CN113322210B

CN113322210B - Bacillus Zhangzhou GX-Y5 strain and application thereof

Info

Publication number: CN113322210B
Application number: CN202110757360.6A
Authority: CN
Inventors: 李瑞芳; 任沛东; 彭建玲; 朱桂宁; 杨萌; 贤小勇; 韦善富
Original assignee: Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Current assignee: Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2023-05-02
Anticipated expiration: 2041-07-05
Also published as: CN113322210A

Abstract

The invention discloses a bacillus zhuzhou bacillus GX-Y5 strain and application thereof, wherein the bacillus zhuzhou bacillus (Bacillus zhangzhouensis) GX-Y5 strain is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 22666 at 2021 and 06 months 07. The bacillus zhangzhouzhanghouensis GX-Y5 has very good antagonism on pathogenic bacteria of rice bacterial leaf streak and rice bacterial leaf blight, and the strain and fermentation liquor thereof can be used for preparing biocontrol microbial inoculum, are easy for industrial production, have low production cost and good market prospect, and simultaneously have important industrial application value.

Description

Bacillus Zhangzhou GX-Y5 strain and application thereof

Technical Field

The invention relates to the technical field of microorganism application, in particular to a bacillus Zhangzhou GX-Y5 strain and application thereof.

Background

Bacterial leaf streak of rice is an important bacterial disease of rice, is widely distributed in Asia, and gradually becomes the fourth disease of rice in China. At present, the common cultivated varieties and rice bacterial leaf blight resistant varieties in China are all susceptible to bacterial leaf streak disease of rice. After the rice is infected, the yield is generally reduced by 5% -10% when the rice is ill, and the yield can be reduced by more than 20% when the rice is ill seriously. Bacterial leaf blight of rice is a major bacterial disease of rice, which was first discovered in Kangda county in 1884, and which occurs in rice farming areas such as Asia, africa, america and Australia. The disease is most serious in Asia, especially in south Asia and southeast Asia where rice is planted throughout the year, and most of China occurs. The disease can reduce the yield of rice by 30% -30%, and the yield can be reduced by more than 50% when the disease is serious.

The pathogen of bacterial leaf streak disease of rice is a pathogenic variety of Xanthomonas oryzae (Xanthomolias oryzae pv. Oryzicola, xoc), and the pathogen of bacterial leaf blight of rice is a pathogenic variety of Xanthomonas oryzae (Xanthomonas oryzae pv. Oryzae, xoo). These two pathogenic bacteria are different pathogenic varieties of the xanthomonas oryzae species. At present, in actual production, chemical agents containing organic copper are mainly used for preventing and treating bacterial leaf streaks of rice. However, the use of chemical agents in large amounts can lead to the development of drug resistance of pathogens on the one hand, and residual pesticides on the other hand can destroy the ecological environment and even threaten the food safety. Therefore, microbial-based biocontrol technologies, i.e., the control of plant diseases by killing or depressing the number of pathogenic organisms with beneficial microorganisms, are receiving attention and are considered to be one of the main directions of development for controlling crop bacterial diseases in the future. Therefore, the use of biocontrol bacteria for preventing and treating bacterial leaf streak and bacterial leaf blight of rice is receiving attention.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Disclosure of Invention

In view of the above, the invention provides a bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain which can be used for preventing and treating bacterial leaf spot and bacterial leaf blight of rice.

The aim of the invention is realized by the following technical scheme:

the invention discloses a bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain which is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22666 and the preservation date of 2021, 06 and 07.

The invention provides a fermentation product of the bacillus alzhuzhou (Bacillus zhangzhouensis) GX-Y5 strain.

The invention provides a biocontrol microbial agent containing the bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain and/or a fermentation product thereof.

