Methods and compositions for treating and controlling plant diseases

Abstract

Provided are a method for controlling plant diseases caused by Xylella fastidiosa and a biological control composition for plant diseases. [Solution] A method for preventing or alleviating symptoms or disease caused by Xylella fastidiosa or Xanthomonas in a plant comprises contacting the plant with a virulent bacteriophage that contains X. fastidiosa and / or Xanthomonas in its host range. The plant disease biocontrol composition formulated for delivery to the plant comprises at least one carrier and at least one bacteriophage selected from the group consisting of Xfas100 phage type and Xfas300 phage type, wherein the bacteriophage is toxic to Xylella fastidiosa and Xanthomonas species.

Description

[Technical field]

[0001] The present invention relates to the field of plant pathology. More specifically, the present invention relates to methods and compositions for the isolation of bacteriophages, including the use of bacterial viruses, and for treating plant diseases caused by Xylella fastidiosa and Xanthomonas axonopodis.

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Application No. 61 / 716,245, filed October 19, 2012, and No. 61 / 785,535, filed March 14, 2013, which are incorporated by reference in their entireties.

[0003] [Statement regarding federally funded research] The U.S. Government has received an Animal and Plant Health Inspection Service (APHIS) Cooperative Agreement Award to AgriLife Research for the Texas Pierce Disease Research and Education Program, Agreement No. 11-8500-0955- Pursuant to Agreement No. 406039 between CA and AgriLife Research and Otsuka Pharmaceutical Co., Ltd., this You have certain rights in the invention.

[0004] [Reference to sequence listing] Measured by Microsoft Windows operating systems, 2013 The sequence listing contained in the 907 KB file entitled "TAMC019WO_ST25.txt" created on October 17th is filed electronically herewith and is incorporated by reference herein. [Background technology]

[0005] Bacteria can cause many diseases in plants, including Pierce's disease in grapevines and citrus canker in citrus plants. The bacteria can infect plant tissues and cause wilting, stunted growth, damaged fruit, and even death. Infections can easily spread to other plants through wind, rain, contaminated equipment, or infestation by insect vectors, resulting in detrimental effects on the plants and extensive crop damage. Effective treatment of these diseases requires plant treatment methods that exclude the bacteria. Summary of the Invention

[0006] In one aspect, the invention provides a method for propagating virulent bacteriophages (phages) that include X. fastidiosa in its host range, comprising infecting a culture of Xanthomonas bacteria with the bacteriophage, propagating the bacteriophage, and isolating bacteriophage particles from the culture. In another embodiment, the Xanthomonas bacteria comprises the species strain EC-12. In another embodiment, the bacteriophage infects the cell by binding to a cell surface feature. In another embodiment, the cell feature is a type IV pilus. In another embodiment, the bacteriophage comprises a tailed bacteriophage selected from the group consisting of podophages, siphophages, and myophages. In another embodiment, the bacteriophage is isolated from the environment, a wastewater treatment plant or sewage, a plant or its surface, or surrounding soil. In another embodiment of the invention, a surrogate host is used to enrich for virulent bacteriophages. In yet another embodiment, the bacteriophage is virulent in Xylella fastidiosa. In another embodiment, an agar overlay is used for growth of the bacteriophage.

[0007] In another aspect, the present invention provides a method for obtaining a candidate control agent for Pierce's disease, comprising contacting X. fastidiosa and Xanthomonas bacteria with a sample containing a population of virulent bacteriophages, and isolating at least a first bacteriophage from the population capable of lysing the X. fastidiosa and Xanthomonas bacteria. In one embodiment, the bacteriophage infects cells by binding to a cell surface feature. In another embodiment, the cell surface feature is a type IV pilus. In yet another embodiment, the cell surface feature is required for pathogenicity / virulence of the bacterial host. Other embodiments include contacting the sample with at least one lawn of X. fastidiosa and Xanthomonas, contacting the sample with X. fastidiosa and Xanthomonas simultaneously, and contacting the sample with X. fastidiosa and Xanthomonas sequentially. In other embodiments, the bacteriophage is isolated from the environment, a sewage treatment plant or sewage, a plant or its surface, or the surrounding soil. In another embodiment, the bacteriophage used is virulent in Xylella fastidiosa. The method may further comprise detecting lysed bacterial host cells or plaque formation after contacting the host bacterium with the virulent bacteriophage. In certain embodiments, the method comprises plating agar overlays or plates of bacterial host cells into which a sample of bacteriophage has been introduced.

[0008] In other embodiments, the bacteriophage are prepared by use of a soft agar overlay containing X. fastidiosa and Xanthomonas, and in further embodiments, a high titer phage plate lysate is prepared by harvesting one or more overlay plates containing X. fastidiosa or Xanthomonas strains exhibiting fusion lysis, e.g., EC-12, followed by maceration and clarification by centrifugation. After filter-sterilization, the resulting lysate can be stored, e.g., at 4°C. The high titer phage lysate is then purified, e.g., by isopycnic CsCl centrifugation and dialysis of the extracted phage solution. The resulting CsCl-purified bacteriophage typically contains approximately 1 x 1011 The titer is expressed in PFU / ml.

[0009] In some embodiments, the rate of bacteriophage in the plant tissue filtrate (PTF) is about 1 ml of PTF to 20 ml of surrogate host (an actively growing culture of the host of choice) for 4 days for X. fastidiosa strain Temecula or 4 hours for Xanthomonas strain EC-12.

[0010] Another aspect of the invention provides a method of preventing or reducing symptoms or diseases associated with X. fastidiosa in a plant, comprising contacting the plant with a bacteriophage that contains X. fastidiosa in its host range, the symptoms or diseases associated with X. fastidiosa including symptoms characteristic of Pierce's Disease (PD), in which leaves exhibit a yellow or red appearance along the leaf margins, eventually accompanied by necrosis of the leaf margins. In one embodiment, the bacteriophage particles may be introduced into the plant. In another embodiment, the plant is selected from the group consisting of grapevine, citrus, almond, coffee, alfalfa, oleander, oak, sweetgum, redbud, elm, peach, apricot, plum, blackberry, mulberry, and Chitalpa tashkentensis. In another embodiment, the bacteriophage is introduced into the plant by an insect vector by injection, or delivered through the root system by injection. In another embodiment, injection includes needle or needleless systems, pneumatic or pressurized injection systems. In another embodiment, the injection is done manually, or in one or more injections. In another embodiment, the insect vector is a glassy winged sharpshooter. In another embodiment, the bacteriophage introduced into the plant is 1P FU / ml (plaque forming units / ml) ~10 12 PFU / ml, 10 4 PFU / ml ~10 11 PFU / ml, and 10 7 PFU / ml ~1010 PFU / ml. In another embodiment, the bacteriophage particles are obtained by a method comprising infecting a culture of Xanthomonas bacteria with a bacteriophage, propagating the bacteriophage, and isolating the bacteriophage particles from the culture. In another embodiment, the method comprises contacting a population of plants with the bacteriophage particles to prevent or reduce symptoms associated with X. fastidiosa. In yet another embodiment, the bacteriophage comprises at least one strain of bacteriophage (phage) selected from the Xfas100 phage type or the Xfas300 phage type described below.

[0011] In another aspect, the invention provides a plant disease biocontrol composition formulated for delivery to a plant, said composition comprising at least one diluent, adjuvant or surfactant, and at least one bacteriophage from the Xfas100 phage type or the Xfas300 phage type described below. In one embodiment, said composition is further defined as being formulated for introduction to a plant via injection, spray, mist, or dusting. In another embodiment, said composition is further defined as being formulated for topical administration to a plant.

[0012] In another aspect, the present invention provides a method for obtaining a candidate biocontrol agent for citrus canker comprising the steps of: a) extracting a Xanthomonas axonopodis pv. citri bacterium from a virulent strain of the bacterium; b) extracting a virulent strain of the bacterium from a virulent strain of the bacterium; and c) extracting a virulent strain of the bacterium from a virulent strain of the bacterium. The present invention provides a method for obtaining a candidate biocontrol agent for citrus canker comprising contacting a sample with a population of bacteriophages and isolating at least a first bacteriophage from the population capable of lysing the Xanthomonas axonopodis bacteria. In one embodiment, the bacteriophage infects the cell by binding to a cell surface feature. In another embodiment, the cell surface feature is a type IV pilus. In yet another embodiment, the cell surface feature is required for pathogenicity / virulence of the bacterial host. Another embodiment comprises contacting the sample with a lawn of Xanthomonas. In another embodiment, the bacteriophage used is virulent in Xanthomonas axonopodis.

[0013] Another aspect of the invention provides a method for preventing or reducing symptoms or disease associated with Xanthomonas axonopodis in a plant, comprising contacting the plant with a bacteriophage that includes Xanthomonas axonopodis in its host range. In one embodiment, the bacteriophage particles may be introduced into the plant. In some embodiments, the plant is a citrus plant selected from the group consisting of a Citrus species, a Fortunella species, a Ponchirus species, lime, lemon, orange, grapefruit, pomelo, or a trifoliate orange hybrid used as a rootstock. In another embodiment, the bacteriophage is introduced into the plant by an insect vector by injection, or delivered through the root system by injection. In some embodiments, injection includes a needle or needle-free system, an air pressure or a pressure injection system. In another embodiment, the injection is performed manually or in one or more instalments. In another embodiment, the insect vector is a leafhopper. In another embodiment, the bacteriophage introduced into the plant is in a concentration between 1 PFU / ml (plaque forming units / ml) and 10 12 PFU / ml, 10 4 PFU / ml ~10 11 PFU / ml, and 10 7 PFU / ml ~1010 In another embodiment, the method comprises contacting a population of plants with the bacteriophage particles to prevent or reduce symptoms associated with Xanthomonas axonopodis and pathovars thereof in the population of plants. In yet another embodiment, the bacteriophage is selected from the following phage types: Xfas100 phage type or Xfas300 phage type: The present invention includes at least one strain of bacteriophage.

[0014] In another aspect, the present invention provides an isolated bacteriophage, Xfas303 bacteriophage, virulent to Xanthomonas axonopodis, a representative sample of which has been deposited under ATCC Accession No. PTA-13099. In yet another aspect, the present invention provides an isolated bacteriophage, Xfas303 bacteriophage, virulent to Xanthomonas axonopodis, a representative sample of which has been deposited under ATCC Accession No. PTA-13099. In yet another aspect, the present invention provides an isolated bacteriophage, Xfas303 bacteriophage, virulent to Xanthomonas axonopodis, a representative sample of which has been deposited under ATCC Accession No. PTA-13099. In yet another aspect, the present invention provides an isolated bacteriophage, Xfas303 bacteriophage, virulent to Xanthomonas axonopodis, a representative sample of which has been deposited under ATCC Accession No. PTA-13099. Isolated bacteriophages virulent to X. fastidiosa are provided, and representative samples of the bacteriophages Xfas103, Xfas106, Xfas302, Xfas303, Xfas304, and Xfas306 have been deposited under ATCC Accession Nos. PTA-13095, PTA-13096, PTA-13097, PTA-13098, PTA-13099, and PTA-13100.

[0015] In certain embodiments, the present invention provides a method for preventing or reducing symptoms or disease associated with or caused by X. fastidiosa or Xanthomonas axonopodis pathovar citri in a plant, comprising the step of contacting said plant with a virulent bacteriophage that contains X. fastidiosa and / or Xanthomonas axonopodis pathovar citri in its host range, further comprising contacting said plant with a virulent bacteriophage selected from the group consisting of Xfas100 phage type, and Xfas300 phage type. At least one bacteriophage is selected, wherein the Xfas100 type phage is characterized in that (a) the bacteriophage is capable of lysing the Xylella fastidiosa and / or Xanthomonas bacteria, (b) the bacteriophage infects cells by binding to type IV pili, (c) the phage belongs to a group of tailed bacteriophages exhibiting a long non-contractile tail with a capsid ranging from 55 mm to 77 mm in diameter, a morphology characteristic of the Siphoviridae family, and (d) the above. and (e) the bacteriophage has at least one property selected from the group consisting of: (a) the bacteriophage is capable of lysing the Xylella fastidiosa and / or Xanthomonas bacteria; (b) the bacteriophage binds to type IV pili to cause cell death. (c) the phage belongs to a group of tailed bacteriophages exhibiting a short non-contractile tail with a capsid in the range of 58 mm to 68 mm in diameter, a morphology typical of the Podoviridae family; (d) the genome size of the bacteriophage is about 43,300 bp to 44,600 bp; and (e) the bacteriophage has activity to prevent or reduce symptoms associated with Pierce's disease in a plant or plants.The present invention provides a method for preventing or reducing symptoms or disease associated with or caused by B. fastidiosa or Xanthomonas axonopodis pathovar citri. In certain embodiments, a single type of virulent bacteriophage is introduced into a plant, while in other embodiments, a combination of two, three, four, five, six or more virulent bacteriophage isolates or types are introduced into a plant, either simultaneously or sequentially. In certain embodiments, the bacteriophage comprises a genome having a DNA sequence selected from the group consisting of SEQ ID NO:11-SEQ ID NO:24, or a DNA sequence at least 90%, 95%, 98%, or 99% identical thereto. Thus, in certain embodiments, the bacteriophage introduced into the plant is Xfas101, Xfas102, Xfas103, Xfas104, Xfas105, Xfas106, Xfas107, Xfas110, Xfas301, Xfas302, Xfas303, Xfas. 304, Xfas305, and Xfas306. Also contemplated are plant disease biocontrol compositions comprising such Xfas100 and / or Xfas300 type bacteriophages formulated for delivery to plants. The biocontrol compositions may further comprise a carrier. In some embodiments, the carrier may comprise a diluent, surfactant, and / or buffer.

