Method for preventing and treating rice blast by using melanogaster peruvieri

Abstract

The invention discloses a method for preventing and treating rice blast by utilizing melanogaster, and relates to the technical field of rice blast prevention and treatment, the melanogaster comprises melanogaster A16, the preservation number of the melanogaster A16 is CCTCC NO: M20242411, the preservation date is October 31, 2024, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation address is Wuhan University, Luojia Hill, Wuchang District, Wuhan City, Hubei Province; the growth of rice moony grains, Nipponbare, Lijiang Xinhua black grains (LTH), land rice Xiangtang grains, old Zizhai black grains and cold spring grains is promoted, the growth of land rice large Y-mouth grains is inhibited, and the resistance of LTH to rice blast is promoted by the permeabilized-sporosorium endophyllum A16; the expression of the OsICS1 gene in the LTH is reduced by the aid of the melanosomes permeatum A16, and the expression of the salicylic acid receptor gene OsNPR1 in the LTH and the expression of the sakuranetin synthetic gene OsNOMT in the phytoalexin are promoted by the melanosomes permeatum A16.

Description

Technical Field

[0001] The present invention relates to the technical field of rice blast control, and particularly relates to a method for controlling rice blast by using [

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[0006] Background Art

[0002] Rice is one of the important food crops for humans. Half of the world's population consumes rice. The total output of rice ranks third among the world's food crops. It is an important cereal widely planted in the tropical regions of Asia, and the southern part of China is the main rice-producing area. Rice blast is a disease that occurs in rice caused by the rice blast pathogen. Rice blast can occur throughout the growth period of rice, damaging seedlings, leaves, ears, nodes, etc.

[0003] The rhizosphere refers to the partial microenvironment that is affected by the activities of plant roots and is different from the soil mass in physical, chemical, and biological properties. The rhizosphere generally refers to the range within a few millimeters from the root axis surface. It is a micro-region of the interaction between soil, roots, and microorganisms, and is also a specific micro-ecosystem formed by different plant species or varieties, soil, and environmental conditions. Many chemical conditions and biochemical processes in the rhizosphere are different from those in the soil mass. The most obvious ones are the changes in rhizosphere pH, Eh, and microbial activity, etc. Active material transformation, circulation, and kinetic processes occur in this micro-region. These processes directly affect plant growth and development, water and nutrient absorption and utilization, the survival and reproduction of beneficial and harmful microorganisms, and the regulatory response of plants to adversity.

[0004] Rhizosphere soil fungi are an important part of the rhizosphere micro-ecosystem. Rhizosphere soil fungi play an important regulatory role in plant nutrient uptake, growth and development promotion, improvement of host plant disease resistance, and maintenance of the balance of the rhizosphere micro-ecosystem. Asian cultivated rice has gradually evolved into different ecotypes during the process of adapting to different agricultural habitats. According to different soil-water conditions and tillage methods, it can be divided into four ecotypes: irrigated rice, lowland rice, upland rice, and deepwater rice. Rhizosphere microorganisms and cultivated rice have a co-evolution history. When crops are dry-seeded, un-domesticated crops have better disease resistance and cold tolerance than domesticated crops, which may be related to the interaction between rhizosphere microbial communities. There are significant differences in the agricultural habitats between upland rice and paddy rice.

[0005] When a plant is infected by a fungus, the plant will have a defense response. The salicylic acid signaling pathway plays an important role in the plant defense response. At the same time, phytoalexins are also an important class of secondary metabolites in the plant defense response. Sakuranetin in phytoalexins and its synthesis gene (OsNOMT) play important roles in rice resistance to rice blast.

[0006] Salicylic acid (SA) is a simple small-molecule phenolic compound that ubiquitously exists in plants. As a type of plant hormone, salicylic acid has important biological functions. Isochorismate synthase (ICS) is the key enzyme for SA synthesis, and the salicylic acid receptor protein NPR1 (non-expressor of pathogenesis-related genes 1) is a key regulatory factor in SA-mediated disease resistance responses.