The invention provides a preparation method of a biocontrol microbial agent containing the bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain and/or a fermentation product thereof, which comprises the following steps:

(1) Seed liquid culture: inoculating the bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain into a liquid culture medium, and fermenting and culturing to obtain seed liquid;

(2) And (3) performing expansion culture: inoculating 1% of seed liquid into a liquid culture medium according to the volume percentage, and fermenting and culturing to obtain fermentation liquor;

(3) Preparing a biocontrol microbial agent: adding a surfactant into the fermentation broth, and uniformly mixing to obtain a biocontrol microbial agent;

wherein, the fermentation culture conditions in the step (1) and the step (2) are as follows: culturing at 26-30 deg.c and 200rpm for 18-22 hr;

wherein the liquid culture medium in the step (1) and the step (2) is NB liquid culture medium; the surfactant in the step (3) is Tween 80, and the addition amount of the surfactant is 1 percent of the addition amount of the surfactant according to the volume percentage of the fermentation liquid.

The invention also provides application of the bacillus zhuzhou (Bacillus zhangzhouensis) GX-Y5 strain or a fermentation product thereof or the biocontrol microbial inoculum in preventing and treating rice bacterial leaf streak and/or rice bacterial leaf blight.

The invention also provides application of the bacillus zhuzhou (Bacillus zhangzhouensis) GX-Y5 strain or a fermentation product thereof in preparing a biocontrol microbial inoculum for preventing and treating rice bacterial leaf spot and/or rice bacterial leaf blight.

The invention also provides an application method of the biocontrol microbial inoculum in preventing rice bacterial leaf streak and/or rice bacterial leaf blight, comprising the following steps: spraying the biocontrol microbial agent on the rice leaves until all the rice leaves are wet; alternatively, the biocontrol agent is infiltrated into the rice leaf using a needleless syringe.

The invention also provides an application method of the biocontrol microbial inoculum in treating rice bacterial leaf streak and/or rice bacterial leaf blight, which is characterized by comprising the following steps: spraying the biocontrol microbial agent on the rice leaves until all the rice leaves are wet; or, injecting the biocontrol microbial agent into the rice leaves in the early stage of the disease occurrence by using a syringe; or, soaking the rice leaves in the early stage of disease occurrence in a biocontrol microbial agent for 20-30 s.

Compared with the prior art, the invention has the following beneficial technical effects:

(1) The invention firstly separates and screens out a new strain bacillus alzhuzhou (Bacillus zhangzhouensis) GX-Y5 which can be used for preventing and treating rice bacterial leaf spot and rice bacterial leaf blight from the natural world, and further widens the strains for preventing and treating rice bacterial leaf spot and rice bacterial leaf blight by utilizing microorganisms.

(2) The bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 has very good antagonism on pathogenic bacteria of bacterial leaf streak and bacterial leaf blight of rice, and the strain and fermentation liquor thereof can be used for preparing biocontrol bacterials, are easy for industrial production, have low production cost, have good market prospect and have important industrial application value.

Description of preservation information

Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 with the preservation number of CGMCC No.22666, the preservation date of 2021, month 06 and 07, the preservation unit of China general microbiological culture Collection center, the preservation address of China center for culture Collection of microorganisms, and the national institute of microbiology, national institute of sciences, national center for sciences, no. 3, beijing, chaoyang.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments, features and aspects of the invention and together with the description, serve to explain the principles of the invention.

FIG. 1 is a phylogenetic tree analysis of the 16S rDNA sequence of Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5;

FIG. 2 is a colony morphology of Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5;

FIG. 3 shows the results of plate antagonism of Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 against pathogenic bacteria of rice bacterial leaf streak;

FIG. 4 shows the results of plate antagonism of Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 against bacterial leaf blight pathogens of rice;

FIG. 5 shows the results of the prevention and treatment of bacterial leaf streak of rice by Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5;

FIG. 6 shows the results of the prevention and treatment of bacterial leaf blight of rice by Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5.

Detailed Description

The invention will now be described in further detail with reference to specific examples, which are intended to illustrate, but not to limit, the invention.

Materials and reagents used in the following examples were obtained commercially, and, unless otherwise specified, experimental methods used in the following examples were conventional.

The NB liquid medium used in the examples of the present invention had the following formulation: 5g/L of polypeptone, 3g/L of beef extract, 1g/L of yeast powder, 10g/L of sucrose and pH 7.0; sterilization conditions: 121 ℃,100kPa,20min.

The formulation of the NA medium used in the examples of the present invention is: 1% agar was added on the basis of NB broth.

