CN101857848B - Biocontrol bacillus subtilis WJ-1 for rice sheath blight, microbial inoculum and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological control of plant diseases, and in particular relates to the isolation and identification of a bacterium used for the biological control of rice sheath blight, a bacterial agent containing the bacterium or a fermentation product thereof, and the prevention and control of rice sheath blight. A biocontrol bacterium-Bacillus subtilis (Bacillus subtilis) WJ-1 strain used for the prevention and treatment of rice sheath blight was obtained by isolation and screening, and was preserved in the China Center for Type Culture Collection with the preservation number: CCTCC NO: M2010069. The invention also discloses the preparation method of the biocontrol bacteria agent and the application of the three bacteria agents of Bacillus subtilis WJ-1 in preventing and treating rice sheath blight. The biocontrol agent of the invention has good biocontrol effects on rice sheath blight and other plant fungal diseases.

Description

Biocontrol Bacillus subtilis WJ-1 of rice sheath blight and its agent and application

technical field

The invention belongs to the technical field of biological control of plant diseases, and in particular relates to the isolation and identification of a bacterium used for the biological control of rice sheath blight, a bacterial agent containing the bacterium or a fermentation product thereof, and the prevention and control of rice sheath blight.

Background technique

Rice sheath blight occurs in all rice areas in the world, and it is more serious in my country mainly in the rice areas of South China and the Yangtze River Basin. In the 1980s, it broke out in a large area in rice planting areas and developed into a kind of rice disease in my country. Main diseases; after the 1990s, the occurrence of the disease was more rampant, and the proportion of the diseased area in southern rice areas generally exceeded more than 50%, becoming the main obstacle of rice production in my country, and the diseased area and damage loss ranked first in rice diseases (Liao Haonian et al., 1997; Meng Qingzhong et al., 2001). According to the statistics of the National Agricultural Technology Extension Service Center, in 2005, rice sheath blight occurred in an area of 248 million mu, and the loss of rice production reached 1.09 million tons, accounting for 7.6% of the loss of major crops in my country due to diseases.

Rice sheath blight is caused by Rhizoctonia solani Kühn of the subphylum Deuteromycetes, Hyphospora, Aspora, and Rhizoctonia genus, and is a stubborn soil-borne disease. Due to the lack of disease-resistant varieties resistant to R. solani, the application of chemical fungicides is still an effective means to control R. solani. Jinggangmycin and Amy are the main pesticide varieties in agricultural production. Jinggangmycin has less environmental pollution, but due to continuous use for more than 30 years, wild strains resistant to Jinggangmycin have been found in the field (Hu Xiurong et al., 2006 ; Xu Wenyao et al., 2007), the large-scale application of Amy has caused serious pollution to the environment, and also increased the probability of drug-resistant mutations of Rhizoctonia solani. Therefore, it is necessary to find new, safe and effective measures to control rice sheath blight.

The use of biocontrol bacteria to prevent and control plant diseases has been reported at home and abroad. So far, four biocontrol agents of Bacillus subtilis have obtained production licenses in the United States; The disease has a good control effect; after retrieval, the applicant retrieved some literature reports and patents about Bacillus subtilis in existing patents and non-patent literature. For example, the patent application No. 03121110.0 discloses "a biological control antagonistic bacteria (BAY-1) and technical application method for preventing postharvest soft rot of fruits and vegetables", using Bacillus subtilis H728 to control tomato gray mold. Patent application No. 98124889.6 discloses "a Bacillus subtilis strain, preparation method, preparation and application", which uses Bacillus subtilis XM16 derived from the surface of apple fruit to prevent and control apple moldy core and branch diseases. The patent application number is 200610200365.4, which discloses "a strain of Bacillus subtilis and its application", which uses Bacillus subtilis M22BH3 to carry out biological control of plant pathogenic fungi such as peach anthracnose, pear black spot, and apple brown rot. The application number is 200410022543, which discloses a method of controlling rice blast by utilizing the Bacillus subtilis Xi-55 strain. In all the patent documents that have been applied for, there is no patent document report related to the subject matter of the present invention.

