CN114262673B

CN114262673B - Bacillus belgii and application thereof in preventing and treating crop diseases

Info

Publication number: CN114262673B
Application number: CN202111551155.0A
Authority: CN
Inventors: 孙敏; 刘琨; 朱其立; 李洪顺; 刘巍; 王子浩
Original assignee: Sinochem Agriculture Linyi Research and Development Center Co Ltd
Current assignee: Sinochem Agriculture Linyi Research and Development Center Co Ltd
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2022-09-02
Anticipated expiration: 2041-12-17
Also published as: CN114262673A

Abstract

The invention provides bacillus belgii and application thereof in preventing and treating crop diseases. The Bacillus velezensis is named as WB-2 and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 23508. The Bacillus belgii can be prepared into microbial agents, composite microbial agents, microbial fertilizers and the like, and has a good inhibiting effect on agricultural fungal pathogens such as potato early blight bacteria, rice bakanae bacteria, wheat gibberellic disease bacteria, rice sheath blight bacteria, potato black nevus bacteria, corn northern leaf blight bacteria and the like. But also can be applied to the prevention and the treatment of potato early blight.

Description

Bacillus belgii and application thereof in preventing and treating crop diseases

Technical Field

The invention relates to the technical field of microorganisms, in particular to Bacillus belgii LY149-1 and application thereof in preventing and treating crop diseases.

Background

The potato (Solanum tuberosum L.) is the fourth most important grain crop next to wheat, rice and corn, and plays an irreplaceable role in guaranteeing national grain safety, planting industry structure adjustment, agricultural industry transformation and upgrade and the like. At present, due to the continuous expansion of the potato planting area, the continuous cropping for many years, the unreasonable long-term continuous cropping mode of a single variety, the insufficient water supply condition and the like, the problem of the potato early blight disease is increasingly serious, and great economic loss is caused.

In agricultural production, aiming at the problem of potato early blight, the potato early blight is mainly prevented and controlled by means of cultivating disease-resistant varieties, using chemical agents, improving agricultural cultivation measures and the like, but the effect is not ideal, and the defects are more. Research shows that the compatibility of the microbial fertilizer and the soil environment is good, the crop disease index can be reduced, and the effect of high quality and high yield can be achieved. Bacillus belgii is a gram-positive bacterium, can produce various antibacterial lipopeptide substances in the growth and colonization process of crops, such as Fengycin (Fengycin), iturin (iturin), surfactin (surfactin) and the like, and has strong inhibition effect on the growth of pathogenic bacteria of agricultural fungi. It is reported that Bacillus velezensis can effectively prevent plant fungal diseases caused by ascomycetes and has good biological control effect on plant diseases caused by phytophthora and bacteria. By screening and enriching the high-quality strain resource library, the method has positive effects on the yield and quality of crops and the soil micro-ecological environment. With the rapid development of biotechnology and the continuous deepening and development of agricultural concepts of green, environmental protection, ecology and sustainable development, biological control methods are widely concerned by people.

Further improvement is needed for disease control in potatoes.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Aiming at the increasing severity of the potato early blight disease and the negative influence of a chemical control method on the environment, the invention provides a Bacillus beijerinckii strain, which is applied to the control of the potato early blight disease and shows excellent effect. Therefore, the invention aims to provide a Bacillus subtilis velezensis, a microbial agent, a complex microbial agent, a microbial fertilizer and a method for preventing and treating diseases.

Specifically, the invention provides the following technical scheme:

the invention provides a Bacillus velezensis, which is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 23508. The provided Bacillus belgii is identified as Bacillus belgii (Bacillus velezensis) through screening, separation and identification, such as 16S rDNA and gyrB gene sequence determination, cell gram staining, colony morphology observation and physiological and biochemical characteristic analysis. And is preserved in the China general microbiological culture Collection center. The Bacillus belgii shows an inhibiting effect on various agricultural fungal pathogenic bacteria, such as potato early blight (Alternaria solani), rice bakanae disease (Fusarium moniliforme), wheat gibberellic disease (Fusarium graminearum), rice sheath blight (Rhizoctonia solani), potato black mole (Rhizoctonia solanikuhn) and corn leaf blight (Exserohilum turcicum), and has a good disease control effect.

In a second aspect of the invention there is provided a microbial agent comprising a bacillus belief in the first aspect of the invention.

The third aspect of the invention provides a complex microbial inoculant, which comprises a first microbial inoculant and a second microbial inoculant, wherein the first microbial inoculant is the microbial inoculant provided in any one embodiment of the first aspect of the invention, and the second microbial inoculant comprises at least one selected from bacillus amyloliquefaciens and other bacillus belgii.

In a fourth aspect, the invention provides a microbial fertilizer comprising a bacillus beijerinckii according to the first aspect of the invention, or a microbial agent according to the second aspect of the invention, or a complex microbial agent according to the third aspect of the invention.