Example 1: isolation, screening and identification of bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5

1. Isolation and screening

1.1 sampling: 10 months and 20 days in 2020, guangxi Zhuang nationality nan Ning City, collecting samples from rhizosphere of corn by experimenters Li Ruifang;

1.2 isolation and screening

1.2.1 preparation of a primary screening plate: culturing indicator bacteria Xoc (Xanthomolias oryzae pv.oryzicola, xoc) and Xoo (Xanthomonas oryzae pv.oryzae, xoo) for 20-24h to logarithmic growth phase, respectively adding into melted NA culture medium, mixing thoroughly, pouring into culture dish, solidifying, and making into primary sieve plate;

1.2.2 preliminary screening: taking 1g of a soil sample collected from the rhizosphere of corn, adding 10mL of physiological saline, fully oscillating to form a soil suspension, carrying out gradient dilution, taking 100 mu L of the soil suspension, coating the soil suspension into the primary sieve plate, culturing for 2-3 d at 28 ℃, picking a colony with a bacteriostasis ring, purifying the colony into a pure culture, numbering and preserving the colony for subsequent experiments;

1.2.3 re-screening: performing a re-screening test by adopting a water agar double-layer diffusion method, inoculating the strain subjected to the primary screening in an NB liquid culture medium, culturing for 24 hours at 28 ℃, and adjusting the OD600 to 1.0 by using physiological saline to serve as a bacteria liquid to be tested for later use;

10mL of 1% (W/V) water agar medium is added into a sterile flat plate, after the water agar is solidified, the water agar is placed into a sterilized oxford cup, indicator bacteria Xoc (Xanthomolias oryzae pv. Oryzicola, xoc) and Xoo (Xanthomonas oryzae pv. Oryzae, xoo) are respectively added into the melted NA medium, the mixture is fully and uniformly mixed, and the mixture is poured into a plate, and after the medium is solidified, the oxford cup is taken out by forceps. 40 mu L of the test bacterial liquid is added into the formed hole, the diameter is measured after the test bacterial liquid is subjected to static culture for 48-72 hours at the temperature of 28 ℃, and the test is repeated three times to obtain an average value. Finally, a strain with the strongest bacteriostasis is obtained, and the number is GX-Y5.

2. Authentication

2.1 Biochemical characterization of strain GX-Y5, table 1 shows the results:

TABLE 1 Biochemical Property test results

Substrate(s)	Detection result	Substrate(s)	Detection result
				Negative control	－	Ammonium gluconate	－
Mannose	－	Urea	－
				Galactose	－	Hydrogen sulfide	－
Maltose	－	Methyl red	－
				D-ribose	W+	Salicylic acid	－
Arabinose (Arabic sugar)	－	Beta-galactoside	+
				Xylose	－	Esculin	+
Lactose and lactose	－	Sorbitol	－
				Fructose	W+	Mannitol (mannitol)	－
Sucrose	W+	Celastrol	－
				Glucose	+	Inositol (inositol)	－
Rhamnose (rhamnose)	－	Adonis amurensis L	－
				Sorbose	－	Ornithine decarboxylase	－
Raffinose	－	Lysine decarboxylase	－
				Nitrate reduction	－	Arginine decarboxylase	－
Simmons citrate	－	Indole compounds	－
				Gluconate salt	－	Oxidase enzyme	－
Malonic acid salt	－	Catalase enzyme	+

In table 1, -: a negative reaction; +: a positive reaction; w+: weak positive response.

2.2 molecular biological characterization of Strain GX-Y5

2.2.1 extraction of DNA of Strain GX-Y5

2.2.2 PCR amplification reaction with the general primers 27F 5 '-AGAGTTTGATCCTGGGCTCAG-3' and 1490R 5'-TACGGCTACCTTGTTACGACTT-3' of bacterial 16S rRNA and total DNA of GX-Y5 as template;

the PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 48℃for 30s, extension at 72℃for 1min for 30 cycles; final extension at 72℃for 10min. The PCR products were checked by 1.5% agarose gel electrophoresis, and after the bands were correct, the PCR products were purified and sequenced.