[0016] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows TEM images of phage Xfas302, phage Xfas303, phage Xfas304 and phage Xfas305 along with morphology and size characteristics of the Podoviridae family. [Diagram 2] FIG. 1 shows TEM images of phage Xfas101, phage Xfas102, phage Xfas103 and phage Xfas104 along with morphology and size characteristics of the Siphoviridae family. [Diagram 3] FIG. 1 shows X. fastidiosa Podoviridae and Siphoviridae bacteriophages isolated from wastewater and capable of forming plaques on XF15 and EC-12. [Figure 4] FIG. 1 shows the genomic maps of Siphoviridae Xfas103 and Xfas106. [Diagram 5] FIG. 1 shows the genomic maps of Siphoviridae Xfas302, Xfas303, Xfas304 and Xfas306. [Figure 6] FIG. 1 shows grapevine plants exhibiting Pierce's disease symptoms 8 weeks after inoculation with strain XF54, which were not subjected to challenge infection with bacteriophage. [Figure 7] FIG. 1 shows a summary of grapevine bacteriophage therapeutic and prophylactic challenge infection studies. [Figure 8] Figure 1. Mobility and persistence of individual bacteriophages in inoculated grapevines 8 weeks (top) and 12 weeks (bottom) after phage inoculation. Left panel: phages present in root tissue. Middle panel: phages present in grapevine hedge 1. Right panel: phages present in grapevine hedge 2. [Figure 9] Figure 1. Levels of XF15 in inoculated grapevines subjected to phage cocktail challenge after 3 weeks. Samples were taken 9 weeks after phage cocktail challenge (12 weeks after bacterial inoculation). Left panel: bacteria present in root tissue. Middle panel: bacteria present in grapevine fence 1. Right panel: bacteria present in grapevine fence 2. Grey bars show levels of XF15 in XF15 inoculated vines. Black bars show levels of XF15 in XF15 inoculated vines subjected to phage cocktail challenge 3 weeks after pathogen inoculation. Arrows indicate the node with the inoculation point. Each bar represents the average CFU / gpt (gram plant tissue) of roots and two fences for three vines. [Figure 10]Figure 1. Cocktail phage levels in grapevine vines initially inoculated with XF15 and challenged with the phage cocktail 3 weeks later. Samples were taken 5, 7 and 9 weeks after phage cocktail challenge (8, 10 and 12 weeks after initial bacterial inoculation). Left panel: phages present in root tissue. Middle panel: phages present in grapevine fence 1. Right panel: phages present in grapevine fence 2. Black bars show phage levels in cocktail inoculated plants. Grey bars show phage levels in vines inoculated with XF15 that were challenged with the phage cocktail 3 weeks after pathogen inoculation. Arrows indicate the nodes with the inoculation points. Each bar represents the average PFU / gpt (gram plant tissue) of the four phages in the cocktail determined from roots and two fences for three vines. [Figure 11] Figure 1. Phage levels in grapevine vines initially inoculated with phage cocktail and challenged with XF15 3 weeks later. Samples were taken 5, 7 and 9 weeks after XF15 challenge (8, 10 and 12 weeks after initial phage inoculation). Left panel: phage present in root tissue. Middle panel: phage present in grapevine fence 1. Right panel: phage present in grapevine fence 2. Black bars indicate phage levels in cocktail inoculated vines. Grey bars indicate phage levels in cocktail inoculated vines challenged with XF15 3 weeks after phage inoculation. Arrows indicate the node with the inoculation point. Each bar represents the average PFU / gpt (gram plant tissue) of the four phages in the cocktail determined from roots and two fences for three vines. [Figure 12] FIG. 1 shows the results of spot titration of phage Xfas303 against Xanthomonas axonopodis pathovar citri strain.

[0018] Brief Description of the Sequence Listing SEQ ID NO:1 X. fastidiosa specific oligonucleotide forward primer designed for X. fastidiosa gyrB. SEQ ID NO:2 X. fastidiosa specific oligonucleotide reverse primer designed for X. fastidiosa gyrB. SEQ ID NO:3 Bacteriophage Xfas304-specific oligonucleotide forward primer designed to the bacteriophage DNA primase gene. SEQ ID NO: 4. Bacteriophage Xfas304-specific oligonucleotide reverse primer designed to the bacteriophage DNA primase gene. SEQ ID NO:5. Bacteriophage Xfas303-specific oligonucleotide forward primer designed to the bacteriophage DNA primase gene. SEQ ID NO:6 Bacteriophage Xfas303-specific oligonucleotide reverse primer designed to the bacteriophage DNA primase gene. SEQ ID NO:7 Bacteriophage Xfas103-specific oligonucleotide forward primer designed to the bacteriophage DNA helicase gene. SEQ ID NO:8 Bacteriophage Xfas103-specific oligonucleotide reverse primer designed to the bacteriophage DNA helicase gene. SEQ ID NO:9 Bacteriophage Xfas106-specific oligonucleotide forward primer designed to the bacteriophage DNA helicase gene. SEQ ID NO:10 Bacteriophage Xfas106-specific oligonucleotide reverse primer designed to the bacteriophage DNA helicase gene. SEQ ID NO:11 Genome sequence of bacteriophage Xfas101. SEQ ID NO:12 Genomic sequence of bacteriophage Xfas102. SEQ ID NO: 13: Genome sequence of bacteriophage Xfas103. SEQ ID NO: 14: Genomic sequence of bacteriophage Xfas104. SEQ ID NO: 15. Genome sequence of bacteriophage Xfas105. SEQ ID NO: 16: Genomic sequence of bacteriophage Xfas106. SEQ ID NO: 17. Genomic sequence of bacteriophage Xfas107. SEQ ID NO:18 Genomic sequence of bacteriophage Xfas110. SEQ ID NO: 19. Genome sequence of bacteriophage Xfas301. SEQ ID NO: 20 Genomic sequence of bacteriophage Xfas302. SEQ ID NO: 21. Genome sequence of bacteriophage Xfas303. SEQ ID NO: 22 Genomic sequence of bacteriophage Xfas304. SEQ ID NO: 23: Genomic sequence of bacteriophage Xfas305. SEQ ID NO: 24: Genomic sequence of bacteriophage Xfas306. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0020] The present invention provides for the first time a method that allows for the efficient propagation and isolation of bacteriophages (phages) capable of infecting, replicating in, and lysing X. fastidiosa and / or Xanthomonas axonopodis (Xa) and pathovars thereof. The present invention also provides a method for controlling bacterial diseases in plants. Plant diseases that may be controlled by the present invention include, but are not limited to, Pierce's disease and citrus canker. Bacterial species useful according to the present invention include, but are not limited to, Xylella species, such as Xylella fastidiosa, or Xanthomonas species, such as Xanthomonas axonopodis and pathovars thereof, such as Xanthomonas axonopodis pathovars citri (Xac).

[0021] As used herein, "bacteriophage" or "phage" refers to a virus of bacteria. As used herein, "Xanthomonas axonopodis" or "Xa" refers to the Xanthomonas axonopodis bacterial species, or a pathotype thereof, which may include Xanthomonas axonopodis pathotype citri (Xac) or any other pathotype of Xanthomonas axonopodis. Currently, growth of bacteriophages capable of lysing X. fastidiosa is laborious in the laboratory using X. fastidiosa host cells and complex, expensive media in solid format. This may require 7-10 days to obtain low amounts of bacteriophage. Therefore, the present invention is a breakthrough that provides for the propagation of bacteriophages capable of infecting X. fastidiosa by growing the bacteriophages in a fast-growing host bacterium such as Xanthomonas sp. EC-12 to rapidly produce the bacteriophages, referred to as the "surrogate host" approach. The technique is rapid, cost-effective, and can be used with conventional media components available in the art. The technique is also amenable to scale-up. The ability to produce virulent phages that lyse (kill) X. fastidiosa and / or Xa in a surrogate host that can replicate every hour under standard conditions, rather than for days, using a host that replicates at most daily in highly complex media, makes it feasible for the first time to produce and practice methods of control and treatment of diseases mediated by X. fastidiosa and / or Xanthomonas axonopodis that involve the use of virulent phages. Cultivation of Xa can be carried out in nutrient medium with a generation time of approximately 2-3 hours. However, Xa is a permissive pathogen and therefore requires biosafety level 2 (BL2) for cultivation, and therefore, like X. fastidiosa, Xa may not be practical for large-scale production.

[0022] Bacteriophages can be isolated by a soft agar overlay method that allows for the isolation of phages from X. fastidiosa and / or Xanthomonas cells, and in a further embodiment, high titer phage plate lysates are prepared by harvesting one or more overlay plates (or plates) of X. fastidiosa or Xanthomonas strains exhibiting fusion lysis, e.g., strain EC-12, followed by maceration, clarification by centrifugation and filter sterilization. The resulting lysates can be stored at 4°C. The high titer phage lysates can then be purified, e.g., by isopycnic CsCl centrifugation, and the extracted phage solution can be dialyzed. As a result, approximately 1 x 10 11 CsCl purified bacteriophage with titers of PFU / ml can thus be obtained. In another embodiment, the bacteriophage in plant tissue filtrate (PTF) may be filtered. A preferred ratio of filtration is 1 ml of PTF to 20 ml of surrogate host culture (actively growing culture of the selected host), for example, grown for 4 days for X. fastidiosa strain Temecula or 4 hours for Xanthomonas strain EC-12.

[0023] X. fastidiosa and / or Xanthomonas axonopodis ("Xa") pathogens Using methods for detection and propagation of virulent bacteriophages against a type, virulent bacteriophages capable of causing lysis of X. fastidiosa and / or Xa can be selected from a desired source, such as plants, wastewater, and / or the environment, including soil water, and propagated according to the invention. Bacteriophages that can be identified according to the invention can be characterized by capsid shape and size, genome size, genetic arrangement and / or genetic modules. , morphology, and life cycle of Casjens et al. (Research in Microbiology, 159:340-348, In one embodiment, the present invention may be defined by the characteristic properties described by The bacteriophage may be virulent, isodense, have a triangulation number of T=7, have a genome size of about 60 kb or within about 15% of 60 kb, and may contain direct terminal repeats in the genome. The virulent bacteriophage may be used to control and prevent diseases caused by, for example, Xylella species and subspecies and / or Xanthomonas species, such as Xanthomonas axonopodis and pathovars thereof, such as Xanthomonas citri.

[0024] Currently, five subspecies of Xylella are recognized to cause plant diseases. Plant species that can be infected by Xylella are listed at www.cnr.berkeley.edu / xylella / control / hosts.htm, as described, for example, in Hernandez-Martinez et al., (American Phytopathological Society, 97(7):857-864, 2007) and Nunney et al., (PLoS ONE, 5(11):e15488, 2010), and Also, commercial crops such as grapevine, citrus, coffee, almond, peach, alfalfa, apricot, plum, blackberry, mulberry, and horticultural plants such as oleander, oak, sweetgum, redbud, elm, and Chitalpa tashkentensis can be included, but are not limited to these. In one embodiment of the present invention, bacteriophages can be isolated from environments where X. fastidiosa cannot grow due to its unique growth requirements. Furthermore, according to the present invention, plant species that can be infected by Xanthomonas axonopodis pathovars can include, but are not limited to, Citrus species, Fortunella species, Ponchirus species, lime, lemon, orange, grapefruit, pomelo, or trifoliate orange hybrids used for rootstocks.

[0025] Thus, the present invention provides methods for the development of bacteriophage-based treatments for the control of plant diseases caused by X. fastidiosa, a xylem-limited, insect-transmitted, Gram-negative bacterium that causes disease in many plants. Most notably, X. fastidiosa is the causative agent of Pierce's Disease (PD) of grapevine, which is currently a limiting factor in the cultivation of high-quality wine grapes in regions of the United States, including Texas and California. One important plant disease caused by X. fastidiosa is Pierce's Disease ("PD") of grapevine, which causes visible symptoms including yellowing or leaves with red margins. Eventually, drying and necrosis of the leaf margins and leaves may occur. Insect vectors, such as the leafhopper G. serrata ("GWSS"), can spread the disease, and phages can infect pathogenic bacteria and be useful for biocontrol efficiency.

[0026] Currently, there are no effective control measures for PD, except for active culling of infected vines. The present invention enables the treatment of such diseases by providing, for the first time, a viable system to generate sufficient bacteriophage quantities in a cost-effective manner to enable treatment of plants. The present invention also provides methods for the development of bacteriophage-based treatments for the control of plant diseases caused by Xa, including Xac, the causative agent of citrus canker. In certain embodiments, the present invention provides methods for controlling Xa disease in plants.

[0027] The term "virulent" as used herein refers to viruses, especially bacteriophages, that are capable of infecting, replicating within, and lysing (killing) a host cell. The term "temperate" refers to viruses that can integrate into the host genome (lysogenize) or lyse the host cell. "Host" refers to a bacteriophage that can infect a pathogen. In one embodiment of the present invention, the phage is propagated in a suitable host as described herein. The term "host" refers to a bacterial cell that can be used to produce large amounts of bacteriophage. One step in the development of the bacteriophage-based control strategies presented herein is the identification and propagation of virulent phages that recognize specific bacterial receptor sites. The production and delivery of virulent bacteriophages against pathogens must be economical to be a viable biocontrol option.