[0007] Rhizosphere soil fungi can trigger plant defense responses. Currently, the types and functions of rhizosphere soil fungi in upland rice and paddy rice during upland cultivation are not clear, and it is necessary to analyze rhizosphere soil fungal resources for biological control against rice blast. Summary of the Invention

[0008] In view of the above problems existing in the prior art, the present invention provides a method for controlling rice blast using Massarina igniaria A16. Massarina igniaria A16 can affect the growth and disease resistance of paddy rice and upland rice during upland cultivation, and induce the expression of OsNPR1 and OsNOMT genes.

[0009] To achieve the above technical objectives and reach the above technical effects, the present invention is realized through the following technical solutions:

[0010] A method for controlling rice blast using Massarina igniaria. In the rhizosphere soil of the local rice variety Yue Liang Gu, 1 culturable fungus was isolated and named Massarina igniaria A16, with the preservation number CCTCC NO: M20242411, the preservation date of October 31, 2024, and the preservation unit being the China Center for Type Culture Collection (CCTCC), with the preservation address: Wuchang District, Wuhan City, Hubei Province, China, Wuhan University, Luojia Mountain.

[0011] The application of Massarina igniaria A16 in controlling rice blast.

[0012] Advantages of the present invention:

[0013] A method for controlling rice blast using Massarina igniaria. From the rhizosphere soil of upland-cultivated rice Yue Liang Gu, it promotes the growth of Yue Liang Gu, Nipponbare, and Lijiangxintuanheigu (LTH) of paddy rice, as well as Xiangtang Gu, Lao Zhai Heigu, and Leng Quan Gu of upland rice, inhibits the growth of Dayakou Gu of upland rice, and Massarina igniaria A16 promotes the resistance of LTH to rice blast. Massarina igniaria A16 reduces the expression of the OsICS1 gene in LTH and promotes the expression of the salicylic acid receptor gene OsNPR1 and the gene OsNOMT for sakuranetin synthesis in phytoalexin in LTH.

[0014] Of course, it is not necessary for any product implementing the present invention to achieve all of the above-described advantages simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0016] Figure 1 Colony and spore morphology of Periconia macrospinosa A16 according to the embodiments of the present invention;

[0017] Figure 2 Effect of Periconia macrospinosa A16 on plant growth according to the embodiments of the present invention;

[0018] Figure 3 Effect of Periconia macrospinosa A16 on blast resistance of rice LTH;

[0019] Figure 4 Effect of Periconia macrospinosa A16 on the expression of OsICS1 and OsNPR1 genes in rice LTH according to the embodiments of the present invention;

[0020] Figure 5 Effect of Periconia macrospinosa A16 on the expression of OsNOMT gene in rice LTH according to the embodiments of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0022] Example 1

[0023] Periconia macrospinosa regulates rice growth;

[0024] Rice Moon Valley, Nipponbare, LTH, upland rice Dayakou Valley, Lao Zhai Black Valley, Lengquan Valley and Xiangtang Valley were inoculated with Periconia macrospinosa A16 ( Figure 2 A - B), and it was found that the growth of upland rice Dayakou Valley was inhibited, but the plant heights of other rice and upland rice were significantly increased ( Figure 2 C).

[0025] Figure 2Among them, A-B: Phenotypes of rice and upland rice plants after watering (A) and watering with the spore suspension of Massarina igniaria (B). C: Statistical results of plant height. X1-X3 = Xiangtanggu, A1-A3 = Yuelianggu, D1-D3 = Dayakougu, LQ1-LQ3 = Lengquangu, L1-L3 = LTH, N1-N3 = Nipponbare, LZ1-LZ3 = LaoZhaiHeigu; H2O = Seedling watering treatment, Massarina igniaria = Seedlings treated with the spore suspension of Massarina igniaria A16. Scale bar = 10 cm; * indicates P < 0.05; ** indicates P < 0.01; ND = No difference (analyzed by Student's t-test).

[0026] Example 2

[0027] Massarina igniaria affects rice blast resistance in rice;

[0028] To detect the effect of Massarina igniaria A16 on plant disease resistance, the blast-susceptible rice variety LTH was inoculated with Massarina igniaria A16, and its resistance to rice blast was observed. It was found that the blast resistance of LTH inoculated with Massarina igniaria was enhanced ( Figure 3 ).