2.2.3 sequencing results are shown as SEQ ID NO.1, BLAST analysis is carried out on the NCBI website by the SEQ ID NO.1, and the similarity between GX-Y5 and bacillus Zhangzhou is 99.79 percent; the phylogenetic tree was constructed by the MEGA7.0 adjacency method (NJ) for the reference sequence downloaded from GenBank, and the reliability of the phylogenetic tree was estimated by Bootstrap test (Bootstrap test) as shown in FIG. 1, with the number of repetitions being 1,000. Finally, the strain is identified as bacillus Zhangzhou (Bacillus zhangzhouensis), the strain is named as bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5, colony morphological characteristics of the strain are shown in figure 2, 2021 is carried out on the 07 th day, and the strain is sent to the China general microbiological culture Collection center for preservation, and the preservation number is CGMCC No.22666.

EXAMPLE 2 pathogen plate antagonism test of Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 against Rice bacterial leaf streak and Rice bacterial leaf blight

1. Preparing a fermentation liquid:

1.1 preparing NB liquid culture medium;

1.2 seed liquid culture: inoculating the bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain into an NB liquid culture medium, and fermenting and culturing to obtain seed liquid, wherein the fermenting and culturing conditions are as follows: culturing at 28 ℃ at 200rpm for 20 hours;

1.3 expansion culture: inoculating 1% of seed liquid into NB liquid culture medium according to volume percentage, fermenting and culturing to obtain fermentation liquid, wherein the fermentation culture conditions are as follows: culturing at 28 deg.C and 200rpm for 20 hr.

2. Plate antagonism test of bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 on pathogenic bacteria of rice bacterial leaf streak

2.1 test method: firstly, 10mL of 1% (W/V) water agar culture medium is added into a sterile flat plate, a sterilized oxford cup is placed after the water agar culture medium is solidified, indicator bacteria Xoc (Xanthomolias oryzae pv. Oryzicola, xoc) are added into the melted NA culture medium, fully and uniformly mixed, the mixture is poured into the flat plate, and after the NA culture medium is solidified, the oxford cup is taken out by forceps; 40. Mu.L of the above-mentioned fermentation broth was added to the resulting wells, and the diameter was measured after 60 hours of stationary culture at 28℃and the test was repeated three times to obtain an average value.

2.2 test results

As shown in FIG. 3, bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 has obvious antagonism to pathogenic bacteria of rice bacterial leaf streak, and the average diameter of the inhibition zone is 3.35cm. Paddy rice bacterial leaf streak pathogens around Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 cannot grow.

3. Plate antagonism test of bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 on pathogenic bacteria of bacterial leaf blight of rice

3.1 test method: firstly, 10mL of 1% (W/V) water agar culture medium is added into a sterile flat plate, a sterilized oxford cup is placed after the water agar culture medium is solidified, indicator bacteria Xoo (Xanthomonas oryzae pv. Oryzae, xoo) are added into the melted NA culture medium, fully and uniformly mixed, the mixture is poured into the flat plate, and after the NA culture medium is solidified, the oxford cup is taken out by forceps; 40. Mu.L of the above-mentioned fermentation broth was added to the resulting wells, and the diameter was measured after 60 hours of stationary culture at 28℃and the test was repeated three times to obtain an average value.

3.2 test results

As shown in FIG. 4, bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 has obvious antagonism to pathogenic bacteria of bacterial leaf blight of rice, and the average diameter of the inhibition zone is 5.68cm. The pathogenic bacteria of bacterial leaf blight of rice around bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 cannot grow.

EXAMPLE 3 prevention and treatment of bacterial leaf streak in Rice by Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5

1. Preparation of biocontrol microbial agent

Adding tween 80 with volume of 1% into the fermentation broth prepared in example 2, and mixing to obtain biocontrol microbial inoculum with viable count concentration of 10 ⁸ ～10 ⁹ CFU/mL。

2. Prevention of bacterial leaf streak of rice by bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5

2.1 test methods

At 4-7 weeks of age, japanese sunny (Oryza. Sativa L. Spp. Japoni)ca) rice was used as a test material, the prepared biocontrol microbial inoculum was infiltrated into rice leaves by a needle-free syringe, and after 3 hours, a microbial inoculum of Xanthomolias oryzae pv.oryzicola, xoc (viable bacteria concentration 10) was used by a needle-free syringe ⁸ ～10 ⁹ CFU/mL) was osmotically pressed to the treated rice leaves at the same position, 20 leaves were treated in total, and the test was repeated three times with rice leaves not treated with the biocontrol agent as a control group.