[0028] Phages infect host cells by recognition of receptors, including but not limited to surface proteins such as OmpA and OmpF, the core and O chain of bacterial LPS of gram-negative bacteria, sex pili and type IV pili (e.g., Roine et al., Mol. Plant Microbe Interact., 11:1048-1056 (1998)), and flagella. Without being limited to any given theory, However, it is believed that bacteriophages can infect X. fastidiosa and Xa cells via type IV pili. Thus, in one embodiment, a host according to the present disclosure may be any type of bacteria, particularly any bacterial species that a virulent temperate bacteriophage, or derivatives thereof, such as passaged phages, can adsorb to and infect via a surface receptor required for virulence and / or pathogenicity, such as type IV pili or TonB-like proteins. "Passaged phages" refers to a phage population that has been propagated for one or more growth periods on a cultured host cell. An exemplary host for use in the present invention may be a bacterial cell, particularly a bacterial species of the family Xanthomonasaceae, which includes both Xylella and Xanthomonas. In some embodiments, strains of X. fastidiosa that may be useful in the practice of the present invention include Temecula 1 (ATCC 700964); Ann-1 (ATCC 700598); Dixon (ATCC 700965); XF53, XF54, and XF95 (Whitehorn et al., Science, 336:351-352 (2012)); XF134, XF136, XF140, Examples of Xanthomonas strains that are susceptible to one or more of the disclosed bacteriophage isolates and may be useful in the present invention include, among others, EC12, Pres-4, and Jal-4 (provided by Dr. N. Wang, University of Florida, Gainesville, Fla.), Noth40, Ft. Basinger, and Block2. 2. Other Xanthomonas bacteria may also be utilized given their susceptibility to Xfas100 and / or Xfas300 bacteriophages.

[0029] The term "isolation" as used herein is defined as the separation and identification of an organism from a solution containing a mixed culture of organisms. Organisms that can be isolated can include viruses, bacteria, plant cells, etc. Bacteriophages can be isolated as described herein and as known in the art. In one embodiment, common laboratory methods for isolating bacteriophages can include, but are not limited to, growth in cultured cells, bacteriophage assays, double agar methods, and plaque assays, among others. The present invention provides a method for isolating bacteriophages by a method comprising overlaying at least a first sample containing different strains of bacterial host cells together to isolate bacteriophages that can infect and grow in both host cell types.

[0030] The present invention also provides a method for propagating a virus (bacteriophage) virulent to Xylella fastidiosa and / or Xa. Methods for propagating bacteriophage are known in the art and may include any method capable of producing a sufficient amount of bacteriophage to treat a plant disease. In one embodiment, propagating a bacteriophage virulent to X. fastidiosa and / or Xac may include growing a bacteriophage in a Xanthomonas bacterium, propagating the bacteriophage, and isolating bacteriophage particles from the culture.

[0031] Bacteriophages virulent to X. fastidiosa may be prepared using the soft agar overlay method. High titer phage plate lysates may be prepared by harvesting overlay plates of, for example, X. fastidiosa strain Temecula or Xanthomonas strain EC-12 exhibiting fusogenic lysis, followed by maceration and clarification by centrifugation. After filter sterilization, the resulting lysates may be stored at 4°C. The high titer phage plate lysates may then be purified by isopycnic CsCl centrifugation and the extracted phage solution dialyzed. For example, 1 x 10 11 CsCl-purified bacteriophage can be obtained with titers of PFU / ml.

[0032] A preferred ratio of bacteriophage in plant tissue filtrate (PTF) for filtration is, for example, 1 ml of PTF to 20 ml of surrogate host culture (actively growing culture of the selected host) grown for 4 days for X. fastidiosa strain Temecula or 4 hours for Xanthomonas strain EC-12.

[0033] The present invention also provides methods of treating or alleviating symptoms associated with X. fastidiosa and / or Xa pathotype in a plant or plants. Typical Pierce's Disease (PD) symptoms include slight yellowing of the leaves or reddening along the leaf margins, respectively, which may eventually result in drying or death of the leaf margins in that area.

[0034] One embodiment of the contemplated method includes administering a bacteriophage(s) virulent to X. fastidiosa and / or Xa to a plant infected with X. fastidiosa and / or Xa in a manner that results in treatment of the plant. Treatment of the plant against infection can be by spraying, misting, dusting, injection, or any other method known in the art. Methods of formulating compositions for such applications are also well known in the art. For example, X. fastidiosa infects the vascular tissue of plants, and therefore, as described herein, the invention may include introducing a purified population of bacteriophage particles virulent to X. fastidiosa into a plant infected with X. fastidiosa by injection, so that the bacteriophage can infect and lyse X. fastidiosa cells, thereby treating the plant infection. However, one skilled in the art will recognize that other methods can be used successfully as well. Xa is a foliar pathogen that naturally infects plant leaves, stems, and fruits by rain directing it to the leaf stomata, or by wounds caused during high winds or by insects, and thus in one embodiment the invention may involve introducing a composition comprising a purified population of bacteriophage particles virulent to Xa into an Xa-infected plant by spraying.

[0035] The terms "treatment", "treating" and "treating" as used herein are defined as acting against a disease, disorder, or condition with an agent to reduce or alleviate the physiological effects of the disease, disorder, or condition and / or its symptoms. As used herein, "treatment" encompasses any treatment of a disease in a host (e.g., plant species, including those for agricultural purposes such as food plants or those used to produce edible products, and ornamental plant species), including (a) reducing the risk of disease development in the plant, (b) preventing disease development, and (c) mitigating the disease, i.e., causing regression of the disease and / or alleviation of one or more disease symptoms. "Treatment" is also meant to encompass the delivery of an inhibitor that provides an effect even in the absence of the disease or condition. For example, "treatment" encompasses the delivery of an inhibitor of a disease or pathogen that provides an enhanced or desired effect in a plant (e.g., reduced pathogen load, reduced disease symptoms, etc.).

[0036] The present invention also provides a plant disease biocontrol composition formulated for delivery to a plant, the composition comprising at least one carrier and at least one bacteriophage toxic to Xylella fastidiosa and Xanthomonas species, such as Xa.

[0037] The active ingredient in the composition of the present invention may be a bacteriophage virulent for Xylella fastidiosa and / or Xanthomonas species, such as Xa, Xfas100 phage type, such as Xfas101, Xfas102, Xfas103, Xfas104, Xfas105, Xfas106, Xfas107, Xfas108, Xfas109, and Xfas110, and / or Xfas301, Xfas302, Xfas303, Xfas304, Xfas305, and Xfas306. and Xfas300 phage types such as Xfas103, Xfas106, Xfas302, Xfas303, Xfas304, and Xfas306, wherein the phage types Xfas103, Xfas106, Xfas302, Xfas303, Xfas304, and Xfas306 have been deposited under ATCC Accession Nos. PTA-13096, PTA-13095, PTA-13098, PTA-13099, PTA-13100, and PTA-13097, respectively.

[0038] The virulent bacteriophage of Xfas100 phage type as an active ingredient in the present invention exhibits at least one of the following properties: (a) the bacteriophage has an activity capable of lysing the Xylella fastidiosa and / or Xanthomonas bacteria; (b) the bacteriophage infects cells by binding to type IV pili; (c) the tailed bacteriophage exhibits a long non-contractile tail with a capsid having a diameter ranging from 55 mm to 77 mm, a morphology typical of the Siphoviridae family; (d) the genome size of the bacteriophage is approximately 55,500 bp to 56,200 bp; and (e) the bacteriophage has an activity to prevent or reduce symptoms associated with Pierce's disease in a plant or plants.

[0039] The virulent bacteriophage of Xfas300 phage type as an active ingredient in the present invention has at least one characteristic, which is that (a) the bacteriophage has an activity capable of lysing the Xylella fastidiosa and Xanthomonas bacteria, (b) the bacteriophage infects cells by binding to type IV pili, (c) the group of tailed bacteriophages exhibits a short non-contractile tail with a capsid ranging from 58 mm to 68 mm in diameter, a morphology typical of the Podoviridae family, (d) the genome size of the bacteriophage is approximately 43300 bp to 44600 bp, and (e) the bacteriophage has an activity to prevent or reduce symptoms associated with Pierce's disease in a plant or plants. The virulent bacteriophage as an active ingredient in the composition of the present invention further comprises at least one bacteriophage selected from the Xfas100 phage type and / or the Xfas300 phage type, wherein the Xfas phage types are Xfas103 and Xfas106, and / or the Xfas300 phage types are Xfas302, Xfas303, Xfas304 and Xfas306.

[0040] Also, the bacteriophages virulent against Xylella fastidiosa and Xanthomonas species, such as Xa, used as active ingredients in the compositions of the present invention may be provided by a combination of phages, such as a cocktail of two, three, four, five, six, or more virulent bacteriophage isolates or phage types, provided simultaneously or sequentially, including in combination with a carrier. The term "carrier" refers to a diluent, adjuvant, surfactant, excipient, or vehicle used to administer the phage. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, and sesame oil. Saline solutions, including phosphate solutions, such as sodium monohydrogen phosphate, potassium dihydrogen phosphate, and aqueous dextrose and glycerol solutions, may also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, Examples include dry skim milk powder, glycerol, propylene glycol, water, and ethanol.

[0041] The plant disease biocontrol compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0042] Protectants such as, but not limited to, casein-based formulations, flour-based formulations, sucrose, Congo Red, N-propyl-gallate, and lignin-based formulations can be added to the plant disease biocontrol composition.

[0043] The phage concentration required for efficient disease control is not limited, but may be, for example, 1×10 PFU / ml to 1×10 12 PFU / ml, 1×10 4 PFU / ml ~ 1 × 10 11 PFU / ml or 1 x 10 7 PFU / ml ~ 1×10 10 It can be expressed as PFU / ml.

[0044] Depending on the age of the tree, the trunk thickness, root size, and dosage are appropriately adjusted. The plant disease biocontrol composition may be a dry product, a substantially dry product, a liquid product, or a substantially liquid product. In some embodiments, the dry or substantially dry product can be reconstituted in a liquid (e.g., water, etc.) and then applied to the plant. In other embodiments, such compositions can be applied in dry or substantially dry form, and liquid already present on the plant, simultaneously applied to the plant, or subsequently generated on the plant (e.g., by application, concentration, etc.) facilitates exposure of the bacteriophage to the target bacteria. In another embodiment, such compositions can be applied to the plant by spraying, misting, or dusting.

[0045] The plant disease biocontrol composition can take the form of a solution, suspension, emulsion, powder, tablet, and the like.

[0046] The timing of application of the plant disease biocontrol composition is not limited, and may be, for example, daily, weekly, or twice a week, monthly, bimonthly, or quarterly.

[0047] The present invention also provides isolated bacteriophages virulent to Xylella fastidiosa, and Xanthomonas species, such as Xa, and pathovars thereof.

[0048] The present invention also provides a bacteriophage isolated as one of the bacteriophages selected from the group consisting of Xfas100 phage types, such as Xfas101 to Xfas110, and / or Xfas300 phage types, such as Xfas301 to Xfas306, wherein Xfas103, Xfas106, Xfas302, Xfas303, Xfas304, and Xfas306 have been deposited under ATCC Accession Nos. PTA-13096, PTA-13095, PTA-13098, PTA-13099, PTA-13100, and PTA-13097, respectively.

[0049] Such bacteriophages can be detected by determining their ability to form plaques on Xylella fastidiosa and / or Xanthomonas species.

[0050] Deposit Information Representative bacteriophage deposits of strains Xfas103, Xfas106, Xfas302, Xfas303, Xfas304 and Xfas306, as well as representative bacterial deposits of X. axonopodis EC-12 disclosed hereinabove and referenced in the claims, are available at Manassas, Virginia, USA 20108. The accessions were deposited with the American Type Culture Collection (ATCC), located at Box 1549, in the U.S. Pat. No. 6,339,411, and the accession numbers of the deposited strains are P The deposits are: PTA-13096, PTA-13095, PTA-13098, PTA-13099, PTA13100, PTA13097, and PTA-13101. All restrictions on such deposits will be removed upon allowance of a patent, and such deposits are intended to satisfy all requirements of 37 C.F.R. §§ 1.801 through 1.809. Such deposits will be maintained in the depository for a period of 30 years, or 5 years from the last claim, or for the life of the patent, whichever is longer, and will be replaced as necessary during such periods. EXAMPLES

[0051] The following examples are included to demonstrate preferred embodiments of the present invention. It will be understood by those skilled in the art that the techniques disclosed in the following examples are techniques that the inventors have found to work well in the practice of the present invention, and thus are believed to constitute preferred modes of its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present invention.

