a recombinant DNA construct of the present invention, or a portion thereof, exhibits enhanced resistance to Botrytis cinerea infection. In various embodiments, for example, when a transgenic plant expresses a recombinant DNA construct of the present invention stacked with other recombinant DNA to confer additional traits, the transgenic plant exhibits the following traits compared to a plant lacking the recombinant DNA construct: (a) Enhanced abiotic stress tolerance; (b) enhanced biotic stress tolerance; (c) altered primary metabolite composition; (d) altered secondary metabolite composition; (e) altered trace element, carotenoid, or vitamin profile; (f) enhanced yield; (g) the ability to use enhanced nitrogen, phosphate, and other nutrients; (h) modified agricultural characteristics; (i) Altered growth or reproductive characteristics; and (j) Enhanced harvesting, storage or processing quality and having at least one additional modified trait selected from the group of traits consisting of:

[0184] In some embodiments, the transgenic plants exhibit improved tolerance to abiotic stress (e.g., tolerance to water deficit or drought, heat, cold, suboptimal nutrient or salinity levels, suboptimal light levels) or biotic stress (e.g., crowding, allelopathy, or wounding), through improved primary metabolite (e.g., fatty acid, oil, amino acid, protein, sugar, carbohydrate) composition, altered secondary metabolite (e.g., alkaloids, terpenoids, polyketides, nonribosomal peptides, and secondary metabolites derived from mixed biosynthesis) composition, altered trace element (e.g., iron, zinc), carotenoid (e.g., β-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e.g., tocopherol) composition, improved yield (e.g., non- improved yield under stress conditions or improved yield under biotic or abiotic stress), improved ability to use nitrogen, phosphorus or other nutrients, altered agronomic properties (e.g. delayed ripening, delayed senescence, earlier or later maturity, altered shade tolerance, improved resistance to lodging of roots or stems, improved resistance to stem "green snap", altered photoperiod response), altered growth or reproductive properties (e.g. intentional dwarfing, intentional male sterility, improved usefulness in, e.g., hybridization procedures, improved vegetative growth rate, improved germination, improved male and female fertility), improved harvesting, storage, or processing qualities (e.g. greater resistance to pests during storage, resistance to bruising, enhanced consumer appeal, etc.), or a combination of these properties.

[0185] In another embodiment, the transgenic seed, or the seed produced by the transgenic plant, has an altered primary metabolite (e.g., fatty acid, oil, amino acid, protein, sugar, carbohydrate) composition, an altered secondary metabolite composition, an altered trace element, carotenoid, or vitamin composition, an altered harvest, storage, or processing quality, or a combination thereof. In another embodiment, it may be desirable to alter the levels of natural components of the transgenic plant or the seed of the transgenic plant, for example, to reduce the levels of allergenic proteins or glycoproteins or the levels of toxic metabolites.

[0186] Typically, screening of a population of transgenic plants regenerated from transgenic plant cells is performed to identify transgenic plant cells that develop into transgenic plants with desired traits. The transgenic plants are assayed to detect enhanced traits, such as enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein, enhanced disease resistance, and enhanced seed oil. Screening methods include direct screening of traits in greenhouse or field trials, or screening of alternative traits. Such analyses aim to detect changes in the chemical composition, biomass, physiological properties, or morphology of the plants. Changes in chemical composition can be detected by analyzing seed composition, protein, free amino acid, oil, free fatty acid, starch, tocopherol, or other nutrient content. Changes in growth or biomass characteristics are detected by measuring plant height, stem diameter, internode length, root and shoot dry weight. Changes in physiological traits are identified by assessing responses to stress conditions (e.g., assays under imposed stress conditions such as water deficit, nitrogen or phosphate deficiency, cold or hot growing conditions, pathogen or insect attack, light deficit, or increased plant density). Other selective traits include days to flowering, days to pollen shedding, days to fruit maturity, fruit quality or yield, leaf elongation rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace root, green hold, stem lodging, root lodging, plant health, fertility, green snap, and pest resistance. Additionally, phenotypic traits of harvested fruit or seeds can be assessed, e.g., in the case of plants, this can include the total number or weight of harvested fruit, or the color, acidity, sugar content, or flavor of such fruit.

[0187] The following examples are presented for purposes of illustration and should not be construed as limiting. EXAMPLES

[0188] overview This example aims to highlight the deployment and effectiveness of exogenous application of dsRNA to control Botrytis cinerea on various fruit-bearing plants and vegetables, including tomato, strawberry, grape, and green bean.

[0189] Example 1 In vitro liquid-based assay fungal culture Botrytis cinerea was plated weekly and cultures were added onto malt extract agar (MEA) from long-term storage on silica stored at 4°C. Three silica pellets were added to the center of MEA agar plates and allowed to grow for 10-14 days at room temperature without a parafilm / tape seal before use. Typically, 12-day-old cultures were used.