Contents of the invention

The object of the invention is to address the defects of the prior art, isolate and screen a bacterial strain that has a significant biological control effect on rice sheath blight caused by rice sheath blight (Rhizoctonia solani); it is prepared as a biocontrol agent, Used to control field rice sheath blight. In this way, the damage of chemical agents to the environment and the impact on human health can be weakened, and at the same time, the emergence of drug-resistant strains of Rhizoctonia solani can be delayed, so as to achieve the harmonious and sustainable development of agriculture, human beings and the environment.

The present invention is realized through the following technical solutions:

The present invention obtains a biocontrol bacterium Bacillus subtilis (Bacillus subtilis) WJ-1 suitable for rice sheath blight prevention and treatment from the vegetable garden soil of Huazhong Agricultural University campus in Wuchang Lion Mountain District, Wuhan City, Hubei Province. The bacterial strain was released in 2010 On March 30, 2010, it was delivered to the China Center for Type Culture Collection (CCTCC) in Wuhan University, Wuhan City, Hubei Province, and its deposit number is CCTCC NO: M2010069.

The applicant has prepared a kind of microbial inoculant with control effect on rice sheath blight by fermentation method, and its steps are as follows:

(1) First activate the strain of Bacillus subtilis WJ-1 with the preservation number CCTCC NO: M2010069 on a PDA plate at 33°C for 1 day, then inoculate a single colony into a conical flask containing 200ml PDB, and inoculate it at 180r/min , shaking culture at 33°C for 48 hours to obtain a fermentation broth A of Bacillus subtilis WJ-1 with a concentration of 1.5×10 ¹⁰ cfu/ml;

(2) Centrifuge the fermentation broth A at 4°C and 6,000r/min for 20min, take the supernatant after centrifugation, and filter the supernatant with a bacterial filter with a pore size of 0.22 μm to obtain the following: Fermentation broth B containing Bacillus subtilis WJ-1, the precipitated thallus obtained is for subsequent use;

(3) Wash the precipitated bacteria three times with sterile water, and then suspend them with sterile water, so that the concentration of Bacillus subtilis WJ-1 reaches 1.5×10 ¹⁰ cfu/ml to obtain bacterial solution C;

(4) Add Tween-20 to the fermentation liquid A or fermentation liquid B or bacterial liquid C respectively, so that the final concentration of Tween-20 in the fermentation liquid A or fermentation liquid B or bacterial liquid C is 0.025 %, that is, the Bacillus subtilis WJ-1 fermentation broth A with a cell concentration of 1.5×10 ¹⁰ cfu/ml; the fermentation broth B without Bacillus subtilis WJ-1; the cell concentration of 1.5×10 ¹⁰ cfu/ml ml of bacterial solution C containing Bacillus subtilis WJ-1;

in

The components and formula of the PDA medium are as follows: 200g of potatoes, 20g of glucose, 10g of agar, supplemented with distilled water to 1000ml, adjusted the pH to 7.0, and sterilized at 121° C. for 30 minutes;

The components and formula of the PDB medium are as follows: 200 g of potatoes, 20 g of glucose, added distilled water to 1000 ml, adjusted the pH to 7.0, and sterilized at 121° C. for 30 minutes.

The applicant has carried out a biological control test on the rice sheath blight under the conditions of in vitro, potted and field cultivation by using the above-mentioned microbial agent, and achieved the desired effect, thereby completing the task of the present invention.

Compared with prior art, positive effect of the present invention is:

1. Bacillus subtilis WJ-1 of the present invention has obvious preventive effect on rice sheath blight, the research of embodiment shows, biocontrol agent of the present invention can reach 96.17% to the control effect of rice sheath blight in pot test. %, in the field test, the control effect on rice sheath blight is equivalent to the control effect of commercial agents Amy and Jinggangmycin;

2. The Bacillus subtilis WJ-1 inoculum provided by the present invention and the method of use are very simple in production and use, and have the advantage of being safe for humans and animals as biological pesticides.

For a more detailed technical solution, refer to the content of "Specific Implementation Modes".

Description of drawings

Sequence listing SEQ ID NO: 1 is the 16S rDNA gene sequence of Bacillus subtilis WJ-1.

Fig. 1. is that Bacillus subtilis WJ-1 of the present invention separates and screens to rice sheath blight bacterium (A), rape sclerotinia bacterium (B), soybean botrytis cinerea (C), cotton fusarium wilt bacterium (D), wheat red Antagonism of important phytopathogenic fungi such as Mildew (E) and Alternaria tabacum (F).