[0052] Example 1 Media, culture conditions and bacterial strains This example describes the isolation, growth, and morphological and genomic characterization of bacteriophages virulent against X. fastidiosa and Xanthomonas species. The medium used in this study differs from the standard medium used for growing X. fastidiosa, which allows rapid growth but affects the infectivity of the bacteriophage. PW broth medium modified by Sherald et al. (Plant Disease 67:849-852, 1983), named PW-M, was used for growing X. fastidiosa isolates, except that the final bovine serum albumin content was 0.3% as modified by Hill and Purcell (Phytopathology 85(12):1368-1372, 1995). For solid medium (PW-MA) and soft agar, PW-M broth was amended with 15 g / l and 7.5 g / l Plant Cell Culture Tested agar (Sigma), respectively. Complex medium TN broth (TNB) was used for routine maintenance. Solid medium (TNA) was identical except that it lacked KNO3 and was supplemented with 20 g / L agar. TN medium was amended with 7.5 g / L agar (TNSA) for soft agar overlay. TNA medium was amended with cycloheximide (40 μg / ml; TNAC) for plating plant extracts to obtain total bacterial counts. All cultures were grown at 28°C and liquid cultures were monitored at λ=600 nm using Nephelo flasks. California X. fastidiosa isolates included in the study included Temecula (XF15), representing X. fastidiosa subsp. fastidiosa; Ann1 (XF108), representing X. fastidiosa subsp. sandyi; and Dixon (XF109), representing X. fastidiosa subsp. multiplex. (XF102) (Hendson et al., Applied and Environmental Microbiology 67(2):895-903, 2001). Texas X. fastidiosa isolates include Platanus occidentalis (XF1), Helianthus annuus (XF2), and Helianthus annuus (XF3). Helianthus annuus (XF5), Iva annua (XF18), Ambrosia psilostachya (XF23), Ratibida columnifera (XF37), Vitis aestivalis (XF39), Vitis mustangensis (XF41) isolates from Ambrosia trifida var. texana (XF16, 40, and 43), two isolates from Nerium oleander (XF93 and 95), and Vitis vinifera vinifera)(XF48, 50, 52, 53, 54, 56, 58, 59, 60, 66, 6 All isolates were single colony purified by streak isolation and stored at -80°C after amending the PW-M broth culture to a final concentration of 20% glycerol (v / v). X. fastidiosa isolates were analyzed using polymerase chain reaction (PCR) analysis as previously described (Hernandez-Martinez et al., Plant Disease 90(11):1382-1388, 2006). Xanthomonas species were identified using the MIDI Sherlock™ Microbial Identification System analyzing fatty acid methyl esters by gas chromatography (GC-FAME).

[0053] Example 2 Processing of plant samples and isolation of bacteria Plant samples of Vitis vinifera, V. mustangensis, and grasses were obtained from vineyards in Brazos and Washington Counties, Texas. Rice (Oryza sativa) plant tissues and grasses were obtained from rice fields in Jefferson and Wharton Counties, Texas. Rice seed samples were obtained from the Texas AgriLife Research Center in Beaumont, Texas. Samples from rose plants (Rosa spp.; knockout) and jalapeno (TAM-mild; Capsicum annuum) were obtained from Brazos County, Texas. )) was obtained. To obtain the plant extract, 10 g of plant tissue was ground in 50 ml of bacteriophage buffer (P-buffer; 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 8 mM MgSO4) using a mortar and pestle, vortexed, and strained through a double layer of cheesecloth to remove large particles. The extract was then dilution plated onto both PW-M and TNAC for isolation of X. fastidiosa and non-X. fastidiosa bacteria, respectively, and incubated at 28°C. Plates were evaluated daily for growth until day 10.

[0054] Example 3 Isolation, purification and titration of bacteriophages from plant samples To obtain plant tissue filtrates (PTFs), the clarified plant extracts were centrifuged twice (10,000×g, 10 min at 4° C.) and the tissue extracts were filtered through 0.22 μm filters (Supor, Pall Life Sciences). The presence of bacteriophages in the PTFs was determined directly by spotting 10 μl of a 10-fold dilution series onto an overlay of a series of X. fastidiosa and Xanthomonas species hosts and observing for the formation of zones or plaques after lawn emergence (6-7 days for X. fastidiosa isolates and 18 hours for Xanthomonas species isolates). Alternatively, bacteriophages were detected by incubating 1 ml of each filtrate with 20 ml of actively growing cultures of X. fastidiosa isolates (4-day cultures; A 600 = 0.30) or A of Xanthomonas spp. 600 Cultures were enriched from PTF by adding 0.01% β-lactam erythrocytes (PW-MA) to the cultures at 0.01% β-lactam ... 600 A host suspension was made in TN broth (A = 0.5), while an 18-h culture of Xanthomonas species isolate grown on medium TNA was used to prepare a host suspension in TN broth (A 600=0.5) were made and used to prepare the overlay. Soft agar overlays used to examine bacterial supernatants for bacteriophage activity were made by mixing 100 μl of the bacterial suspension with 7 ml of molten PW-M or TN soft agar and pouring the mixture onto PW-MA or TNA plates and allowing it to solidify and dry. Spotted overlays that showed either plaques or clear zones formation were further examined by plating as above, except that the PTF dilutions were mixed directly with the individual host indicator suspensions before overlaying. Individual plaques formed on either X. fastidiosa or Xanthomonas species host overlays were excised, suspended in P-buffer and titered. The procedure was repeated three times to obtain single plaque isolates. 10 PFU / ml were prepared by harvesting the overlay of plates exhibiting fusion lysates with 5 ml of P-buffer, softening the soft agar overlay, clarifying the lysates by centrifugation (10,000 x g, 15 min at 4°C), and filter-sterilizing through a 0.22 μm filter. Lysates were stored at 4°C.

[0055] Plant extracts were plated on both PW-M and TNAC for selection of X. fastidiosa and to obtain total bacterial counts, respectively. Plant extracts from all plants assayed did not yield any traces of X. fastidiosa isolates. However, non-selective plating yielded a wide variety of bacterial colony types. Representative single colonies of yellow-stained bacteria were selected from the plates and streak-purified to obtain stocks. The stocks were used to generate overlays on which PTFs were spotted to observe zone or plaque formation. Bacterial isolates Presidio-4, Jal-4, and EC-12, obtained from extracts of rice seeds, jalapeno leaves, and rice tissue, respectively, were all identified as Xanthomonas species using gas chromatography-based fatty acid methyl ester analysis (GC-FAME) and used as hosts for evaluation of PTFs. Other Xa strains may also be utilized, taking into account the virulence of one or more bacteriophages disclosed for such strains.

[0056] After 5-6 days of incubation, several dilutions of the PTFs produced plaques on isolates XF15, XF53, XF54, and XF95, indicating that bacteriophages capable of forming plaques on these hosts were present in the plant tissues. Initial titers observed from extracts were 5 × 10 1 PFU / gram of tissue ~7×10 5 The titers ranged from 100 PFU / gram of tissue. Since all PTFs showed the same production pattern for XF15, XF53, XF54 and XF95, strain XF15 was used as the host for plaque purification and production. The high titers found in this unenriched PTF indicate that the natural bacterial host associated with plant tissue may serve as a host for bacteriophages capable of producing plaques on an overlay of X. fastidiosa. Successive plating of the PTFs yielded individual plaques that were uniform in size. Individual plaques were excised and plaque purified three times using XF15 as the host to obtain clonal isolates. Figure 1 shows that bacteriophage Xfas302 grew on X. fastidiosa strain XF15 (Jones et al., 2007) or X. fastidiosa strain Temecula (ATCC700964) with a titer of 100 PFU / gram of tissue. 9 PFU (plaque forming units / ml) and Xanthomonas species isolates Pres-4 and EC-12 (both 5 × 10 7 Culture plates of bacteriophage Xfas302 titrated for CFU (colony forming units) / ml were able to form plaques on Xanthomonas species strains Pres-4 and EC12 (EC12 deposited under ATCC Accession No. PTA-13101), providing evidence that bacteriophage grown on X. fastidiosa can adsorb, replicate, and plaque on Xanthomonas strains. Host range studies presented below further substantiate these results.

[0057] Phages Xfas101 to Xfas105 all produced small clear plaques on XF15 lawns, whereas phages Xfas301 to Xfas305 produced large clear plaques on the same host. TEM images of Xfas302 to Xfas305 and Xfas101 to Xfas104 are shown in Figures 1 and 2, respectively. High titer lysates produced using XF15 were used to obtain CsCl purified preparations of each bacteriophage and were used to perform transmission electron microscopy (TEM) studies. Phages Xfas101 to Xfas105 all exhibited long noncontractile tails with capsids ranging from 55 nm to 64 nm in diameter and were therefore determined to belong to the Siphoviridae family. Xfas301 to Xfas305 all exhibited a short, noncontractile tail with capsids ranging from 58 nm to 65 nm in diameter, a morphology typical of the Podoviridae family.

[0058] Example 4 Enrichment of bacteriophages from wastewater. Clarified wastewater samples were obtained from a treatment facility in the Bryan, Texas area. Samples were obtained from the Still Creek, Carter Creek, Turkey Creek, and Barton Creek facilities. Samples were centrifuged twice (10,000×g, 10 min at 4° C.) and tissue extracts were filtered through a 0.22 μm filter. Phages were enriched by adding 1 ml of each filtrate to 20 ml of actively growing cultures of the selected host described above. After 72 and 24 hours of growth for enrichment of X. fastidiosa or Xanthomonas species, respectively, the cultures were centrifuged and filter sterilized. The enriched filtrates were spotted onto an overlay and titered as described above.

[0059] Phages Xfas106-109 and Xfas306 were isolated individually from enrichment samples obtained from four wastewater treatment plants using host EC-12 as a host. TEM studies of purified bacteriophage concentrates morphologically identified bacteriophage Xfas306 as a Podoviridae by a short non-contractile tail with a capsid of 68 nm diameter (Figure 3), whereas isolated phage Xfas106-109 exhibited a long non-contractile tail with a capsid of approximately 77 nm diameter, characteristic of Siphoviridae (Figure 3). Bacteriophage Xfas306 produced large clear plaques on both hosts EC-12 and XF15, whereas phage Xfas106-109 produced small clear plaques on the same host (Figure 3). Thus, the method used in this study allowed for the isolation of X. fastidiosa bacteriophages from environmental samples.

[0060] Example 5 CsCl purification The filter-sterilized bacteriophage suspension was concentrated by centrifugation (90,000×g, 2.5 h at 5° C.) using a 60Ti rotor in a Beckman L8-70M ultracentrifuge. The pellet was resuspended in P-buffer and treated with DNase I and RNase A (Sigma) at a final concentration of 1 μg / ml for 2 h at 25° C. A final concentration of 0. CsCl was added to the bacteriophage suspension at 75 g / ml and centrifuged in a VTi65.2 rotor (300,000 × g, 18 h at 5 °C). Visible bacteriophage bands were extracted using an 18-gauge syringe needle and dialyzed overnight at 4 °C against P-buffer amended to 1 M NaCl and twice for 4 h at 25 °C against P-buffer, and eluted at 1 × 10 11 A suspension of PFU / ml was obtained. The CsCl purified bacteriophage was stored at 4°C.

[0061] Example 6 Transmission electron microscopy Prepare CsCl-purified bacteriophage (1 x 10) by diluting in P-buffer and applying 5 µl for 1 min onto freshly glow-discharged formvar carbon-coated grids. 11 Electron microscopy was performed on 100 cells / ml of 1000 PFU. The grids were then washed briefly on a drop of deionized water and stained with 2% (w / v) aqueous uranyl acetate. Specimens were observed on a JEOL 1200EX transmission electron microscope operating at an accelerating voltage of 100 KV.

[0062] Example 7 Plate efficiency and host range Plating efficiency (EOP) was obtained by calculating the ratio of bacteriophage plaque titers obtained using a heterologous (non-growing) host to the bacteriophage plaque titers obtained for the homologous (growing) host. 100 μl of bacteriophage stock dilutions and individual host indicator suspensions (A) in soft agar (7 ml) were plated prior to overlaying on solid medium. 600 = Bacteriophage stocks were titered against either X. fastidiosa or Xanthomonas species hosts using appropriate media by mixing with 0.05% PBS and 0.05% PBS.

[0063] Studies comparing the EOP of Xfas phages are shown in Table 1. The EOP for phages isolated from plant samples grown using Xanthomonas sp. strain EC-12 and then titrated using X. fastidiosa strain XF15 as the host was 1 × 10 -1 ~1×10 -3 Similar results were seen when bacteriophage grown using strain XF15 were subsequently titrated using EC-12 as the host. Similar studies with phage isolated from wastewater filtrates and grown on strain EC-12 yielded titers in the range of 1 × 10 -1 ~5×10 -1 Phages Xfas106-109 were propagated on strain XF15 and plated on host EC-12, resulting in an EOP ranging from 1 × 10 -1 ~3×10 -1EOPs were obtained, indicating that while DNA restriction and modification barriers may be present, phages propagated in the fast-growing strain EC-12 in 1 day are able to adsorb, replicate, and form plaques on X. fastidiosa, a process that may take up to 10 days in X. fastidiosa alone.

[0064] [Table 1]

[0065] Host lawns were generated by overlaying plates of the appropriate medium, PW-M (for XF15) or TNA (for EC-12), with homogenous soft agar seeds containing the individual hosts. High titer lysates (1 × 10 9 Spot titrations were performed on individual lawns by spotting 10 μl of a 10-fold dilution series onto an overlay of host X. fastidiosa or Xanthomonas species. After incubating the plates at 28°C for the appropriate time (24 hours for EC-12 or 5-7 days for XF15), the plates were assessed for zone and plaque formation.