[0190] Spore and medium preparation A pure culture of Botrytis cinerea was filled with 0.01% Triton x-100 solution and the conidia were scraped off with a sterile hockey stick. The suspension was transferred to two 1.5mL centrifuge tubes and centrifuged at 8,000 rpm for 2 minutes. The supernatant was discarded and the pelleted spores were washed twice with sterile water and resuspended in sterile water. Conidia were quantified using a hemocytometer and diluted to a final concentration of 5,000 conidia / mL stock. For the assay setup, a stock solution of 1.25X minimal medium was prepared using the following components: Once the stock solution was made, the pH was adjusted to 6.0 using NaOH or KOH, then filter sterilized by passing through a 0.2um filter tower and vacuum pump and stored at 4°C. Kao&Michayluk Vitamins(100X) is a filter sterilized stock solution. As described by Kao&Michayluk, the stock solution contains vitamins and amino acids. After filter sterilization, 20 mg of 4-aminobenzoic acid in 100 ml of solution was added.

[0191] Minimal medium (MM) formulation Prepare 800 mL of 1.25X MM. [Table 1]

[0192] To make the nitrate stock solution, the two parts described below were prepared separately, autoclaved at 121° C. for 20 minutes, cooled and then mixed.

[0193] 20X Nitrate 250 mL of 20X nitrate was prepared in two portions.

[0194] 1st portion 200mL [Table 2]

[0195] 2nd portion 50mL [Table 3]

[0196] Experimental setup Screening assays were set up in 96-well plates containing conidia that had been co-incubated with treatments (control and dsRNA) for 3 days before measuring fungal biomass. Each treatment was replicated 8 times in a single plate. Each well consisted of 12.5 μl of conidial suspension, 25 μl of treatment, 12.5 μl of sterile deionized water, and 200 μl of minimal medium. Each plate was covered with Breathlite film and placed in a Percival growth chamber at 23° C. on a shaking platform set at 120 RPM with 24-hour lighting. Fungal growth was measured after the plates had been incubated for 72 hours.

[0197] Read the assay plate Fungal biomass was determined by reading the plates on a Cytation 5 machine. To measure biomass, the program was set up to compile eight photographic slices per well under a bright-field microscope equipped with a 2.5X lens.

[0198] Data analysis The analysis was performed automatically in Cytation5 to determine the final biomass.

[0199] The results are shown in Table 1B and represent the average reduction in biomass relative to the control for the sequences tested over the several runs described above.

[0200] Example 2 Detached Leaf Assay

[0201] plant tissue Tomato seedlings of the Brandywine cultivar were grown in the greenhouse for 3 weeks before harvesting the first true leaves for the assay. Nodes 2-4 were prioritized for harvesting, and only the lateral tips were used for the assay. Leaflets of approximately equal size were used each week and randomized between treatments. Peeled leaflets were brought immediately to the laboratory for processing and used within 4 hours of harvesting. Peeled leaflets were stored at 4°C until processed and placed on 1% water agar medium for incubation.

[0202] fungal culture Botrytis cinerea isolate (B05.10) was extracted from these tubes stored on silica for long-term storage and stored at 4 °C for growth on nutrient medium. Three silica pellets were added to the center of a malt extract agar (MEA) agar plate and grown at room temperature without a parafilm / tape seal for approximately 14 days or until abundant conidia were formed before use.

[0203] Leaflet Treatment Leaflets were treated 24 hours prior to inoculation using an atomizer to obtain a fine mist of small droplets that completely covered the leaflets. Silwet L-77 was used as a spreading agent and was added to a final concentration of 0.03% (Silwet L-77 brand Phyto Technology Laboratories, S7777 Lot#14D7777001F). Inoculated controls were sprayed with the same volume of sterile deionized water containing Silwet L-77 in the absence of fungicide or other experimental materials. Leaflets were spray treated by placing them on a paper towel and applying the atomizer approximately 6 inches away from the leaflets. Treated leaves were placed on 1% water agar (dissolve 10 g Bacto Agar, Difco in 1 L deionized water, sterilize at 121 °C for 30 min, and pour into 100 × 20 mm Petri dishes once a temperature of 55 °C was reached) and the Petri dish lid replaced before placing the Petri dish under a plastic bag to maintain humidity prior to inoculation the next day. The Petri dishes of treated leaflets were left on the laboratory counter until the next day. Each treatment was replicated 10 times, with each replicate being a single treated leaflet with two inoculation sites, one on each side of the midvein of the leaf.