Fig. 2 is the inhibitory effect of different concentrations of Bacillus subtilis WJ-1 fermentation liquid B of the present invention on rice sheath blight mycelia growth. In the picture:

A: PDA medium with 15% fermentation broth B; B: PDA medium with 13% fermentation broth B;

C: PDA medium with 9% fermentation broth B; C: PDA medium with 5% fermentation broth B;

E: PDA medium added with 1% fermentation broth B; F: PDA medium control;

Fig. 3 shows the destructive effect of Bacillus subtilis WJ-1 fermentation liquid B isolated and screened by the present invention on rice sheath blight mycelium.

Figure 4 is the in vitro control effect of Bacillus subtilis WJ-1 bacterial agent on rice sheath blight. In the picture:

A sprays the bacterial agent first, and then inoculates the rice sheath blight strain WH-1 after 12 hours; B sprays the bacterial agent first, and then inoculates the rice sheath blight bacterial strain WH-1 after 24 hours;

C is inoculated with the rice sheath blight bacteria strain WH-1 first, and then sprays the bacterial agent after 12 hours; C is inoculated with the rice sheath blight bacteria WH-1 strain first, and then sprays the bacterial agent after 24 hours;

Fig. 5 is the living pot test control effect of the isolated and screened Bacillus subtilis WJ-1 microbial agent of the present invention on rice sheath blight.

In the figure: A is the clean water control; B is the fermentation broth A (the concentration of the bacteria is 1.5×10 ¹⁰ cfu/ml); C is the fermentation broth C (the concentration of the bacteria is 1.5×10 ¹⁰ cfu/ml); D is the fermentation Solution B; E is Jinggangmycin; F is Amy.

Detailed ways

In the following examples, the applicant has studied the morphological characteristics, physiological and biochemical characteristics and genetics characteristics of the Bacillus subtilis WJ-1 bacterial strain, has verified that the WJ-1 bacterial strain is effective to rice sheath blight (Rizoctonia solani), rapeseed fungus The antagonism of important plant pathogenic fungi such as Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium oxysporum, Gibberella zeae and Alternaria alternata, etc. The strain was fermented and cultivated, and the prepared biocontrol agent was tested in vitro, potted and field control of rice sheath blight, and its control effect on rice sheath blight was confirmed.

Example 1: Isolation and identification of Bacillus subtilis WJ-1 and research on its bacteriological characteristics

1. Isolation and identification of Bacillus subtilis WJ-1

A biocontrol bacterium Bacillus subtilis (Bacillus subtilis) WJ-1 suitable for the prevention and treatment of rice sheath blight of the present invention was isolated and screened on the vegetable garden soil of Huazhong Agricultural University campus in Shizishan District, Wuchang, Wuhan City, Hubei Province. Its separation and identification methods are carried out according to the method reported (Edited by Zhou Deqing, "Microbiology Experimental Technology Manual", Shanghai Science and Technology Press, 1986 edition), which is a routine microbial isolation and identification method. The bacterial strain has been submitted to the Chinese Type Culture Collection Center (CCTCC) located in Wuhan University, Wuhan City, Hubei Province for patent procedure preservation on March 30, 2010, and its preservation number is CCTCC NO: M2010069.

2. Mycological characteristics of Bacillus subtilis WJ-1

(1) Morphology of Bacillus subtilis WJ-1

Bacillus subtilis WJ-1 in the potato dextrose agar (PDA) medium (recipe: potato 200g, glucose 20g, agar 10g, add distilled water to 1000ml, adjust the pH to 7.0, and sterilize at 121°C for 30min) The colonies are white, opaque, and finally gradually turn beige. The bacterium is rod-shaped, the size is 0.7-0.8×2-3μm, and the flagella are perinatal. Colony edges are not smooth.

The preservation of the bacterial strain is carried out according to the method introduced in "Microbiology Experimental Technology Manual", Shanghai Science and Technology Press, 1986 edition, edited by Zhou Deqing.