[0066] Initial host range studies, shown in Table 2, demonstrated that all phages capable of forming plaques on the X. fastidiosa host XF15 also formed plaques on the host EC-12. On the other hand, hosts Jal-4 and Pres4 were not susceptible to most siphophages. The reasons for resistance could be lack of adsorption or inhibition of bacteriophage genome uptake, superinfection immunity, restriction-modification, and other mechanisms such as clustered regularly interspaced short palindromic repeats (CRISPR). This extends to post-adsorption mechanisms.

[0067] [Table 2]

[0068] Example 8 Preliminary identification of Xfas adsorption sites Based on the observation that X. fastidiosa phages obtained or enriched from either plant tissues or wastewater samples formed plaques on X. fastidiosa, we aimed to determine whether cell surface components could serve as adsorption sites. Known adsorption sites for phages include surface proteins such as OmpA and OmpF, the core and O-chain of bacterial LPS of Gram-negative bacteria, sex and type IV pili, and flagella. Wild-type and derived mutants with a deletion of pilA resulting in a derivative lacking type IV pili were evaluated as hosts for Xfas phages. All bacteriophages tested formed plaques on the XF15 wild-type strain but not on the XF15ΔpilA mutant. The results indicate that type IV pili may be the primary site of attachment for Xfas phages. Suggested.

[0069] Based on the results obtained with XF15-ΔpilA, we aimed to determine whether a pilA deletion variant of Xanthomonas sp. strain EC-12 could be rendered insensitive to the Xfas phages Xfas103, Xfas106, Xfas302, Xfas303, Xfas304 and Xfas306. Strain EC-12ΔpilA was insensitive to the phages in plate titer assays and no adsorption to the host was observed in adsorption experiments with phage Xfas303. EC-12ΔpilA complemented in trans to pilA was sensitive to all phages tested. This further demonstrated that type IV pili are the primary attachment site for phages as observed for X. fastidiosa.

[0070] Example 9 Bacteriophage DNA isolation and genome sequencing Modification of Promega Wizard DNA clean-up kit (Promega) Using filter-sterilized high titer (1 x 10 9 Bacteriophage genomic DNA was prepared from 10-20 ml of CsCl-purified bacteriophage lysate (> PFU / ml). Briefly, 10-20 ml of bacteriophage lysate was digested with 10 μg / ml each of DNase I and RNase A (Sigma) at 37°C for 30 min and diluted with 10% (w / v) polyacrylamide gel electrophoresis. The bacteriophage was precipitated in the presence of polyethylene glycol 8000 and 1M NaCl for 16-20 h at 4 °C. The precipitate was centrifuged at 10000 × g for 10 min at 4 °C and the pellet was resuspended in 0.5 ml of P-buffer. 1 ml of DNA purification resin provided by the Wizard kit was added to the resuspended bacteriophage, packed into a mini-column and washed with 2 ml of 80% (v / v) isopropanol. DNA was eluted from the resin by adding 100 μl of water preheated to 80 °C followed immediately by centrifugation of the mini-column. DNA integrity was confirmed by running on a 0.8% agarose gel and staining with ethidium bromide, and DNA was quantified by band densitometry. Bacteriophage genome sizes were estimated by pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA on a 1% agarose gel (Pulsed-Field agarose, BioRad) and comparison with a size marker (MidRange Marker I, NEB).

[0071] "454" pyrosequencing (Roche / 454 Life Sciences, Branford, CT, USA; Emory GRA Genomics Core, Emory University, Atlanta, GA, USA) Phages were sequenced using the FLX Titanium Sequencer (454 Life Sciences). Bacteriophage genomic DNA was prepared from bacteriophage isolates as described above and mixed in equimolar amounts to a final concentration of approximately 100 ng / μl. The pooled DNA was sheared and ligated with multiplex identifier (MID) tags specific to each of the four pools and sequenced by pyrosequencing using a full plate reaction on a Roche FLX Titanium sequencer according to the manufacturer's protocol. The pooled bacteriophage DNA was present in two sequencing reactions. The reactions contained genomic DNA for a total of approximately 3331 kb of genomic sequence representing 39 genomic elements, and the sequencing run generated 987815 reads with an average length of 405 bp, providing a total coverage of 120-fold for the entire pool. The shaped FLX Titanium flowgram output corresponding to each of the four pools was analyzed using Newbler assembler version 2.5.3 (454 Life Sciences). Contigs were individually integrated using a PCR primer set that was adjusted to include only reads containing a single MID identifier per integration. Identification of individual contigs was determined by PCR using primers generated against the contig sequences and individual bacteriophage genomic DNA preparations as templates, and individual phages were matched to their contigs using generation of products of predicted size derived from the bacteriophage DNA template. A sequencer (Gene Codes Corporation) was used for sequence integration and editing. Protein coding regions were predicted using Genemark (opal.biology.gatech.edu / GeneMark / gmhmm2_prok.cgi) and hand-edited in Artemis (www.sanger.ac.uk / Software / Artemis / ) (Lukashin et al., Nucleic Acids Research 26(4):1107-1115, 1998; Rutherford et al., Bioinformatics 16(10):944-945, 2000). Dot plots were generated using DOTTER (Brodie et al., Bioinformatics 20(2): 279-281, 2004). Predicted proteins were compared to proteins in the GenBank database using BLAST (www.ncbi.nlm.nih.gov / blast / Blast.cgi). Conserved domains, lipoprotein processing signals, and transmembrane domains (TMDs) were identified using InterProScan (www.ebi.ac.uk / Tools / webservices / services / interproscan), LipoP (www.cbs.dtu.dk / services / LipoP / ), and TMHMM (www.cbs.dtu.dk / services / TMHMM / ), respectively.

[0072] [Table 3]

[0073] Example 10 Genomic analysis of Xfas phages and description of the Xfas100 and Xfas300 phage types The phages isolated for their ability to attack Xanthomonas EC-12 and X. fastidiosa and subsp., all require type IV pili for infection and all are virulent, since no lysogenic colonies could be isolated from the infections and no genes associated with a temperate lifestyle (repressor, integrase) were found in the genome sequences. The phages are classified as type IV pili, as defined by Casjens et al. (Research in Microbiology, 159:340-348, 2008) They can be further divided into two phage types.

[0074] (1) Xfas100 phage type: The Xfas100 phage type is composed of virulent siphophages of Xanthomonas and Xylella (ICTV Siphoviridae), the prototypes of which are phage Xfas101, phage Xfas102, phage Xfas103, phage Xfas104, phage Xfas105, phage Xfas106, phage Xfas107, phage Xfas108, phage Xfas109, and phage Xfas110 (Table 12), further examples of which are listed in Table 3 as any phage designated "Xfas1nn", where n is any number (referred to as the Xfas100 series). This flexible naming system is necessary because it is expected that further varieties of the Xfas1nn phage type will be isolated. Xfas100 type phages are siphophages, virulent in their mode of life, and require type IV pili for infection of Xylella and Xanthomonas species. Xfas100 type phages have an icosahedral capsid head measuring approximately 55 nm to 77 nm in diameter, and a flexible tail measuring approximately 200 nm to 262 nm in length, both dimensions determined by standard precision of negative staining electron microscopy (see Figures 2 and 3). Xfas100 series viral DNA has cohesive (cos) ends characterized by single-stranded DNA overhangs (Casjens, et al., Methods Mol Biol 502:91-111, 2009), which is important for phages used in antibacterial applications, as cos DNA packaging avoids the generation of generalized transducing particles that may enhance the delivery of virulence determinants. The Xfas100 genome consists of two distinct gene clusters, A and B. L and A R and B L and B. R The Xfas100 phage type has a characteristic overall organization (see FIG. 4) with the genes aligned in the right-handed gene cluster B. The Xfas100 phage type has the essential structural and lysis genes of the phage aligned in the right-handed gene cluster B. LThe Xfas100 series phage types are also distinguished by encoding their own unimolecular DNA polymerases (Xfas103gp71 and Xfas106gp66), primases (Xfas103gp76 and Xfas106gp71) and helicases (Xfas103gp69 and Xfas106gp64).

[0075] (2) Xfas300 phage type: The Xfas300 phage type is composed of virulent podophages of Xanthomonas and Xylella (ICTV Podoviridae), the prototypes of which are phage Xfas301, phage Xfas302, phage Xfas303, phage Xfas304, phage Xfas305 and phage Xfas306, further examples of which are listed in Table 3 and refer to any phage designated as "Xfas3nn", where n is any number (referred to as the Xfas300 series). This flexible naming system is necessary since it is expected that further varieties of the Xfas300 phage type will be isolated. Xfas300 type phages have an icosahedral capsid head with dimensions of approximately 58 nm to 68 nm in diameter, as determined by standard precision in negative staining electron microscopy (see Figures 1 and 3). The Xfas302-306 genome encodes a single subunit RNA polymerase flanked by structural protein domains. The Xfas300 series genomes have a characteristic overall organization (see FIG. 5) with the genes arranged on a single strand, including the phage replication, structural and lysis genes. The Xfas300 phage types are also distinguished by encoding their own single-molecule DNA polymerases (Xfas302gp18, Xfas303gp17, Xfas304gp17, and Xfas306gp17), single-subunit RNA polymerases (Xfas302gp31, Xfas303gp28, Xfas304gp27, and Xfas306gp30, respectively) and helicases (Xfas302gp15, Xfas303gp14, Xfas304gp15, and Xfas306gp14) (see FIG. 5 for a schematic of the phage genome).

[0076] Example 11 Transfer, challenge and protection studies in grapevines using bacteriophage Xfas304 Bacteriophage Xfas304 is a member of the Podoviridae family and was isolated from an environmental sample with a host range that includes both X. fastidiosa and Xanthomonas species. In the studies presented here, the movement and persistence of Xfas304 in grapevine vines in the absence of susceptible hosts was determined to determine whether treatment of plants with the bacteriophage could prevent subsequent infection by X. fastidiosa. Furthermore, to determine whether the bacteriophage could therapeutically control the development of Pierce's disease, grapevines initially inoculated with X. fastidiosa were subsequently challenged with bacteriophage Xfas304 4 weeks after pathogen inoculation.

[0077] For the prevention study, grapevines were inoculated with 40 μl of a bacteriophage Xfas304 suspension (1 × 10 10 The bacteria were inoculated with 1000 PFU / ml of X. fastidiosa and then challenged with X. fastidiosa 4 weeks after bacteriophage inoculation. The X. fastidiosa suspension used for inoculation was spectrophotometrically adjusted (A 600 =0.4;1×10 9 CFU / ml. Using the needle inoculation technique described by Hopkins (Plant Dis. 89:1348-1352, 2005), individual fences were inoculated between the second and third nodes on opposite sides (two points per fence) with 40 μl of the bacterial suspension. Control vines were mock inoculated with phosphate buffer at the same inoculation points as above.

[0078] The results showed that bacteriophage Xfas304 can be used to treat and prevent Pierce's disease caused by X. fastidiosa subsp. fastidiosa in grapevines. Thus, bacteriophage Xfas304 and other virulent Xylella-Xanthomonas phages identified from these studies have potential use in plant protection and treatment against diseases caused by other X. fastidiosa subsp. and Xanthomonas species.

[0079] Bacteria used in this study included X. fastidiosa strains Temecula (XF15) and XF54, which are associated with Pierce's disease of grapevine in California and Texas, respectively. Cultures of X. fastidiosa were maintained on PW-M agar medium (Summer et al., J Bacteriol 192(1): 179-190, 2010) at 28°C for 5-7 days. Five-day-old cultures of X. fastidiosa isolates grown on PW-MA were used to make bacterial suspensions in phosphate buffer (0.125 M, pH 7.1) for vine inoculation.

[0080] Dormant V. vinifera cv. Cabernet Sauvignon clone 08 on 1103P rootstock was purchased from Vintage Nurseries (Wasco, CA, USA). Plants were planted in 7-gallon pots using 101 Sunshine Mix 1 (Sun Gro Horticulture, Vancouver, BC, Canada) under 16 h light (26 °C, 300 μE / m) with supplemental lighting from sodium lamps. 2 s~400μE / m 2Plants were grown in a greenhouse with a (1·s) / 8 h dark (18°C) cycle. Plants were watered with tap water every other day. Vines were fertilized every 15 days using Peter's General Purpose 20-20-20 fertilizer and micronutrients. Plants were pruned aggressively to provide uniform plants as follows: upon producing two unbranched single lateral branches measuring 100cm-150cm, two lateral branches were pruned back to 80cm. Lateral branches and buds were removed. The two fences were supported by stakes and allowed to grow until each fence was approximately 2.5m-7.5m in length before using the vines in the above experiments.

[0081] Standard qRT-PCR line plots were obtained for X. fastidiosa strains XF15 and XF54, as well as for bacteriophage Xfas304, all with R values ​​greater than 0.9. 2 values, with efficiencies of 157%, 130% and 123%, respectively. Quantitative assessment of duplicate hedges from triplicate samples of XF15 and XF54, respectively, showed distribution of the pathogen throughout all nodes assayed, with typical visible Pierce's Disease (PD) symptoms such as slight yellowing of the leaves or reddening along the leaf margins, and eventually drying of the leaf margins or zone death by 8 weeks post-inoculation (Figure 6). In vines inoculated with bacteriophage Xfas304, in the absence of a permissive host, a progression of bacteriophage distribution was observed at 2, 4, and 6 weeks post-inoculation, which declined between weeks 8 and 12 with no vine symptoms observed.