[0204] inoculation Conidial suspensions were prepared by pouring a 7% white grape juice solution onto culture plates and scraping the plates with a sterile spatula to remove spores and mycelium. Grape juice solution was prepared by mixing Welch's brand organic white grape juice with sterile deionized water. After removing the spores and mycelium of the cultures, the fungal mycelium was removed by pouring the suspension through two layers of cheesecloth and capturing the conidial suspension in a clean 50 mL conical tube. Approximately 3 μL of TWEEN 20 was added to the conidial suspension to prevent adhesion to the conical tube. Spore concentrations were measured using a Neubauer modified hemocytometer and a spore concentration of 1x10 was determined. 5The inoculum was further diluted with 7% grape juice until it reached 100 conidia / mL. The inoculum was used to inoculate host tissues within 2 h of preparation. Each tomato leaflet was inoculated twice, with one inoculation site on either side of the midvein of the leaflet. A repeater pipette was used to place a 10 μL volume of spore suspension at each inoculation site. Special care was taken to avoid touching the leaflet surface tips to avoid contamination between treatments.

[0205] incubation The Petri dishes were placed on a lunch tray, six dishes high, and transferred to the incubator. The incubator was humidity controlled to keep the relative humidity at approximately 75%. The incubator was kept on a 12 / 12 light / dark schedule with the temperature set at 23°C.

[0206] evaluation Measurements were taken 5 days after inoculation (DPI). To help determine lesion diameter, each developed lesion was measured in two directions at right angles to each other using electronic calipers.

[0207] Data analysis Data were analyzed using the SAS JMP statistical program (version 13.2.1) and compared to untreated inoculated controls. If the mean lesion diameter of the inoculated controls was less than 16 mm, the assay was considered "failed" and the assay was repeated to obtain greater virulence and more consistent lesion development.

[0208] The results are shown in Table 2 and represent the average percent reduction in lesion size relative to the control for the sequences tested across the several runs described above.

[0209] Example 3. Greenhouse (whole plant assay) plant tissue Tomato seedlings of the cultivar "Lanai" were planted and then thinned to leave one seedling per pot in 18-cell trays. Seedlings 3 weeks after germination were used to evaluate dsRNA treatments. Plants were grown at a day / night temperature of 23°C / 17°C with 10 hours of light every 24 hours. RH was ambient and plants were fertilized twice a day, every day except Sundays, when they were only watered. Fafard brand medium (70-80% Sphagnum peat moss, perlite, vermiculite, dolomitic limestone and water sprinklers) was used to grow plants throughout the experiment.

[0210] fungal culture Botrytis cinerea B05.10 isolate was maintained and spores were harvested as previously described for the detached leaf assay, except that no grape juice additive was used in the preparation of the inoculum.

[0211] Plant treatment Whole tomato seedlings were treated approximately 24 hours prior to inoculation using a spray booth. A 0.07% solution of Silwet L-77 was used as the wetting agent, added to a final volume of 0.07% in water (Silwet L-77 brand Phyto Technology Laboratories, S7777 Lot#14D7777001F). The spray booth deck was positioned 20 inches above the crowns of the seedlings, and a Twin Jet 8002EVS nozzle with a 100 mesh filter was placed in the spray mechanism. The spray boom was calibrated prior to treatment application with the following specifications to obtain a collection volume of 133mL (+ / - 6mL) in 10 seconds: 50GPA rate, 1.25MPH speed, 6' spray length, single pass, and 45psi. Ten replicates of these treatments were used per experiment.

[0212] Manual inoculation Plants were inoculated using a mist-o-matic applicator pressurized by pumping. Immediately prior to inoculation in the GH space, the inoculum suspension was poured into the mist-o-matic. The container was only half-full and the inoculum was applied evenly to all leaf surfaces with an even coverage.

[0213] incubation Plants were incubated in a 40-inch-tall humidity chamber supported by PVC pipes and covered with 3M translucent plastic. A humidifier was located inside the chamber and set to low, continuous humidity (filled with tap water). Temperature was D: 24-20°C / N: 20-18°C, photoperiod was 15 hours with 9 hours of supplemental lighting. RH was ambient, watering was daily by subirrigation, and benches were flooded for 10 minutes before draining.

[0214] evaluation Tomato seedlings were evaluated three times over the course of a two-week experiment, beginning 4 days after inoculation and every 3-4 days from the first evaluation date. Plants were evaluated for number of lesions and disease severity, which is the percentage of plants showing symptoms including necrotic lesions, pathogen sporulation, and chlorosis.

[0215] Data analysis Data shown for each sequence are the average of two biological runs (10 replicates per treatment) in the greenhouse. Percent disease severity (%DS) was determined by the percent area of ​​tomato leaves with symptoms including lesions, chlorosis, necrosis, and wilting. Disease severity was assessed on three dates (assessment numbers) during the 12-day test period. Treatments were compared to untreated inoculated controls and chemical standards. Statistical analysis was performed using SAS JMP version 13.2.1, and treatments were compared to untreated inoculated controls and chemical standards using Tukey-Kramer and student's T tests.