[0029] Figure 3 Among them, A: Leaf lesion phenotypes of rice LTH inoculated with Magnaporthe oryzae GUY11 on the seventh day after being treated with Massarina igniaria and water for two days. B: Expression level of the MoPot2 gene. H20 = Watering treatment of rice LTH, Mi = Treatment of rice LTH with Massarina igniaria; Scale bar = 1 cm; ** indicates P < 0.01; ND = No difference (analyzed by independent-samples t-test in SPSS software).

[0030] Example 3

[0031] Step1: Growth of plant materials and isolation of fungi;

[0032] The upland rice varieties LaoZhaiHeigu, LengQuanGu, XiangTangGu, DayaKouGu and the rice varieties YueliangGu, Nipponbare, LijiangXintuanHeigu (LTH) are preserved in the National Key Laboratory of Yunnan Agricultural University;

[0033] Isolation of rhizosphere soil fungi: The rice variety YueliangGu plants were planted in the experimental base at the back mountain of Yunnan Agricultural University (25°8′26″N, 102°45′29″E) and grown in a drought environment, with watering twice a week. After the plants grew for 4 months, the rhizosphere soil of the roots was collected; The rhizosphere soil was put into a triangular flask containing 45 mL of sterile water, shaken at 160 r / min and 28 °C for 20 min, left standing for 10 min, and the supernatant was diluted to 10 -1 、10 -2 、10 -3 、10 -4, 10 -5 5 concentration gradients; 300 μL of the diluted solution was respectively aspirated and evenly spread on Rose Bengal medium (Beijing Solarbio Science & Technology Co., Ltd.), and cultured in an inverted position at 28 °C for 5 - 7 d. The single colonies grown were picked and the single spores were cultured on PDA medium.

[0034] Step 2: Morphological and molecular identification of fungi

[0035] The isolated strains were morphologically identified with reference to "Fungal Identification Handbook". For molecular identification of the strains, first, fungal genomic DNA was extracted by the CTAB method. The internal transcribed spacer sequence (ITS) of fungi was PCR amplified using the universal primers ITS1 and ITS4 (Table 2). At the same time, the large subunit (LSU) sequence of fungal ribosome was PCR amplified using the primers NL1 and NL4 (Table 2). PCR reaction system: 10 μL of Power Up TMSYBRTM Green Master Mix enzyme (Thermo Fisher Scientific Inc.), 1 μL each of 10 μM forward primer and reverse primer, 7 μL of ddH2O, and 1 μL of DNA template. Amplification program: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 45 s, annealing at 50 °C for 30 s, extension at 72 °C for 1 min, for a total of 30 cycles, and extension at 72 °C for 5 min. The PCR products were sequenced bidirectionally (Kunming Tsingke Biotechnology Co., Ltd.), and the sequencing results of ITS and LSU genes were subjected to BLAST sequence alignment in the NCBI database.

[0036] Step 2.1: Through the observation of the colony and spore morphology of the fungi ( Figure 1 ), and the sequencing analysis of ITS and LSU sequences (Table 1), this strain of fungus was identified as Periconia byssiseda A16. Figure 1 Among them, the colony (A) and spore (B) morphology of Periconia byssiseda growing on PDA medium for 7 days, scale bar: 1 cm (colony) and 10 μm (spore).

[0037] Table 1 ITS and LSU sequences of Periconia byssiseda

[0038]

[0039]

[0040] Step 3: Treat rice with rhizosphere soil fungi;

[0041] To observe the role of rhizosphere soil fungi in the growth of upland rice and lowland rice, seeds of upland rice varieties 'Laozhaiheigu', 'Lengquangu', 'Xiangtanggu', 'Dayakougu' and lowland rice varieties 'Yuelianggu', 'Nipponbare', 'LTH' were planted in sterilized soil. The soil was irrigated with a mixture of mycelia and spores of 7 rhizosphere soil fungi (Periconia byssoides, Phoma sp., Phialophora sp., Sporidesmium ovatum, Metarhizium anisopliae, Absidia sp., Paecilomyces sp.) at a rate of 14 mL of the fungal solution each time, and irrigation was carried out once every two weeks. After 20 days of plant growth, the plant heights of upland rice and lowland rice were measured.