[0082] Example 12 Inoculation of grapevines with bacteria and bacteriophages. For therapeutic evaluation of bacteriophage treatment, 15 vines (2 hedges each) were inoculated with X fastidiosa strains XF15 or XF54. The bacterial suspension used for inoculation was adjusted spectrophotometrically (A 600 =0.4;1×10 9CFU / ml. CFU / gram plant tissue (gpt) and PFU / gpt were determined using the average of qRT-PCR results from three similarly located nodes (e.g., 1 / 1a, 1 / 1b, 1 / 1c) from triplicate vines. Individual vines were inoculated between the second and third nodes on opposite sides (two points per vine) with 40 μl of bacterial suspension using the needle inoculation technique described by Hopkins (Plant Dis. 89:1348-1352, 2005). Control vines were mock inoculated using phosphate buffer following the same protocol. Four weeks after pathogen inoculation, 15 vines from each treatment were inoculated with 40 μl of bacteriophage Xfas304 suspension (1 × 10 10 Vines were challenged with 40 μl (A PFU / ml) of X. fastidiosa and bacteriophage. Vines were scored twice weekly for 12 weeks for the development of symptoms and assayed in triplicate for X. fastidiosa and bacteriophage at the time of inoculation and at weeks 8, 10, and 12 as described below. To determine whether the bacteriophage acts in a preventative manner, nine vines (two vines each) were inoculated with 40 μl of bacteriophage Xfas304 using the same inoculation protocol and inoculation technique as above. Four weeks after inoculation with bacteriophage, 40 μl (A 600 =0.4;1×10 9 The vines were challenged with 1000 CFU / ml of strain XF15.

[0083] To assess bacterial and bacteriophage movement in grapevines, 15 vines each were inoculated with either XF15 or XF54 and 24 vines were inoculated with bacteriophage Xfas304 alone using the same inoculation protocol described above. Vines inoculated with XF15 or XF54 were assayed in triplicate immediately after inoculation and at 8, 10, and 12 weeks after inoculation. Vines inoculated with bacteriophage were assayed in triplicate immediately after inoculation and every 2 weeks for 12 weeks. The assay method is described below.

[0084] To determine how bacteriophages may affect pathogen populations and disease development in vines, X. fastidiosa-inoculated vines were challenged with bacteriophage Xfas304 4 weeks after pathogen inoculation. Eight weeks after inoculation with XF15, 4 weeks after challenge with Xfas304, vines did not show any PD symptoms, and bacterial populations were 1-3 logs lower in bacteriophage-challenged vines compared to unchallenged vines. Plants not challenged with bacteriophage showed PD symptoms (Figure 7, panel 2), whereas bacteriophage-challenged vines did not show any PD symptoms after 5 weeks (Figure 7, panel 6). Between 8 and 12 weeks after inoculation with XF15 (4 and 8 weeks after Xfas304 challenge), no PD symptoms were observed in bacteriophage-challenged vines, and XF15 populations continued to decline to nearly undetectable levels compared to vines not challenged with bacteriophage.

[0085] Quantitative assessment of bacteriophage populations in the presence and absence of the introduced host (XF15) demonstrated that the bacteriophage could replicate in susceptible hosts growing on vines. , and declined in the absence of a susceptible host. Experiments with strain XF54 challenged with Xfas304 4 weeks after pathogen inoculation showed results similar to those observed for vines challenged with XF15. XF54 populations in vine extracts, as measured by CFU / ml of extract, declined from 8 to 12 weeks in bacteriophage-challenged vines compared to those observed in vines not challenged with bacteriophage. No PD symptoms were observed in bacteriophage-challenged vines from 8 to 12 weeks after XF54 inoculation (4 to 8 weeks after Xfas304 challenge) (Figure 7, panel 7). The bacteriophage populations, which increased over the challenge period in the presence of XF54 and declined in the absence of a host, again indicated that the bacteriophage was able to replicate in the vines when a susceptible host was present. A summary of the challenge study is presented in Figure 7 , showing that in vines inoculated with XF15 or XF54 challenged with bacteriophage Xfas304 (4 weeks after inoculation with the pathogen), no further PD symptoms were observed after 5 weeks (Figure 7 , columns 6 and 7), whereas in vines not challenged with bacteriophage, inoculated with strains XF15 or XF54, respectively, symptoms developed between weeks 9 and 10 (Figure 7 , columns 2 and 3).

[0086] To determine the protective potential of bacteriophage treatment, further studies were performed with vines inoculated with bacteriophage Xfas304 and then challenged with XF15 4 weeks after bacteriophage inoculation. Vines did not show any PD symptoms at 4 and 8 weeks after challenge with XF15 (Figure 7, panel 8), whereas vines not treated with phage developed symptoms (Figure 7, panel 2). Bacteriophage populations increased in the presence of XF15 from week 8 to week 12 after the challenge period and decreased in the absence of host.

[0087] These results confirm that bacteriophage treatment prevents or reduces X. fastidiosa PD symptoms in plants and that the phage treatment does not cause any side effects on the plants.

[0088] Example 13 Sampling and processing Duplicate hedges from each vine were divided into 5–6 nodes and the nodes were numbered from bottom to top. Each node was homogenized in 15 ml of P-buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 8 mM MgSO4) using a PRO250 homogenizer with a 20 mm × 115 mm generator (PRO Scientific, Connecticut, USA) and filtered through sterile cheesecloth (Fisher Scientific, USA) to remove plant tissue debris. The filtrate was centrifuged (10,000×g for 15 min) and filter-sterilized to assay for bacteriophage. The filtrate was used for bacteriophage DNA extraction. The same protocol was used for the bacterial assay, except that the pellet was resuspended in 1 ml of Milli-Q water for bacterial DNA extraction.

[0089] Example 14 Propidium monoazide (PMA) treatment of samples The PMA protocol described by Nocker (J Microbiol Meth 67:310-20, 2006) was used to exclude dead X. fastidiosa cells from the assay and from the assay used to generate a standard curve for assaying vine tissue extracts. Briefly, PMA (Biotium Inc., Hayward, CA) was dissolved in 20% dimethyl sulfoxide (Sigma-Aldrich, Germany) to obtain a 20 mM stock solution and stored in the dark at -20°C. A volume of 1.25 μl of PMA stock solution was added to 500 μl of X. fastidiosa cell suspension (A 600 =0.4;1×10 9 CFU / ml) or extracts from control and inoculated vines. Preparations were incubated in clear microcentrifuge tubes in the dark for 5 min with repeated inversion. Following incubation, the microcentrifuge tubes were placed on ice and incubated for 20 The tubes were exposed to a 650W halogen light source (Ushio, USA) at a distance of 1 cm for 1 min. They were briefly spun by hand every 5 seconds and flipped after 30 seconds of illumination to ensure complete crosslinking of available DNA and conversion of free PMA to hydroxyaminopropidium. After light-induced crosslinking, viable cells were harvested by centrifugation (12000×g, 2 min at 25° C.), washed in 500 μl of sterile distilled water, and resuspended in Mili-Q water for DNA extraction.

[0090] Fungal / Bacterial DNA was extracted from PMA-treated cell preparations and vine extracts using ZR Fungal / Bacterial DNA Minipreps (Zymo Research, USA) according to the manufacturer's instructions. Bacterial DNA was extracted.

[0091] Wizard DNA Clean-up system (Promega, Wisconsin, USA) with modifications as described by Summer (Methods Mol. Biol. 502:27-46, 2009). was used to extract bacteriophage DNA from control preparations and plant extracts.

[0092] Example 15 Detection of X. fastidiosa and bacteriophage Xfas304 using real-time PCR SYBR-green-based real-time PCR (RTPCR) was performed on a 7500 Real-Time PCR System (Applied Biosystems, CA, USA) using X. fastidiosa-specific primers INF2 5'-GTTTGATTGATGAACGTGGTGAG-3' (SEQ ID NO: 1) and INR1 5'-CATTGTTTCTTGGTAGGCATCAG-3' (SEQ ID NO: 2) (Bextine and Child, FEMS Microbiology Letters 276: 48-54, 2007), designed for gyrB, and DNA primers The bacteriophage Xfas304 specific primers 304-PrimF 5'-AAGAAGCGTGGTTTGTTTGC-3' (SEQ ID NO: 3) and 304-PrimR 5'-CTACCGGCTTCCCTAACTCC-3' (SEQ ID NO: 4) designed to the Xfas304 gene were used. A master mix was made using 10 μl of Express SYBR GreenER SuperMix (Invitrogen), 0.4 μl of both primers (at a concentration of 10 μM), 8.56 μl of sterile molecular grade water, 0.04 μl of ROX reference dye (Invitrogen), and 1 μl of DNA template per reaction. Standard conditions were used for all reactions with an initial denaturation step at 95°C for 3 min, followed by 40 cycles of the following parameters: 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. At the end of the PCR, the temperature was increased from 72°C to 99°C at a rate of 0.5°C / 10 s and the fluorescence emission was measured every 10 s. Each DNA sample was analyzed in triplicate. As a positive control, DNA was extracted from X. fastidiosa cells and bacteriophage Xfas304 lysates using the method described above. The cycle threshold (Ct), describing the PCR cycle number at which fluorescence emission rises above baseline, was determined using a software package provided by Applied Biosystems.

[0093] To obtain a standard curve for absolute quantification, 1 × 10 of XF15 and XF54 in 1 ml volumes were 8 CFU / ml cell suspensions were treated using the PMA protocol. Bacterial DNA was extracted as described above and 1 × 10 -1 ~1×10 -5 The bacteriophage DNA was serially diluted to 1 × 10 in a 1 ml volume and subjected to the real-time PCR assay described above. 9 Extracted from the above bacteriophage Xfas304 at PFU / ml, 1 × 10 -1 ~1×10 -6 The DNA was diluted to 100 µL and subjected to real-time PCR assay. Standard curves were generated for X. fastidiosa and bacteriophage Xfas304 using triplicate samples. X. fastidiosa DNA concentrate (A260 The standard was determined by plotting the Ct values ​​generated from real-time PCR against the Log DNA concentration per μl (calculated by A curve was constructed. The efficiency (E) was calculated as follows: E = 10 (-1 / 傾き) -1 (Klein et al., Electrophoresis 20:291-299, 1999).

[0094] Example 16 Lysogen formation assay studies To assay for phage lysogen formation, survivors of phage infection were ) were tested for the presence of prophage. For each phage, bacteria were infected at an input MOI of approximately 3 and plated on soft agar overlay. Plates were monitored for colony growth (10-15 days for X. fastidiosa strain Temecula and 2-3 days for Xanthomonas strain EC-12). Individual colonies that emerged were selected, purified (in triplicate) and tested again for phage sensitivity by spotting dilutions of the same phage on soft agar overlay. Phage-insensitive isolates were then tested for the presence of prophage sequences using primer pairs specific for the Xfas103 and Xfas106 helicase genes, or the Xfas303 and Xfas304 primase genes (Table 5). Wild-type bacterial DNA was used as a negative control and wild-type bacterial DNA spiked with phage DNA served as a positive control.

[0095] To test whether evidence of premature lysogeny (i.e., establishment of inhibition) could be found, we performed the same procedure as Gill et al. (Gill JJ, et al., J. Bacteriol., 193:5300-5313 (2011)) except that reversibly bound phages were removed by three successive washes. The procedure was followed. A logarithmically growing liquid culture of Xanthomonas strain EC-12 was grown to an OD600 of approximately 0.3-0.5. A 1 ml aliquot was pelleted by centrifugation and resuspended in 0.20 ml of phage lysate (recovered in TNB) or sterile TNB. After incubation at 25°C for 30 min, the cell-phage mixture was centrifuged, the supernatant removed, and titration was performed to identify adsorbed phage. Preliminary experiments determined that phage were reversibly bound, which affected the calculation of the MOI estimate (Kasman, LM, et al., J. Virol., 76:5557-5564 (2002)). To circumvent this issue and to obtain an accurate MOI estimate, cells were resuspended in sterile TNB, incubated at 25°C for 5 min, centrifuged, and the supernatant removed. This procedure was repeated three times to remove unbound phage. Each supernatant was titrated to determine PFU. To enumerate the viable bacteria remaining after phage exposure, the cell pellet was resuspended in 0.20 ml of sterile TNB and serially diluted and plated. From these data, the actual MOI (i.e., the ratio of the number of adsorbed phages to the number of CFUs in the phage-free control) was calculated. These actual MOIs were used to calculate the expected proportion of uninfected cells using a Poisson distribution. This experiment was repeated three times using both Xfas103 and Xfas303.

[0096] Lysogenic. To examine the possibility of lysogeny, 40 phage-insensitive isolates each of X. fastidiosa strain Temecula and Xanthomonas strain EC-12 were recovered after challenge infection with phages Xfas103, Xfas106, Xfas303 or Xfas304. PCR using phage-specific primers did not detect the presence of phage lysogens in the resistant isolates, indicating that resistance was not due to lysogeny. Furthermore, the possibility of premature lysogeny was investigated by using infection at a high MOI and measuring survival (Gill et. al (2011)). As shown in Table 4, there was no significant difference between predicted and actual surviving cells after infection, indicating that phage infection at a high MOI did not result in the establishment of inhibition. Together, these results indicate no evidence of lysogeny or inhibition, supporting the conclusion that the four phages are virulent.