[0216] The results are shown in Tables 3 to 14 and Figures 1 to 5.

[0217] Example 4: Field test Example 4.1 Control of Botrytis Infection in Strawberries with GS349 and GS730 Compositions RNAi compositions containing dsRNA, each containing a trigger sequence (GS349 or GS730) described herein, were evaluated for their ability to control Botrytis in strawberry in an outdoor field trial. Unformulated (dissolved in water), GS349 and GS730 were tank mixed with water and standard adjuvant Silwet L-77 0.1% v / v, respectively. GS349 was tested at concentrations of 25 grams active ingredient per hectare (g ai / ha) and 50 g ai / ha, and GS730 was tested at a concentration of 100 g ai / ha. Serenade ASO, 85 g ai / ha, a standard biological fungicide, was used as a positive control. A second positive control group was treated with alternating applications of chemical standards Teldor Plus 750 g ai / ha, Signum 600 g ai / ha, and Switch 500 g ai / ha. The study also includes an untreated group as a negative control.

[0218] Strawberry seedlings were sprayed with the test compositions and positive control in 12 applications. The seedlings were observed starting 14 days after the first application, and disease progression was assessed by measuring the percentage of fruit surface area affected by disease (% disease severity). Disease severity was plotted over the entire study observation period, and the area under the symptom development curve (AUDPC) was calculated to measure the cumulative effect of the various treatments across multiple evaluations. Means were separated according to Fisher's LSD, alpha (p) ≦ 0.05.

[0219] The results are shown in Figure 6. Statistical significance was indicated by letters on each bar. GS349 and GS730 at both concentrations showed significant control of Botrytis versus the untreated control. GS349 25 g ai / ha and GS730 100 g ai / ha were not significantly different from the biological standard, Serenade. GS349 at 50 g / h ai showed significantly improved control versus the biological standard and was not significantly different from the chemical standard.

[0220] Example 4.2 Control of Botrytis Infection in Grapes with GS730 Composition RNAi compositions, each containing dsRNA containing a trigger sequence (GS730) described herein, were evaluated for their ability to control Botrytis in grapevine in an outdoor field trial. Unformulated and formulated compositions were tested. Unformulated compositions were tank mixed with water and Silwet L-77 0.1% v / v. Formulated samples were formulated with RO water, propylene glycol, linear alcohol ethoxylate, lignosulfanate, sodium citrate dihydrate, polyacrylic acid polymer, two biocide compositions, anhydrous citric acid, and antifoam, all of which are readily available on the market. Unformulated and formulated GS730 were tested at concentrations of 50 g ai / ha and 100 g ai / ha, respectively. A standard biological program of Serenade 9.4 l / ha alternating with JMS Sylet Oil 1% v / v was used as a positive control. Chemical standards Miravis Prime 392 g ai / ha ai and Switch 610 g ai / ha were applied to a second positive control group in alternating applications. An untreated group was used as a negative control.

[0221] The positive control was applied to grapevine plants at anthesis, bunch closure, veraison, and 7-10 days prior to harvest. Test applications were timed the same as the positive control, with additional applications made 7 days after anthesis, bunch closure, veraison, and 7-10 days prior to harvest. Symptoms were assessed over a 3-4 week period, starting 2 days after the last application (slide 3) or 18 days prior to the last application. Percent disease severity was assessed and AUDPC calculated in the same manner as described in Example 4.1.

[0222] The results of two different trials are shown in Figures 7 and 8. Statistical significance is indicated by letters in each bar. In both trials, unformulated GS730 at both concentrations and formulated GS730 at 100 g ai / ha showed significant reductions in Botrytis disease compared to the untreated control and were not statistically different from the biological reference programs of Serenade and JMS Stylet Oil. In the second trial, unformulated GS730 at both concentrations and formulated GS730 at 100 g ai / ha showed results comparable to the chemical programs of Miravis Prime and Switch.

[0223] Example 4.3 Control of Botrytis infection in green beans with GS349, GS2280, GS2303, and GS2297 compositions RNAi compositions containing dsRNA, each containing a trigger sequence (GS349, GS2280, GS2303, or GS2297) described herein, were evaluated for their ability to control Botrytis in green beans in field trials. All test samples were formulated with RO water, propylene glycol, linear alcohol ethoxylate, lignosulfanate, sodium citrate dihydrate, polyacrylic acid polymer, two biocide compositions, anhydrous citric acid, and antifoam, all of which are readily available on the market. The formulated samples were tank mixed with Activator 90 0.125% v / v, a standard adjuvant and non-ionic surfactant. The compositions were tested at concentrations of 10 g ai / ha, 20 g ai / ha, and 40 g ai / ha. Positive controls were the chemical standard Switch 525g ai / ha and the biological standard Double Nickel LC 9.4l / ha, both tank mixed with Activator 90 0.125% v / v. An untreated control was used as the negative control, as above.