(2) Genetic characteristics of Bacillus subtilis WJ-1 strain

The WJ-1 strain was cultured overnight at 33°C at 180r/min, and 1ul was used as a template to amplify the 16S rDNA fragment by PCR. The general primers and standard method (seeing: Weisburg et al., 1991) that primer relates to and amplification condition with reference to bacterial 16S rDNA, the DNA sequence of described primer is as follows:

Forward primer: 5′ACG GTT ACC TTG TTA CGA CT 3′

Reverse primer: 5′CCT GAG CCA GGA TCA AAC TCT 3′

Sequence analysis showed that the 16S rDNA amplified fragment was 1513bp in length, and its sequence was shown in SEQ ID NO: 1 in the sequence table. BLAST analysis was carried out on the NCBI website (http://www.ncbi.nlm.nih.gov), and it can be known that its homology with the corresponding sequence of Bacillus subtilis C7-2 strain is 99%.

(3) Physiological and biochemical characteristics of Bacillus subtilis WJ-1 strain

The suitable growth temperature of WJ-1 strain is 33℃, and the suitable pH value of the growth environment is 7.0. Can utilize carbon sources such as glucose, sucrose, maltose, sorbitol, starch, L- arabinose and inositol; can utilize nitrogen sources such as yeast extract, beef extract and yeast powder; biochemical tests such as gelatin liquefaction and starch hydrolysis are identified as positive , positive for Gram staining, positive for VP assay, positive for catalase assay, positive for glucose oxidative fermentation assay, positive for nitrate reduction reaction, positive for malonate reaction, positive for citrate utilization , When the NaCl concentration exceeds 10%, it cannot grow, the indole production is negative, the oxidase assay is negative, the methyl red (MR) assay is negative, and the phenylalanine deaminase reaction is negative.

(4) Other characteristics of Bacillus subtilis WJ-1: WJ-1 strain is effective against rice sheath blight (Rizoctonia solani), rape sclerotiorum (Sclerotinia sclerotiorum), soybean gray mold (Botrytis cinerea), cotton wilt ( It has a strong inhibitory effect on important plant pathogenic fungi such as Fusarium oxysporum, Gibberellazeae and Alternaria alternata. It has obvious destructive effect on rice sheath blight hyphae, and the fermented substances produced by it can make rhizoctonia solanum hyphae deform, swell, and internal substances ooze out, as shown in Figure 1, Figure 2 and Figure 3.

Embodiment 2: the preparation of bacillus subtilis WJ-1 bacterial agent

(1) Activation culture of the WJ-1 strain: first activate the WJ-1 strain with the preservation number CCTCC NO: M2010069 on a PDA medium (formulation as described in Example 1) plate at 33°C for 1 day.

(2) Liquid culture of the WJ-1 strain: Inoculate the single colony prepared in step 1 into a 500ml Erlenmeyer flask containing 200ml of PDB (the formula is the same as PDA, except that no agar is added), 180r/min, 33°C shaking culture for 48h, that is It is fermentation broth A, in which the concentration of Bacillus subtilis WJ-1 is 1.5×10 ¹⁰ cfu/ml.

(3) Preparation of WJ-1 bacterial agent: Centrifuge the fermentation broth prepared in step 1 at 6,000r/min for 20min at 4°C, take the centrifuged supernatant, and filter it with a common bacterial filter (pore size 0.22μm) supernatant to obtain a sterile fermentation filtrate (abbreviated as fermentation broth B). The precipitated bacteria were washed 3 times with sterile water, then suspended in sterile water, oscillated and shaken until the concentration reached 1.5×10 ¹⁰ cfu/ml (bacteria solution C for short). Add Tween (Tween)-20 to fermentation liquid A or fermentation liquid B, or bacterial liquid C respectively, so that the final concentration of their respective Tween-20 is 0.025%, that is, the bacterial cell concentration is 1.5×10 ¹⁰ cfu/ ml of fermentation broth A containing Bacillus subtilis WJ-1; fermentation broth B without Bacillus subtilis WJ-1; bacterial broth containing Bacillus subtilis WJ-1 with a cell concentration of 1.5×10 ¹⁰ cfu/ml c.