[0097] [Table 4]

[0098] Example 17 Phage cocktail protection and prophylaxis studies Bacterial strains, phages, and inoculum preparation: The bacterial isolates used in this study were X. fastidiosa strains Temecula (XF15) and XF54 (see Example 3). Cultures of X. fastidiosa were maintained on PW-M as described in Example 1. Inocula of XF15 and XF54 were prepared as described in Example 11. High titer phage lysates (1×10 10 PFU / ml) were prepared and titrated as described in Example 3. Phage cocktails were prepared by mixing four phages each, with 1 × 10 10 A final concentration of PFU / ml was obtained.

[0099] Inoculation of grapevines with bacteria and phage cocktail: Grapevines were inoculated with either X. fastidiosa strain XF15 or XF54 to assess bacterial translocation in the grapevines. Grapevines were assayed in triplicate immediately after inoculation (0 min) and at 8 and 12 weeks post-inoculation. Additionally, grapevines inoculated with XF15 or XF54 were challenged 3 weeks post-inoculation with phage cocktail to assess therapeutic efficacy. Grapevines inoculated with phage cocktail were challenged 3 weeks post-inoculation with either XF15 or XF54 phage to assess preventative efficacy of the cocktail. Grapevines from each treatment were scored twice weekly for symptom development. To identify the distribution of individual phages comprising the cocktail, grapevines were assayed at 0, 2, 4, 6, 8, 10 and 12 weeks post-inoculation as described below. Grapevines inoculated in either the phage or pathogen challenge infection studies were assayed for phage and / or X. fastidiosa infection at weeks 0, 6, 8, 10, and 12 as described below. Control grapevines were assayed at weeks 0, 8, and 12 after inoculation to monitor pathogen distribution and disease development. All grapevine assays were performed in triplicate with vines containing two fences. Each fence (e.g. fence 1=S1, or fence 2=S2) was divided into 5-6 nodes (5 inches). Vine nodes were numbered from the inoculation point (0) and at the 5 inch nodes as below (-) or above (+) the inoculation point. Root sections were divided into three nodes: R1, R2, or R3.

[0100] Sample collection and processing: For quantification of cocktail phages and pathogens, samples were obtained as described in Example 13. For phage assays, the filtrates were centrifuged (10,000×g, 15 min at 4° C.) and filter-sterilized. The filtrates were used for phage DNA extraction to accomplish quantitative real-time PCR (qRTPCR) (see below). The same protocol was used for bacterial assays, except that the pellets were resuspended in 1 ml Milli-Q water for isolation of bacterial DNA used. CFU and PFU were determined using the average of qRTPCR results from three similarly positioned nodes (e.g., 0a, 0b, 0c) from triplicate vines. To determine whether phage-resistant X. fastidiosa emerged as a result of phage challenge infection experiments, samples taken from grapevines 12 weeks after inoculation with the pathogen were processed as described in Example 13. Briefly, 100 μl of a suspension of pellets in 1 ml Milli-Q water were plated on PW-MA (Example 1) supplemented with 40 μg / ml cycloheximide (PW-MAC) and incubated at 28°C. After 10-12 days, individual colonies were selected and streak-purified in triplicate on PW-MAC. Representative individual colonies from each grapevine sample (20 colonies in total) were identified for species and genotype using PCR analysis as described by Hernandez-Martinez et al. (Example 1). Confirmation was performed at the subspecies level. Phage susceptibility of confirmed isolates was determined by serial dilution spot assay on overlay and soft agar overlay methods as described in Example 3.

[0101] PMA treatment and qRTPCR: PMA treatment and SYBR-green based qRTPCR protocol was performed as described in Example 15 using X. fastidiosa specific primers INF2 (SEQ ID NO: 1) and INR1 (SEQ ID NO: 2) and bacteriophage Xfas303 specific primers 303-PrimF (SEQ ID NO: 5) and 303-PrimR (SEQ ID NO: 6), Xfas304 specific primers 304-PrimF (SEQ ID NO: 3) and 304-PrimR (SEQ ID NO: 4), Xfas103 specific primers 103-HelF (SEQ ID NO: 7) and 103-HelR (SEQ ID NO: 8); and Xfas106 specific primers 106-HelF (SEQ ID NO: 9) and 106-HelR (SEQ ID NO: 10) listed in Table 5.

[0102] [Table 5]

[0103] Movement of X. fastidiosa and disease development in grapevines: XF15 or XF54 Quantitative evaluation of duplicate segments from triplicate samples of inoculated grapevines indicated distribution of the pathogen in the grapevine segments assayed. qRTPCR revealed an average of 1 × 10 virulence in segments (seg) S1 / 1 (segment 1, 5-inch segment above the inoculation point) and S2 / 1, respectively. 4 CFU / gram plant tissue (gpt) and 1×10 5 The presence of XF15 CFU / gpt was detected, with an average of 1 × 10 4 CFU / gpt of XF54 were detected. By 8 weeks post-inoculation, grapevines not challenged with the cocktail showed typical Pierce's disease symptoms, such as slight yellowing or reddening of the leaves and dry or necrotic leaf margins. At 12 weeks post-inoculation, an average of 1 x 10 4 CFU / gpt and 1×10 6 CFU / gpt of XF15 were detected in S1 / 3 and S2 / 2, respectively. At the same assay interval, grapevines exhibiting PD symptoms were used to detect an average of 1 × 10 5 CFU / gpt and 1×10 4 CFU / gpt of XF54 was detected in S1 / 3 and S2 / 1, respectively, with an average of 1 × 10 1 CFU / gpt ~1×10 2 Both pathogens (XF15 and XF54) were detected in the root system of grapevines 8 and 12 weeks after pathogen inoculation at CFU / gpt.

[0104] Phage movement and persistence in grapevines: Standard qRTPCR line plots were obtained for phages Xfas303, Xfas304, Xfas103, and Xfas106 with R2 values ​​greater than 0.9 and efficiencies of 127%, 123%, 129%, and 120%, respectively. Quantitative evaluation of duplicate roots from triplicate samples of grapevines inoculated with the phage cocktail (Xfas303, Xfas304, Xfas103, and Xfas106) showed the distribution of all phages individually in grapevine nodes assayed 2 to 8 weeks after cocktail inoculation (Figure 8). By weeks 8 and 12, individual phages were no longer detectable in roots, and an average of 1 × 10 phages were detected by week 12 without any grapevine symptoms being observed in the nodes assayed. 1 PFU / gpt~1×10 2 The number of PFU / gpt decreased to 100 (Fig. 8).

[0105] Therapeutic effect of phages against X. fastidiosa in grapevines: Grapevines inoculated with XF15 were challenged with the phage cocktail 3 weeks after pathogen inoculation. At week 8 (5 weeks after cocktail challenge), XF15 populations in non-challenged vines were 2-3 logs higher on average than challenged vines. Non-therapeutic grapevines showed typical PD symptoms, whereas challenged vines did not show PD symptoms. At week 12 after XF15 inoculation (9 weeks after cocktail challenge), bacterial populations in non-challenged vines were 2-3 logs higher on average when compared to phage cocktail challenged vines (Figure 9). No PD symptoms were observed in phage challenged vines throughout the study (12 weeks), whereas non-cocktail treated vines showed symptoms as early as week 4, which progressed over 12 weeks. Similarly, bacterial populations in grapevines challenged with the XF54 inoculum cocktail were significantly reduced from weeks 8 to 12 compared to unchallenged grapevines, and no symptoms were observed in the cocktail-challenged grapevines. Plating of plant extracts from cocktail-challenged grapevines at week 12 yielded an average of 1 × 102 CFU / gpt were obtained. Representative isolates (20ea) identified as X. fastidiosa from each of the three grapevines were susceptible to all four phages that comprised the cocktail.

[0106] Prophylactic efficacy of cocktail treatment to prevent PD in grapevines: The prophylactic efficacy of the phage cocktail was evaluated by first inoculating grapevines with the above cocktails and then challenging them with X. fastidiosa strains XF15 or FX54 3 weeks after cocktail inoculation. Prophylactically treated grapevines did not show any PD symptoms at 8 and 12 weeks after cocktail inoculation. In cocktail-inoculated grapevines challenged with XF15, pathogen populations averaged up to 1 × 10 in the examined grapevine nodes at 8 and 12 weeks. 3 CFU / gpt, averaging 1 × 10 6 CFU / gp Similar results were observed in grapevines challenged with XF54 3 weeks after inoculation with the phage cocktail after prophylactic treatment with the cocktail. Plating of plant extracts from grapevines challenged with the cocktail at 12 weeks yielded an average of 3 × 10 2 CFU / gpt were obtained. Representative isolates (20ea) identified as X. fastidiosa from each of the three grapevines were susceptible to all four phages that make up the cocktail.

[0107] Phage persistence and replication in grapevines: We aimed to determine phage populations in grapevines in the presence or absence of introduced hosts (XF15 and XF54). Quantification of phage populations in the presence or absence of hosts confirmed that in both therapeutic and preventative studies, cocktail phages were able to replicate and maintain larger populations when a susceptible host was present in the grapevine, and then declined in the absence of a susceptible host (Figures 10 and 11). Phage populations in host-free grapevines decreased between weeks 8 and 12, whereas in grapevines inoculated with XF15 or XF54 and challenged with the phage cocktail (therapeutic treatment), phage populations increased by an average of 1-2 logs during the same period (Figure 10). Similar results were obtained in the preventative study, where phage populations increased by an average of 1-2 logs relative to phage populations observed in host-free grapevines (Figure 11). These results confirmed that bacteriophage treatment prevented or reduced PD symptoms caused by X. fastidiosa in plants and did not show any side effects on the treated plants.

[0108] Example 18 Transmission of X. fastidiosa by leafhoppers The leafhopper (GWSS), Homalodisca vitripennis GWSS are xylem-feeding leafhoppers that transmit X. fastidiosa. GWSS are prevalent throughout the grape-growing regions of Southern California and Texas. To investigate the uptake of X. fastidiosa or phage by GWSS, laboratory-grown GWSS without X. fastidiosa were fed cowpea (Vigna unguiculata subsp. unguiculata) plants carrying either X. fastidiosa or virulent phage Xfas304 for 48 h in three trials. To determine the ability of GWSS to transmit bacteria or phage to plants, GWSS with bacteria or phage were fed plants without bacteria or phage. A subset of GWSS with bacteria were challenged by feeding plants with phage for 48 or 96 h. GWSS and plants were assayed separately in all experiments and qRTPCR was used to assess uptake, transmission or persistence of bacteria and / or phages. GWSS were able to uptake and transmit X. fastidiosa and / or phages. In GWSS with X. fastidiosa and challenged with phages, the titer of phage Xfas304 was increased 2-fold compared to the titer observed in GWSS without X. fastidiosa. A 2-fold decline in bacterial population was observed in GWSS when challenged with phage Xfas304 compared to those not challenged. GWSS transmitted X. fastidiosa and / or phages to plants. This is believed to be the first report of phage transmission by GWSS.

[0109] Bacterial strains, phages and inoculum preparation: X. fastidiosa strain XF54 (see Example 1) and phage Xfas304 (see Example 3) were used in this study. Cultures of XF54 were grown on PW-M as described in Example 1. Day 5 cultures of XF54 grown on PW-MA were used to make bacterial suspensions in phosphate buffer (0.125 M, pH 7.1). Xfas304 (1 × 10 10High titer phage lysates (PFU / ml) were prepared and titered in sterile deionized water (SDW) as described in Example 3.

[0110] Plant growth conditions and preparation: Cowpea (Vigna unguiculata subsp. unguiculata) plants were grown in 3-inch pots using Metro-Mix and maintained at 24°C to 29°C (16 h light and 8 h dark, respectively) and watered as needed.

[0111] Leafhoppers: Laboratory-raised GWSS were originally raised for multiple generations in greenhouses at one of two locations: (i) the California Department of Agriculture and Food (CDFA) in Irvine, or (ii) the University of California Cooperative Extension in San Marcos, California. All GWSS used in this study were adult males and females and delivered from the two locations. After receipt, the insects were fed cowpea plants maintained at 24°C to 29°C (16 hours light and 8 hours dark, respectively) for 2 days to acclimate to the new climate before use in experiments.

[0112] Experimental design: Each experimental unit (i.e., cage) contained a 15 cm long stem of cowpea at the 3-4 leaf stage and a 50 ml flat-bottom tube containing 50 ml of phage or bacterial suspension in SDW as appropriate. Cowpea stems with leaves at the 3-4 leaf stage (cut stems) were taken from 2- or 3-week-old plants inserted through a hole in the lid and fixed in place with parafilm (fixed cut stems). GWSS (3 GWSS / cut stem / cage) were placed in the cages and fed as needed.

[0113] Uptake of X. fastidiosa and phages by GWSS: To determine uptake of X. fastidiosa and / or phages by GWSS, cut cowpea stems with leaves were inoculated with X. fastidiosa (1 × 10 9 CFU / ml) or phage Xfas304 (1 × 10 10Cut stems were fixed in tubes filled with a suspension of 1000 PFU / ml of X. fastidiosa or phage for 4 hours to allow capillary uptake of X. fastidiosa or phage. Control cut stems were placed in SDW. After uptake of the appropriate suspension for 4 hours, a subset (3 cut stems) was assayed to quantify X. fastidiosa or Xfas304. After the 4 hour uptake period, GWSS (3 GWSS / cut stem / cage) were fed the cut stems. Each experimental set was performed in triplicate (1 cut stem x 3 GWSS x 3 cages). After 48 hours, all cowpea cut stems and GWSS were assayed to quantify the presence of X. fastidiosa and / or phage by qRTPCR. Water uptake controls under the same conditions were performed for all experiments and assayed for X. fastidiosa and phage.