[0224] The compositions were applied to green bean seedlings using a plot sprayer delivering 50 gallons of water per acre. A total of three applications were made for each sample and each positive control. Symptoms were assessed from 8 days after the first application to 37 days after the third and final application. Percent disease severity was assessed and AUDPC was calculated in the same manner as described in Example 4.1.

[0225] The results are shown in Figure 9. Statistical significance was indicated by letters in each bar. All sequences at all tested concentrations significantly reduced Botrytis cinerea disease compared to the negative control and were not significantly different from the biological standard, Double Nickel. GS349 at 20g ai / ha and 40g ai / ha, GS2280 at 10g ai / ha and 40g ai / ha, GS2303 at all tested concentrations, and GS2297 at 20g ai / ha further showed no significant difference in symptoms compared to the chemical standard treatment.

[0226] Example 4.4 Control of Botrytis Infection in Strawberries with GS349 Composition RNAi compositions containing dsRNA, each containing a trigger sequence (GS349) described herein, were evaluated for their ability to control Botrytis in strawberry in an outdoor field trial. All test samples were formulated as described in Example 4.3 and tank mixed with standard adjuvant Activator 90 0.25% v / v. Compositions were tested at concentrations of 10 g ai / ha, 20 g ai / ha, 30 g ai / ha, and 40 g ai / ha. A biological standard program of Regalia 470 g ai / ha and Stylet Oil 6800 g ai / ha was used as a positive control. A chemical standard program of Captan 2240 g ai / ha and Switch 525 g ai / ha was used as a second positive control. An untreated control was used as a negative control.

[0227] The composition was applied to strawberry plants using a plot sprayer delivering 50 gallons of water per acre. Five applications were made at weekly intervals. Symptoms were assessed from 6 days after the first application to 12 days after the fifth and final application. Percent disease severity was assessed and AUDPC was calculated in the same manner as described in Example 4.1.

[0228] The results are shown in Figure 10. Statistical significance was indicated by letters in each bar. GS349 at all tested concentrations significantly reduced Botrytis cinerea disease and showed no significant difference from chemical or biological standard programs.

[0229] Table 1A. Universal ID numbers and SEQ identification of exemplary trigger sequences used in this study [Table 4]

[0230] Table 1B. In vitro liquid-based assay results Activity was confirmed by determining the percent biomass reduction after treatment compared to untreated controls. Two concentrations of dsRNA were co-incubated with Botrytis cinerea conidia for 3 days before measuring biomass using a Cytation5 machine. Average biomass reduction is the average of several biological runs, each with eight replicates of each treatment. [Table 5]

[0231] Table 2. Results of the detached leaf assay Activity was confirmed by comparing the average lesion size of each treatment at two different concentrations to the untreated inoculated control. The average percent reduction in lesion size was calculated by averaging two or more biological runs for each dsRNA sequence. [Table 6]

[0232] Tables 3 to 14. Greenhouse, whole plant assay results

[0233] Table 3 Mean percent segregation of treatments using Tukey-Kramer analysis for GS349 on the final evaluation date (Evaluation #3) and mean percent control compared to untreated inoculated treatments. Data from two runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 3 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 7]

[0234] Table 4 Mean percent segregation of treatments using Tukey-Kramer analysis for GS413 on the final evaluation date (Evaluation #3) and mean percent control compared to untreated inoculated treatments. Data from two runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 3 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 8]

[0235] Table 5 Mean percent segregation of treatments using Tukey-Kramer analysis for GS686 on the final evaluation date (Evaluation #3), and mean percent control compared to untreated inoculated treatments. Data from two runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 3 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 9]

[0236] Table 6 Mean percent segregation of treatments using Tukey-Kramer analysis for GS728 on the final evaluation date (Evaluation #3), and mean percent control compared to untreated inoculated treatments. Data from two runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 3 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 10]

[0237] Table 7 Mean percent segregation of treatments using Tukey-Kramer analysis for GS730 on the final evaluation date (Evaluation #3), and mean percent control compared to untreated inoculated treatments. Data from two runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 3 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 11]

[0238] Table 8 Mean percent segregation of treatments using Tukey-Kramer analysis for GS2280 on the final evaluation date (Evaluation #3), and mean percent control compared to untreated inoculated treatments. Data from three runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 4 weeks after emergence. p-values ​​for Tukey-Kramer analysis were less than 0.05. [Table 12]