Embodiment 3: In vitro and indoor living test of bacillus subtilis WJ-1 bacterial agent for controlling rice sheath blight

(1) Indoor in vitro inoculation control test:

The rice sheath blight strain WH-1 of the present invention was activated on a PDA medium plate at 28° C. (method refers to the method reported by Xie et al., 2008). Cut 7.5 cm leaves of rice seedling stage (5-leaf stage), place them in a sterile glass petri dish (diameter 9 cm), put absorbent paper in the petri dish to keep moist, and each dish has 3 leaves. Setup processing includes:

① Spray clean water, fermentation broth A, fermentation broth B and bacteria fluid C until there is a layer of liquid film on the surface of the rice leaves, and after 12 hours, connect the activated 5mm rice rhizoctonia (WH-1) bacteria block to the rice leaves , a distance of about 2cm;

②Spray clear water, fermentation broth A, fermentation broth B, and bacteria fluid C until a layer of liquid film is formed on the surface of rice leaves, and connect activated 5mm R. A distance of about 2cm;

③ Connect the 5mm rice sheath blight (WH-1) block to 2cm from one end of the rice leaf, and spray water, fermentation liquid A, fermentation liquid B and bacterial liquid C after 12 hours until there is a layer of liquid film on the surface of the rice leaf ;

④ Connect the 5mm rice sheath blight bacteria (WH-1) block to 2cm from one end of the rice leaf, and spray water, fermentation liquid A, fermentation liquid B and bacterial liquid C after 24 hours until there is a layer of liquid film on the surface of the rice leaf ;

Five repetitions were set up for each treatment, and after moisturizing for 48 hours, the disease state of rice sheath blight in each treatment was investigated according to the following grading standards for rice sheath blight.

Grading standards for rice sheath blight are as follows:

Level 0: leaf health

Grade 1: 1%-25% leaf disease

Grade 2: 26%-50% leaf disease

Grade 3: 51%-75% leaf disease

Grade 4: More than 76% of the leaves are yellowed and festered

(2) Indoor live inoculation control experiment:

Rice (9311) was sown in plastic pots (40cm×30cm), and then the rice at the seedling stage was transplanted into the plastic pots, and 12 (3 rows×4 columns) plants were transplanted in each pot. During the pregnancy stage of rice, inoculate the sclerotia of Rhizoctonia solani WH-1 strain with uniform size to the stem base of rice, and then wrap the sclerotia with absorbent paper to keep moisture. After 18 hours, spray fermentation liquid A, fermentation liquid B, bacterial liquid C, Jinggangmycin (produced by Tonglu Zhengzhong Biochemical Trade Co., Ltd., Zhejiang Province, 20% soluble powder) and Amy (Hubei Tradewind Crop Protection Co., Ltd.) respectively. production, 300g/L emulsifiable oil) and sterile water until a uniform water film is formed on the surface of rice leaves. There were 12 seedlings in each treatment, and there were 4 replicates in total. Then the rice was kept moist and cultivated for one week, and after one month, the disease state of each treatment rice sheath blight was investigated according to the following grading standards of rice sheath blight.

Grading standards for rice sheath blight:

Grade 0: The whole plant is disease-free;

Grade 1: The fourth leaf and the following leaf sheaths and leaves are infected (the flag leaf is the first leaf);

Grade 3: The third leaf and the following leaf sheaths and leaves are infected (the flag leaf is the first leaf);

Grade 5: The second leaf and its following leaf sheaths and leaves are diseased (the flag leaf is the first leaf);

Grade 7: The blade of the flag leaf and the following leaf sheaths and leaves are infected (the flag leaf is the first leaf);

Grade 9: The whole plant becomes ill and dies prematurely.

According to the disease conditions in step (1) and step (2), calculate the disease index and the relative control effect of Bacillus subtilis WJ-1 on rice sheath blight.

Disease index = ∑ [number of disease grades × number of diseased plants at this level]/(total number of investigations × highest number of disease grades) × 100

Relative control effect (%) = (aseptic water treatment disease index - treatment disease index) / sterile water treatment disease index × 100%

The test results are shown in Table 1, Figure 4 and Figure 5. No matter whether the WJ-1 three bacterial agents are inoculated in vitro or in vivo to control rice sheath blight, they can obtain ideal control effects, and their control effects are comparable to those of traditional commercial preparations " Jinggangmycin" had no significant difference, indicating that the biocontrol agent of the present invention has both preventive and therapeutic effects on rice sheath blight.

Table 1 In vitro control effect of WJ-1 bacterial agent on rice sheath blight

Table 2 Control effect of WJ-1 bacterial agent on rice sheath blight indoor live inoculation

Embodiment 4: the field test of control rice sheath blight with Bacillus subtilis WJ-1 bacterial agent of the present invention

(1) The preparation of the Bacillus subtilis microbial bacterial agent of the present invention (ie fermentation liquid A, fermentation liquid B and bacterial liquid C, the same below) is the same as described in "Summary of the Invention".