Blade observation and measurement of the length of the water spot are respectively collected at the 7d and the 14d, the result is averaged, and the formula is adopted: control effect= (control group lesion length-treatment group lesion length)/control group lesion length x 100%, control effect is calculated.

2.2 test results

As shown in fig. 5, the lengths of lesions caused on the treated leaves were significantly shortened after the treatment at 7d and 14d, compared with the control group, and the control effects of the control group were 43.5% and 40.0%, respectively.

3. Treatment of bacterial leaf streak of rice by bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5

3.1 test methods

Using Nippon rice of 4-7 weeks old as test material, and culturing pathogenic bacteria Xanthomolias oryzae pv. Oryzicola, xoc (viable bacteria concentration of 10) ⁸ ～10 ⁹ CFU/mL) was infiltrated into rice leaves with a needleless syringe, and after 3 hours, the prepared biocontrol microbial inoculum was infiltrated into the positions treated with the pathogenic bacteria liquid with a needleless syringe, 20 leaves were treated in total, the results were averaged, and the test was repeated three times.

Taking rice leaves which are not treated by the biocontrol microbial agent as a control group; blade observations were collected at 7d and 14d, respectively, and the length of the water spot was measured according to the formula: control effect= (control group lesion length-treatment group lesion length)/control group lesion length x 100%, control effect is calculated.

3.2 test results

As shown in fig. 5, 7d and 14d after treatment, the length of the lesions induced on the leaves of the treatment was significantly shorter than that of the control group, the effects of the treatment groups were 30.6% and 38.3% respectively,

EXAMPLE 4 prevention and treatment of bacterial leaf blight of Rice by Bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5

1. Prevention of bacterial leaf blight of rice by bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5

1.1 test methods

Dipping the biocontrol microbial inoculum prepared in example 3 with sterile scissors by taking Japanese sunny rice with the age of 4-7 weeks as a test material, cutting off the biocontrol microbial inoculum at a position of 1-2 cm of the tip of the rice, and carrying out 3h treatment on a bacterial solution (the living bacterial concentration is 10) of pathogenic bacteria Xanthomonas oryzae pv ⁸ ～10 ⁹ CFU/mL) was inoculated to the wound of the treated rice leaf, 20 leaves were treated in total, and the test was repeated three times with the rice leaf not treated with the biocontrol agent as a control group.

Leaf observation and measurement of the length of the yellow spot were collected at 7d and 14d, respectively, and the results were averaged and according to the formula: control effect= (control group lesion length-treatment group lesion length)/control group lesion length x 100%, control effect is calculated.

1.2 test results

As shown in fig. 6, the lengths of lesions caused on the treated leaves were significantly shortened after the treatment at 7d and 14d, compared with the control group, and the control effects of the control group were 93.5% and 51.9%, respectively.

2. Treatment of bacterial leaf blight of rice by bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5

2.1 test methods

The bacterial solution of the pathogenic bacteria Xanthomonas oryzae pv. Oryzae, xoo (living bacterial concentration 10) is dipped in sterile scissors by using the Japanese sunny rice of 4-7 weeks old as a test material ⁸ ～10 ⁹ CFU/mL), cutting at 1-2 cm of the rice leaf tip, soaking the rice leaf wound for 25s by using the biocontrol microbial agent prepared in the example 3 after 3 hours, treating 20 leaves in total, and repeating the test three times by using the rice leaf which is not treated by the biocontrol microbial agent as a control.

2.2 test results

As shown in fig. 6, the lengths of lesions induced on the leaves of the treatment were significantly shorter after treatment at 7d and 14d compared to the control group, and the effects of the treatment groups were 83.3% and 40.5%, respectively.