[0239] Table 9 Mean percent segregation of treatments using Tukey-Kramer analysis for GS2297 on the final evaluation date (Evaluation #3) and mean percent control compared to untreated inoculated treatments. Data from three runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 4 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 13]

[0240] Table 10 Mean percent segregation of treatments using Tukey-Kramer analysis for GS2303 on the final evaluation date (Evaluation #3) and mean percent control compared to untreated inoculated treatments. Data from three runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 4 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 14]

[0241] Table 11 Mean percent segregation of treatments using Tukey-Kramer analysis for GS245 on the final evaluation date (Evaluation #3) and mean percent control compared to untreated inoculated treatments. Data from three runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 4 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 15]

[0242] Table 12 Mean percent segregation of treatments using Tukey-Kramer analysis against GS659 on the final evaluation date (Evaluation #3) and mean percent control compared to untreated inoculated treatments. Data from three runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 4 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 16]

[0243] Table 13 Mean percent segregation of treatments using Tukey-Kramer analysis for GS793 on the final evaluation date (Evaluation #3) and mean percent control compared to untreated inoculated treatments. Data from three runs were combined, with each run containing 10 replicates, each evaluating 'Lanai' tomato seedlings 4 weeks after emergence. The p-value for the Tukey-Kramer analysis was less than 0.05. [Table 17]

[0244] Table 14 Mean percent segregation of treatments using Tukey-Kramer analysis for GS2291 on the final ev...

Claims

1. A composition for controlling Botrytis cinerea, comprising: (a) a bactericidal amount of a polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 contiguous nucleotides that are essentially complementary to, or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, a DNA or target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or a fragment of RNA transcribed from said DNA or target gene; or (b) a bactericidal effective amount of at least one polynucleotide comprising at least one silencing element that is essentially complementary to, or has at least about 85%, at least about 90%, or at least about 95% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides of DNA or a target gene or RNA transcribed from said DNA or target gene, wherein said DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12; or (c) a bactericidal effective amount of at least one RNA comprising at least one fragment that is essentially complementary to, or has at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides of, a DNA or target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or a fragment of an RNA transcribed from said DNA or target gene; or (d) an RNA molecule that, when transfected into or contacted with said Botrytis cinerea, causes death, growth inhibition, reduced virulence or pathogenicity, or reduced proliferation / fertility of Botrytis cinerea, wherein said RNA molecule is essentially complementary to, or comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides that have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, a DNA or target gene or a fragment of RNA transcribed from said DNA or target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12; or (e) a double-stranded RNA molecule that, when transfected into or in contact with said Botrytis cinerea, causes death, growth inhibition, reduced virulence or pathogenicity, or reduced proliferation / fertility of Botrytis cinerea, wherein at least one strand of said double-stranded RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides that are essentially complementary to, or have at least 85%, 90%, 95%, 98%, or 100% sequence identity to, a DNA or target gene or a fragment of RNA transcribed from said DNA or target gene, and said DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12; or (f) a bactericidal amount of at least one double-stranded RNA comprising at least one strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to said nucleotide sequence; or (g) a bactericidal amount of a polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to said nucleotide sequence; or (h) a bactericidal effective amount of at least one RNA comprising at least one fragment that is essentially complementary to, or has at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60; or (i) an RNA molecule that, when transfected into or contacted with said Botrytis cinerea, causes death, reduced growth, reduced virulence or pathogenicity, or reduced reproductive / proliferative potential of Botrytis cinerea on the plant, wherein said RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides that are essentially complementary to, or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60; or (j) a double-stranded RNA molecule that causes death, growth inhibition, reduced virulence or pathogenicity, or reduced reproductive / proliferative potential of Botrytis cinerea on Vitis vinifera when transfected into or contacted with said Botrytis cinerea, wherein at least one strand of the fungicidal double-stranded RNA molecule is essentially complementary to, or comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 consecutive nucleotides having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60; or (k) a double-stranded RNA molecule that, when transfected into or contacted with said Botrytis cinerea, causes death, reduced growth, reduced virulence or pathogenicity, or reduced reproductive / proliferative ability of Botrytis cinerea on the plant, wherein at least one strand of the fungicidal double-stranded RNA molecule has at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60.

2. 10. The composition of claim 1, wherein the composition is in at least one form selected from the group consisting of a solid, a liquid, a powder, a suspension, an emulsion, a spray, an encapsulation, a microbead, a carrier particle, a film, a matrix, a seed treatment, a soil drench, and an implantable formulation.

3. 3. The composition of claim 1, further comprising at least one component selected from the group consisting of a carrier agent, a surfactant, an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide insecticide, a polynucleotide insecticide, a safener, and a pathogen growth regulator.