[0042] To observe the effect of rhizosphere soil fungi on the blast resistance of lowland rice, the local lowland rice variety LTH, which is susceptible to blast, was selected. The basal soil of LTH lowland rice plants at the three-leaf and one-heart stage was irrigated with a mixture of mycelia and spores of the rhizosphere soil fungus Periconia byssoides (OD 600 = 2.1). Two days after the rice plants grew, the leaves of the rice plants were sprayed with a spore suspension of Magnaporthe oryzae GUY11 (1×10 4 spores / mL). Seven days after the rice plants grew, the disease incidence of the leaves was observed.

[0043] Step4: Extraction of DNA and RNA from rice leaves and cDNA synthesis;

[0044] On the seventh day after inoculating the leaves of lowland rice LTH with Magnaporthe oryzae, leaves with a 1-cm area before and after the lesions were cut off. The leaf DNA was extracted by the CTAB method, and the total leaf RNA was extracted by the Trizol method. The integrity of RNA and DNA was detected by 1% agarose gel electrophoresis. cDNA was synthesized using a reverse transcription kit (TransGen Biotech Co., Ltd., Beijing). The reaction system included 4 μL of total RNA, 1 μL of Random Primer (0.5 μg / μL), 10 μL of 2×ES Reaction Mix, 1 μL RT / RI EnzymeMix, 1 μL of gDNA Remover, and 23 μL of RNase-free water.

[0045] Step5: Detection of gene expression levels by Real-time PCR;

[0046] Detect the expression levels of OsNPR1, OsNOMT and MoPot2 genes in rice leaves. Use primer pairs OsNPR1-rFP / OsNPR1-rRP, OsNOMT-rFP / OsNOMT-rRP, MoPot2-rFP / MoPot2-rRP (Table 2) to amplify OsNPR1, OsNOMT and MoPot2 genes respectively. Use the cDNA of LTH rice leaves as a template to detect the expression of OsNPR1 and OsNOMT genes, and use the DNA of LTH rice leaves as a template to detect the expression of MoPot2 gene of Magnaporthe oryzae in rice leaves. Carry out real-time PCR reaction with ABI QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, USA). The reaction system includes: 0.4 μL of each of the forward primer and reverse primer at 10 μM, 3.5 μL of ddH2O, 0.5 μL of cDNA template or DNA template, 0.2 μL of Universal Passive Reference Dye, 5 μL Green qPCR SuperMix (TransGen Biotech Co., Ltd., Beijing). In the real-time PCR reaction, the actin gene Actin (Os11g0163100) is used as an internal reference gene and amplified with the primer pair OsActin-FP / OsActin-RP. The PCR reaction is carried out under the following conditions: pre-denaturation at 95 °C for 10 s, then denaturation at 95 °C for 45 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min for 40 cycles. Use 2 -ΔΔCt -ΔΔCt method to calculate the relative expression level. Each PCR result has at least 3 biological replicates, and the data are mean ± SD. Use software SPSS version 19.0 (IBM, Inc., Armonk, NY, USA) to analyze the differences in gene expression. P < 0.05 indicates a statistical difference, and P < 0.01 indicates a significant difference.

[0047] Table 2 Gene-specific primer sequences

[0048]

[0049] In the description of this specification, the description referring to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0050] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, according to the content of this specification, many modifications and variations can be made. These embodiments are selected and specifically described in this specification in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A method for preventing and controlling rice blast using Psoralea penetrata, characterized in that: Named as Black Soil Pseudomonas aeruginosa Massarina igniaria )A16, the deposit number is CCTCC NO:M20242411, the deposit date is October 31, 2024, the depositor is China Center for Type Culture Collection (CCTCC), and the deposit address is Wuhan University, Luojia Mountain, Wuchang District, Wuhan City, Hubei Province, China.

2. Use of the Black Soil Pseudomonas aeruginosa as claimed in claim 1 in preventing and controlling rice blast and regulating rice growth.

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