[0114] The first experiment was designed to determine whether GWSS could acquire X. fastidiosa or phages from cut stems carrying the pathogen or phages, and if so, whether GWSS could transmit X. fastidiosa or phages to other cut stems. After 48 h, cut stems and GWSS had an average of 2 × 10 8 ±1×10 8 CFU / gram plant tissue (gpt) and 1×10 6 ±0.7×10 6 CFU / GWSS and GWSS as previously reported (Bextine et al., Biotechniques 38:184, 186, 2005). In a parallel experiment to determine whether GWSS acquired phages by feeding on cut stems, GWSS assayed after 48 h had a phages concentration of 2 × 10 8 ±1×10 8 An average of 2 × 10 obtained from cut stems containing PFU / gpt 6 ±0.9×10 6 The results showed that the GWSS could acquire the phages by feeding on the cut stems.

[0115] Phage uptake and transmission by GWSS: To evaluate phage uptake and transmission by GWSS, cut cowpea stems (9 stems) were soaked in a phage Xfas304 suspension (1 × 10 10 The stems were placed in a 50 ml tube filled with 1000 ng / ml of phage (PFU / ml). A control (3 cut stems) was placed in SDW. Both sets of cut stems were loaded with the respective media. After 4 hours, the 3 cut stems loaded with phage were assayed to determine the phage concentration. The remaining 6 cut stems were Each GWSS was placed in an individual cage containing GWSS (3 GWSS / cut stem / cage). After 48 hours, 9 GWSS and their respective 3 cut stems were assayed for phage content, and the remaining 9 GWSS were transferred to new cowpea cut stems fixed in SDW (3 GWSS / cut stem x 3 cages) and fed cowpea for another 48 hours to determine phage transmission to the cut stems. The cut stems (3 stems) and GWSS (9 individuals) were assayed for phage after the indicated time periods. Controls taking up water under the same conditions were performed for all experiments and assayed for phage.

[0116] Having determined that both phages and bacteria could be acquired by GWSS, we aimed to determine whether GWSS that acquired phages from cut stems could transmit phages and / or bacteria to other cut stems. A subset of phage-harboring GWSS were transferred to and fed on fresh cut cowpea stems in SDW. After 48 h, the cut stems and GWSS each had an average of 3 × 10 2 ±2.5×10 2 PFU / gpt and average 3 × 10 3 ±1.6×10 3 PFU / GWSS, indicating that the GWSS was able to transmit the phage.

[0117] Phage challenge of X. fastidiosa with GWSS: To determine whether phages could affect X. fastidiosa populations in GWSS, GWSS with X. fastidiosa were challenged with phages. Briefly, cut stems containing X. fastidiosa were fed using the method described above using triplicate replicates, and GWSS confirmed to contain X. fastidiosa were transferred and fed to cut cowpea stems loaded with phage Xfas304. After 48 or 96 hours of feeding, the cut stems and GWSS were assayed for phage and / or X. fastidiosa. For X. fastidiosa uptake, cut cowpea stems (15 stems) were soaked in XF54 suspension (1×10 ) prior to introducing GWSS. 9 CFU / ml) for 4 hours. At 4 hours, 3 cut stems were assayed for X. fastidiosa. Each of the 12 remaining cut stems was placed in a cage containing 3 GWSS per cut stem and the GWSS were allowed to feed on the X. fastidiosa-containing cut stems for 48 hours. After 48 hours, the X. fastidiosa-fed GWSS and host cut stems were subdivided into 3 groups: Group 1 was assayed for X. fastidiosa (3 cut stems and 9 GWSS); Group 2 (9 GWSS) was transferred to fresh cowpea cut stems (3 stems) placed in SDW and allowed to feed on the GWSS and cut stems for 48 hours before assaying for X. fastidiosa; Group 3 (18 GWSS) was assayed for X. fastidiosa with XFas304 suspension (1 × 10 10 The GWSS and cut stems were then transferred to cut cowpea stems (3 stems) in 1000 μg / ml water (Table 1) and allowed to feed for 48 or 96 h before assaying for X. fastidiosa and phages. Controls taking up water under the same conditions were performed for all experiments and assayed for both X. fastidiosa and phages.

[0118] Thirty-six GWSS were fed cowpea cut stems in a X. fastidiosa suspension and then assayed to determine X. fastidiosa uptake, X. fastidiosa and / or phage transmission, and the effect of phage and / or X. fastidiosa on the GWSS. X. fastidiosa strain XF54 (3 × 109 GWSS (group 1) fed cut stems in a suspension of 1 × 10 CFU / ml for 48 hours had an average of 1 × 10 6 ±0.7×10 6 CFU / GWSS were identified, and the cut stem-feeding hosts had an average of 2 × 10 8 ±1×10 8 CFU / gpt. GWSS with X. fastidiosa (group 2; 1 × 10 6 ±0.7×10 6 After feeding on freshly cut stems in SDW for 48 h, the average number of cut stems was 1 × 10 3 ±1.3×10 3 CFU / gpt, and GWSS was 2 × 10 3 ±1×10 3 CFU / GWSS residual X. fastidiosa, reaffirming previous results of X. fastidiosa transmission by GWSS. 10 GWSS carrying group 3 X. fastidiosa transmitted to cut stems at 100 PFU / ml and fed for 48 h showed phage uptake and persistence of X. fastidiosa. GWSS assayed showed an average of 3 × 10 4 ±1.8×10 4 PFU / GWSS Xfas304, 2 × 10 3 ±1.1×10 3 CFU / GWSS of XF54 were maintained. Cut stems assayed at the same time intervals had an average of 3 × 10 8 ±2×10 8 PFU / gpt and 2×10 3 ±0.6×10 3 CFU / gpt. GWSS fed for 96 h contained an average of 2 × 10 5 ±1.2×10 5 PFU / GWSS Xfas304 and 1×10 2 ±0.9×10 2 CFU / GWSS for XF54, showing a decrease in XF54 and an average 6-fold increase in Xfas306.

[0119] Harvesting and assay of cowpea cut stems and GWSSs: GWSSs were sacrificed by freezing at -20°C for 5 min, and cowpea cut stems were harvested by cutting the tube cap seam with a sterile razor. Each triplicate GWSS was placed in a 1.5 ml microcentrifuge tube containing 0.5 ml P-buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 8 mM MgSO4), homogenized using a sterile plastic micro pestle (Fisher), and filtered through sterile cheesecloth (Fisher Scientific, USA) for assembly. Tissue debris was removed. Each triplicate cut stem was weighed, commuted using a sterile razor blade, homogenized in 1 ml of P-buffer using a mortar and pestle, and filtered through sterile cheesecloth (Fisher Scientific, USA) to remove tissue debris. The filtrate was centrifuged (10,000×g for 15 min) and filter sterilized to assay the phages. A portion of the filtrate was used for phage DNA extraction as in Example 9 and then for qRTPCR as described below. The remainder of the filtrate was used to titer the phages as described in Example 3. The same protocol was used for the bacterial assay (CFU), except that the pellet was resuspended in 0.5 ml of sterile Milli-Q water for PMA treatment, bacterial DNA extraction and qRTPCR as described below.

[0120] PMA treatment and qRTPCR: PMA treatment and SYBR green-based qRTPCR protocol were performed as described in Example 17 using primers specific for X. fastidiosa and phage.

[0121] Example 19 Phage activity against Xanthomonas axonopodis pathovar citri Previous studies have evaluated the use of phages for the control of citrus canker, but no conclusive data confirmed the virulence characteristics of the phages (Balogh et al., Plant Disease, 92:1048-1052, 2008). Only virulent, non-transducing phages should be used to evaluate and implement sustainable phage biocontrol systems. We tested the susceptibility of three Xac field strains obtained from Florida (North 40, Block 22, Fort Basinger) to two fully characterized virulent phages representative of the Podoviridae family (Xfas303) and Siphoviridae family (Xfas103), respectively. The results show that the three Xac strains tested were only susceptible to phage Xfas303 (Figure 12). Phage Xfas303 was able to form clear plaques on the three Xac strains tested. The Xfas303 genome was subjected to 454 pyrosequencing and the predicted genes were fully annotated. strain EC-12 as the primary receptor site for phage Xfas303. Both phage Xfas303 and phage Xfas103 adsorb to form clear plaques on strain EC-12, but not on the ΔpilA derivative, and we only show results for plating phage Xfas303 on EC-12 or EC-12ΔpilA. Also, since type IV pili are the primary receptor site for phage Xfas303, it is possible that this phage has a different, second receptor site required for infection. We identified that the phage DNA was restricted by Xac-independent vectors, and the results indicate that the developed Xac-independent method can be used to isolate phages toxic to Xac without compromising plating efficiency (EOP 0.75).

[0122] Example 20 Evidence for the expression of type IV pili in Xac. Reports in the literature are contradictory regarding the expression and role of type IV pili in the infection process and pathogenesis of Xac (Brunings et al., Mol. Plant. Pathol. 4:141-57, 2003; Li et al., PLoS ONE6:e21804, 2011; Yang et al., Curr. Microbiol. 48:251-61, 2004). The results shown indicate that all three Xac strains tested express functional type IV pili, as pili must be retracted to facilitate phage adsorption and infection. Using light microscopy studies, the three Xac strains were evaluated for twitching motility, an indicator of functional type IV pili. Pseudomonas aeruginosa strain PAO1 and Xanthomonas subsp. strain EC-12 were used as positive controls, and E-12ΔpilA was used as a negative control. Three Xac strains (North 40, Block 22, and Fort Basinger) were evaluated for twitching motility. PAO1, EC-12, and the three Xac strains exhibited twitching motility, whereas EC-12ΔpilA did not. Microscopy studies confirmed the results obtained by phage susceptibility testing and showed that the three Xac strains possessed functional type IV pili that acted as adsorption sites for phage Xfas303. The results confirm the observations of others (Brunings et al., Mol. Plant. Pathol. 4:141-57, 2003; Li et al., PLoS ONE 6:e21804, 2011; Yang et al., Curr. Microbiol. 48:251-61, 2004) that type IV pili are expressed by Xac. [Accession number]

[0123] PTA-13095 PTA-13096 PTA-13097 PTA-13098 PTA-13099 PTA-13100 PTA-13101

Claims

1. 1. A method for propagating a virulent bacteriophage that includes Xylella fastidiosa and / or Xanthomonas in its host range, comprising: (a) infecting a culture of Xanthomonas bacteria with the virulent bacteriophage; (b) propagating the virulent bacteriophage; and (c) isolating virulent bacteriophage particles from the culture; Including, the virulent bacteriophage is selected from the group consisting of Xfas100 phage-type bacteriophage and Xfas300 phage-type bacteriophage; The Xfas100 phage type has the following characteristics: (a) the bacteriophage is capable of lysing the Xylella fastidiosa and / or Xanthomonas bacteria; (b) the bacteriophage infects a cell by binding to a type IV pilus; (c) the bacteriophage contains a non-contractile tail, a morphology typical of the Siphoviridae family, and has a capsid size ranging from 55 nm to 77 nm in diameter; (d) the genome size of the bacteriophage is about 55,500 bp to 56,200 bp; and (e) the bacteriophage prevents or reduces symptoms associated with Pierce's disease and / or citrus canker in a plant(s); indicates, The Xfas300 phage type has the following characteristics: (f) the bacteriophage is capable of lysing the Xanthomonas bacterium; (g) the bacteriophage infects a cell by binding to type IV pili; (h) the bacteriophage contains a non-contractile tail, a morphology typical of the Podoviridae family, and has a capsid size in the range of 58 nm to 68 nm in diameter; (i) the genome size of the bacteriophage is about 43,300 bp to 44,600 bp; and (j) the bacteriophage prevents or reduces symptoms associated with Pierce's disease and / or citrus canker in a plant or plants; Showing, a method.

2. 2. The method of claim 1, further comprising: said Xfas100 phage-type virulent bacteriophage particles comprising a genome having a DNA sequence at least 90% identical, but less than 99% identical, to a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18; and said Xfas300 phage-type virulent bacteriophage particles comprising a genome having a DNA sequence at least 90% identical, but less than 99% identical, to a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:

24.

3. The method of claim 1 , wherein the virulent bacteriophage is isolated from plants, wastewater, and / or soil water.

4. 2. The method of claim 1, wherein the culture of Xanthomonas bacteria comprises Xanthomonas strain EC-12 deposited under ATCC Accession No. PTA-13101.

5. 10. The method of claim 1, further comprising the use of an agar overlay for growth of said virulent bacteriophage.

6. 2. The method of claim 1, wherein the virulent bacteriophage particles of the Xfas100 phage type comprise a genome having a DNA sequence at least 95% identical, but less than 99% identical, to a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and the virulent bacteriophage particles of the Xfas300 phage type comprise a genome having a DNA sequence at least 95% identical, but less than 99% identical, to a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:

24.

7. 2. The method of claim 1, wherein the virulent bacteriophage particles of the Xfas100 phage type comprise a genome having a DNA sequence at least 98% identical, but less than 99% identical, to a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and the virulent bacteriophage particles of the Xfas300 phage type comprise a genome having a DNA sequence at least 98% identical, but less than 99% identical, to a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.

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