4. 3. The composition of claim 1, wherein the polynucleotide, the RNA, or the dsRNA comprises a double-stranded RNA molecule that, when transfected into or contacted with the Botrytis cinerea, causes death, growth inhibition, reduced virulence or pathogenicity, or reduced fertility (sporulation) of the Botrytis cinerea on the plant, and the double-stranded RNA molecule comprises at least one fragment that is essentially complementary to, or has at least 95% sequence identity with, at least 21 consecutive nucleotides of a DNA or target gene having a sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12, or an RNA transcribed from the DNA or target gene, and the length of the double-stranded RNA molecule is at least 100 base pairs in length, or is between about 100 and about 600 base pairs in length.

5. 3. The composition of claim 1, wherein the polynucleotide, or the RNA, or the dsRNA comprises a dsRNA comprising a first strand comprising a nucleotide sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48.

6. 6. The composition of claim 5, wherein the first strand comprises a nucleotide sequence having at least about 98% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48.

7. The composition of claim 5 , wherein the polynucleotide, or the RNA, or the dsRNA further comprises a second strand that is complementary to the first strand.

8. 2. The composition of claim 1, wherein the nucleotide sequence of (f), (g), (h), (i), (j) or (k) is selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48.

9. 2. The composition of claim 1, wherein the DNA or target gene listed in (a) to (e) has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 7, 9, 11, and 12.

10. A dsRNA that suppresses expression of a target gene in Botrytis cinerea, wherein a first strand of the dsRNA has a length of at least 100 nucleotides and comprises an RNA sequence that is 85% to 100% complementary to at least 100 consecutive nucleotides of an RNA encoded by a sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, and 24.

11. 11. The dsRNA of claim 10, wherein the second strand of the dsRNA is complementary to the first strand.

12. 11. The dsRNA of claim 10, wherein the first strand of the dsRNA comprises a nucleotide sequence that is 95% to 100% complementary to an RNA encoded by a sequence selected from the group consisting of 14, 19, 21, 23, and 24.

13. 13. The dsRNA of claim 12, wherein the dsRNA comprises a nucleotide sequence that is at least about 98% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48.

14. 14. The dsRNA of claim 13, wherein the dsRNA comprises the nucleotide sequence of SEQ ID NO:

26.

15. A dsRNA that suppresses expression of a target gene in Botrytis cinerea, wherein a first strand of the dsRNA comprises an RNA sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least 95% identical, at least about 98% identical, about 100% identical, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48.

16. 16. The dsRNA of claim 15, further comprising a second strand complementary to the first strand.

17. 16. The dsRNA of claim 15, wherein the first strand of the dsRNA comprises an RNA sequence that is at least about 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48.

18. 18. The dsRNA of claim 17, wherein the first strand of the dsRNA comprises an RNA sequence that is at least about 98% identical to SEQ ID NO:

26.

19. 19. A composition comprising the dsRNA of any one of claims 10 to 18, formulated for application in a form selected from the group consisting of a sprayable solution, an emulsion, a tank mix, and a powder.

20. 20. The composition of claim 19, further comprising one or more additional ingredients selected from the group consisting of a carrier agent, a surfactant, an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a polynucleotide insecticide, a non-polynucleotide insecticide, a polynucleotide fungicide, a non-polynucleotide fungicide, a polynucleotide insecticide, a non-polynucleotide insecticide, a safener, and a pathogen growth regulator.

21. 3. The composition of claim 1, wherein the at least 18 consecutive nucleotides listed in (a) to (e) and (g) to (j) is at least 400 consecutive nucleotides.

22. 19. A method for controlling Botrytis cinerea infection in a plant, comprising contacting the Botrytis cinerea with any of the compositions of claims 1, 2, 8 or 9, or with the dsRNA of any one of claims 10 to 18.

23. 19. A method for controlling Botrytis cinerea infection in plants, comprising topically applying to the plant any of the compositions of claims 1, 2, 8 or 9, or the dsRNA of any one of claims 10 to 18.

24. 24. The method of claim 23, wherein the topical application comprises spraying the composition onto the leaves, stems, flowers, or fruits of the plant.

25. 25. The method of claim 24, wherein the topical application comprises spraying the composition onto the leaves of the plant.

26. 24. The method of claim 23, wherein the plant is selected from the group consisting of a fruit-bearing plant, a vegetable, or an ornamental plant, optionally such plant is selected from the group consisting of a strawberry, a grape, a tomato, or a bean.

27. The method according to claim 23, wherein RNA interference is induced, resulting in death, growth inhibition, reduced virulence, reduced pathogenicity, or reduced growth / fertility (sporulation) of Botrytis cinerea.