(2) The experiment was carried out in two areas at the same time: Songyang Village, Meichuan Town, Wuxue City, Hubei Province, the rice variety was Kaiyou No. 8 (a rice variety publicly promoted); Village, the variety is Jinyou 288 (a rice variety publicly promoted); the growth period is the pregnancy period of rice. The paddy field is set up as a plot, 120m ² / plot. Fermented bacterial liquid A, fermented liquid B, bacterial liquid C, Jinggangmycin (Tonglu Zhengzhong Biochemical Trade Co., Ltd., Zhejiang Province, 20% soluble powder), Amy (Hubei Province Tradewind Crop Protection Co., Ltd., 300g/L EC) and The clean water control has six treatments in total, 12-15ml/m ² , which can be diluted with clean water when spraying. For each treatment, 4 plots are set to repeat, and the 4 plots are randomly distributed. After 20 days, the incidence of rice sheath blight was investigated, and the disease index and relative control effect were calculated as described above.

The field test results are shown in Table 3. WJ-1 three kinds of bacterial agents have achieved high control effects on rice sheath blight, and there is no significant difference between the control effects and the chemical agents Aimi and Jinggangmycin.

Table 3 WJ-1 bacterial agent of the present invention is to rice sheath blight field control effect

references:

1. Liao Haonian, Xiao Lingsheng, Wang Huasheng. A brief analysis of the history and evolution of rice sheath blight. Guangxi Plant Protection, 1997, 3: 35-38.

2. Meng Qingzhong, Liu Zhiheng, Wang Heying, Zhang Shushen, Wei Songhong. Research progress on rice sheath blight. Journal of Shenyang Agricultural University. 2001, 32(5): 376-381.

3. Hu Xiurong, Xu Wenyao, Lu Weicheng. Detection of resistance of rice sheath blight to Jinggangmycin in Fujian Province. China Agricultural Science Bulletin, 2006, 22 (Supplement): 160-162.

4. Xu Wenyao, Lu Weicheng, Hu Xiurong. Biological characteristics of Jinggangmycin-resistant mutants of rice sheath blight. Journal of Fujian Agriculture and Forestry University (Natural Science Edition). 2007, 36(3): 230-233.

5. Editor-in-Chief Zhou Deqing, Microbiology Experimental Technique Manual, Shanghai Science and Technology Press, 1986

6. Weisburg WG, Barns SM, Pelletier DA, Lane HJ. 16S ribosomal DNA amplification for phylogenetic study. JBacteriol. 1991, 173(2): 697-703.

7. Xie J, Fu Y, Jiang D, et al. Intergeneric transfer of ribosomal genes between two fungi. BMC Evolutionary Biology. 2008.8: 87.

sequence listing

<110> Huazhong Agricultural University

<120>Bacillus subtilis WJ-1 and its agent and application for biocontrol of rice sheath blight

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<141>2010-03-29

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<170>PatentIn version 3.1

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<221> gene

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cgagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60

ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa 120

cctgcctgta agactgggat aactccggga aaccggggct aataccggat ggttgtctga 180

accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcggcg 240

cattagctag ttggtggggt aacggctcac caaggcgacg atgcgtagcc gacctgagag 300

ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 360

gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt 420

cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt 480

gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540

gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 600

gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca 660

gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 720

agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 780

aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 840

tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 900

aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960

ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag 1020

gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080

agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1140

tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg aggtcaaatc 1200

atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggcagcg 1260

aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac 1320

tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380

tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt 1440

gaggtaacct tttaggagcc agccgccgaa ggtgggacag atgattgggg tgaagtcgta 1500

acaaggtaac cgt 1513

Claims (2)

1. a strain separation screening has the biocontrol strain of preventive and therapeutic effect to rice sheath blight disease; It is characterized in that; This bacterial strain is subtilis (Bacillus subtilis) WJ-1, and its preserving number is CCTCC NO:M2010069, and its 16S rDNA sequence is shown in sequence table SEQ ID NO:1.

2. what comprise the said biocontrol strain of claim 1 has the microbiobacterial agent of preventive and therapeutic effect to rice sheath blight disease.

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