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Sequence listing

<110> Guangxi Zhuang nationality academy of agriculture science

<120> a Bacillus Zhangzhou GX-Y5 strain and use thereof

<130> JC

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1446

<212> DNA

<213> Bacillus zhangzhouensis

<400> 1

gtggggggtg ctataatgca gtcgagcgga cagaagggag cttgctcccg gatgttagcg 60

gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac 120

cggagctaat accggatagt tccttgaacc gcatggttca aggatgaaag acggtttcgg 180

ctgtcactta cagatggacc cgcggcgcat tagctagttg gtggggtaat ggctcaccaa 240

ggcgacgatg cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc 300

cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag 360

caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca 420

agtgcgagag taactgctcg caccttgacg gtacctaacc agaaagccac ggctaactac 480

gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa 540

gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc 600

attggaaact gggaaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga 660

aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac 720

gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 780

aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa 840

gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg 900

cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg 960

acatcctctg acaaccctag agatagggct ttcccttcgg ggacagagtg acaggtggtg 1020

catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080

cttgatctta gttgccagca tttagttggg cactctaagg tgactgccgg tgacaaaccg 1140

gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc 1200

tacaatggac agaacaaagg gctgcaagac cgcaaggttt agccaatccc ataaatctgt 1260

tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc 1320

ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac 1380

gagagtttgc aacacccgaa gtcggtgagg taacctttat ggagccagcc gccgaaggtg 1440

ggcaaa 1446

Claims

1. A bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain is characterized in that the strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 22666 and the preservation date of 2021, 06 and 07.

2. The fermentation broth of bacillus Zhuzhou (Bacillus zhangzhouensis) GX-Y5 strain of claim 1, wherein the preparation method of the fermentation broth comprises the following steps:

(1) Preparing NB liquid culture medium;

(2) Seed liquid culture: inoculating the bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain into an NB liquid culture medium, and fermenting and culturing to obtain seed liquid, wherein the fermenting and culturing conditions are as follows: culturing at 28 ℃ at 200rpm for 20 hours;

(3) And (3) performing expansion culture: inoculating 1% of seed liquid into NB liquid culture medium according to volume percentage, fermenting and culturing to obtain fermentation liquid, wherein the fermentation culture conditions are as follows: culturing at 28 deg.C and 200rpm for 20 hr.

3. A biocontrol microbial agent comprising the fermentation broth of bacillus alzhuz (Bacillus zhangzhouensis) GX-Y5 strain and/or bacillus alzhuz (Bacillus zhangzhouensis) GX-Y5 strain of claim 1, wherein the preparation method of the fermentation broth comprises the following steps:

(1) Preparing NB liquid culture medium;

4. The method for preparing the biocontrol microbial agent as claimed in claim 3, which is characterized by comprising the following steps:

adding tween 80 with volume of 1% into the fermentation broth prepared in claim 3, and mixing to obtain biocontrol microbial inoculum with viable count concentration of 10 ⁸ ～10 ⁹ CFU/mL。

5. Use of bacillus Zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain according to claim 1 or the fermentation broth according to claim 2 or the biocontrol microbial inoculum according to claim 3 for controlling bacterial leaf streak and/or bacterial leaf blight of rice.

6. Use of bacillus zhangzhou (Bacillus zhangzhouensis) GX-Y5 strain according to claim 1 or the fermentation broth according to claim 2 for preparing biocontrol microbial inoculum for preventing and treating rice bacterial leaf spot and/or rice bacterial leaf blight.

7. The application method of the biocontrol microbial inoculum prepared by the preparation method of claim 4 in preventing bacterial leaf streak and/or bacterial leaf blight of rice, which is characterized by comprising the following steps: spraying the biocontrol microbial agent on the rice leaves until all the rice leaves are wet; alternatively, the biocontrol agent is infiltrated into the rice leaf using a needleless syringe.

8. The application method of the biocontrol microbial inoculum prepared by the preparation method of claim 4 in treating rice bacterial leaf streak and/or rice bacterial leaf blight is characterized by comprising the following steps: spraying the biocontrol microbial agent on the rice leaves until all the rice leaves are wet; or, injecting the biocontrol microbial agent into the rice leaves in the early stage of the disease occurrence by using a syringe; or, soaking the rice leaves in the early stage of disease occurrence in a biocontrol microbial agent for 20-30 s.