28. 1. A method for controlling Botrytis cinerea infection in plants, comprising: (a) contacting the Botrytis cinerea with at least one polynucleotide comprising a nucleotide sequence that is essentially complementary to, or has at least 85%, 90%, or 95% sequence identity to, at least 18 consecutive nucleotides of a DNA or target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or an RNA transcribed from the DNA or target gene; or (b) topically applying to said plant a composition comprising at least one polynucleotide comprising a nucleotide sequence that is essentially complementary to, or has at least 85%, 90%, or 95% sequence identity to, at least 18 consecutive nucleotides of a DNA or target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12, or an RNA transcribed from said DNA or target gene; or (c) expressing in said plant at least one polynucleotide comprising at least one fragment that is essentially complementary to, or has at least 85%, 90%, or 95% sequence identity to, at least 18 consecutive nucleotides of a DNA having a sequence selected from the group consisting of SEQ ID NOs: 1-12; (d) contacting the Botrytis cinerea with a fungicidally effective amount of double-stranded RNA, at least one strand of which is essentially complementary to, or comprises a fragment having at least 85%, 90%, or 95% sequence identity to, at least 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48; or (e) topically applying to the plant a fungicidally effective amount of double-stranded RNA, at least one strand of which is essentially complementary to, or comprises a fragment having at least 85%, 90%, or 95% sequence identity to, at least 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21, 23, 24, 26, 31, 33, 35, 36, 38, 43, 45, 47, and 48.

29. 29. The method of claim 28, wherein the polynucleotide is double-stranded RNA.

30. 30. The method of claim 28 or 29, wherein the double-stranded RNA is chemically or enzymatically synthesized, or produced by expression in a microorganism or expression in a plant cell.

31. 30. The method of claim 28 or 29, wherein the double-stranded RNA comprises a strand having a nucleotide sequence comprising at least 21 consecutive nucleotides of SEQ ID NO:

26.

32. 30. The method of claim 28 or 29, wherein the double-stranded RNA comprises a strand comprising SEQ ID NO:

26.

33. 29. The method of claim 28, wherein the nucleotide sequence of claim 28(d) or (e) is SEQ ID NO:

26.

34. 29. The method of claim 28, wherein the method comprises topically applying to the plant a composition comprising at least one polynucleotide comprising a nucleotide sequence that is essentially complementary to, or has at least 85%, 90%, or 95% sequence identity to, at least 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO:

26.

35. 35. The method of claim 28 or 34, wherein the method comprises contacting the Botrytis cinerea with an effective amount of a solution comprising double-stranded RNA, wherein at least one strand of the double-stranded RNA is essentially complementary to at least 18, 19, 20 or 21 consecutive nucleotides of DNA having the nucleotide sequence of SEQ ID NO: 2 or a target gene, or has at least 85%, 90% or 95% sequence identity, and wherein RNA interference is induced, resulting in death, reduced growth, reduced virulence or pathogenicity, or reduced growth / fertility (sporulation) of Botrytis cinerea.

36. 36. The method of claim 35, wherein the solution further comprises one or more components selected from the group consisting of organosilicone surfactants, carrier agents, organosilicones, organosilicone surfactants, polynucleotide herbicide molecules, non-polynucleotide herbicide molecules, polynucleotide insecticides, non-polynucleotide insecticides, safeners, and pathogen growth regulators.

37. 29. The method of claim 28, wherein the plant is a grape, a tomato, a strawberry, or a bean.

38. 29. The method of claim 28, wherein the at least 18 contiguous nucleotides listed in (a) through (e) are at least 400 contiguous nucleotides.

39. 29. A plant, or a fruit, seed or propagable part of said plant, having enhanced resistance to Botrytis cinerea infection provided by the method of claim 28.

40. 40. The plant of claim 39, wherein the plant is selected from the group consisting of a fruit-bearing plant, a vegetable, or an ornamental plant, optionally such a plant is selected from the group consisting of grapes, strawberries, tomatoes, or beans.

41. 1. A composition comprising a fungicidally effective amount of a polynucleotide that, when transfected into or contacted with Botrytis cinerea on a plant, inhibits expression of a target gene in Botrytis cinerea, wherein the target gene is selected from the group consisting of Sec18 / Cdc48, CDC42, NBP35, SDH, Bcrrp1, XM_024691746.1, Bcsec15, Bcswd2, Bcmcm4, Bcgpi2, Bcrpb5, and Bcded1.

42. 42. The composition of claim 41, wherein the polynucleotide is a double-stranded RNA molecule comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, or 600 contiguous nucleotides that are essentially complementary to, or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-60.

43. 3. The composition of claim 2, wherein the composition is in the form of a liquid mixture selected from the group consisting of a soluble liquid concentrate, a suspension, a colloid, a micelle, and an emulsion.

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