In a fifth aspect, the invention provides use of bacillus belgii in preparation of a microbial agent, a complex microbial agent or a microbial fertilizer, wherein the bacillus belgii is the bacillus belgii in the first aspect of the invention.

A sixth aspect of the present invention provides a method for preventing and treating diseases, comprising:

the method comprises the step of applying an effective amount of bacillus belgii, a microbial agent, a composite microbial agent or a microbial fertilizer to plants, wherein the bacillus belgii is the bacillus belgii in the first aspect of the invention, the microbial agent is the microbial agent in the second aspect of the invention, the composite microbial agent is the composite microbial agent in the third aspect of the invention, and the microbial fertilizer is the microbial fertilizer in the fourth aspect of the invention.

Information on the preservation of strains

Bacillus subtilis velezensis with the preservation number of CGMCC No.23508, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is the microorganism research institute of China academy of sciences No. 3 of Xilu No. 1 North Chen in the Chaoyang area of Beijing, and the preservation date is 2021, 09 and 29 days.

Drawings

Fig. 1 is a graph showing the antagonistic effect of bacillus belgii on various agricultural fungi according to an embodiment of the present invention, in which fig. 1 shows a case where a is potato early blight (Alternaria Solani), B is rice bakanae disease (Fusarium moniliforme), C is wheat scab disease (Fusarium graminearum), D is rice sheath blight disease (Rhizoctonia Solani), E is potato black nevus (Rhizoctonia Solani), and F is corn northern leaf blight (exoHircium turcicum).

FIG. 2 shows the amplification results of the genes for synthesizing B.belgii WB-2 lipopeptides according to an embodiment of the present invention, wherein M in FIG. 2 is DL2000 DNA marker, 1 is a positive control strain (LJ), and 2 is B.belgii WB-2.

FIG. 3 is a diagram showing the results of observing the colonies and microscopic morphology of Bacillus belgii WB-2 according to an embodiment of the present invention.

FIG. 4 is a phylogenetic tree of the antagonistic strain WB-2 established against B.belgii based on the 16s rDNA sequence provided in accordance with an embodiment of the present invention.

FIG. 5 is a phylogenetic tree of Bacillus beleisi WB-2 established based on the gyrB gene sequences according to an embodiment of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, which are intended to be illustrative and not to be construed as limiting the invention.

Herein, when the content of a certain substance is expressed, the mass of the substance is referred to as a percentage of the total substance weight, unless otherwise specified.

Herein, when referring to "prevention" of a disease, "prevention" includes not only prevention of occurrence of a certain disease but also treatment of a certain disease, thereby alleviating, reducing, alleviating symptoms of the disease.

The invention provides a Bacillus velezensis, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 23508. The strain is selected from rhizosphere soil of potato plants, the soil depth is 0-20cm, the strain is identified as Bacillus velezensis through colony morphology observation, cell gram staining, physiological and biochemical characteristic detection and 16S rDNA and gyrB gene sequence determination analysis, and the Bacillus velezensis WB-2 provided by the invention is used as a microbial inoculum product and has better inhibiting effect on agricultural fungal pathogens such as potato early blight bacteria, rice seedling blight bacteria, wheat gibberellic disease bacteria, rice sheath blight bacteria, potato black nevus bacteria, corn northern leaf blight bacteria and the like. In addition, the WB-2 metabolite can amplify the synthetic genes of the antibacterial lipopeptide substances fenD, ituC, srfAA and srfAB. The Bacillus belgii WB-2 provided by the invention is used as a microbial agent product, is applied to a potato potting test in a spraying, irrigating and other modes, can respectively reduce the disease indexes of early blight of potato plants by 50.65 and 7.99, and has a relative prevention effect of 60.93%. The Bacillus belgii WB-2 provided by the invention is used as a green multi-effect functional strain, is compounded with Bacillus amyloliquefaciens and other Bacillus belgii, and is added into a compound fertilizer, so that the incidence rates of potato early blight are respectively reduced by 53.33 and 19.44; the relative control effect is respectively improved by 76.19 percent and 27.78 percent; the yield is respectively increased by 18.69 percent and 12.19 percent, and the content of the soluble starch is respectively increased by 3.5 percent and 26 percent. Moreover, the compound has certain inhibiting effect on 6 agricultural fungal pathogenic bacteria such as potato early blight (Alternaria solani), rice bakanae disease (Fusarium moniliforme), wheat gibberellic disease (Fusarium graminearum), rice sheath blight (Rhizoctonia solani), potato black nevus (Rhizoctonia solanikuhn) and corn northern leaf blight (Exserohilum turcicum), the plate inhibition rates are respectively 87%, 65.19%, 59.44%, 57.22%, 66.30% and 63.89%, and synthetic genes of antibacterial lipopeptide substances fenD, ituC, srfAA and srfAB can be amplified in the metabolite of the strain WB-2. The provided Bacillus belgii WB-2 can be developed into a biological control strain resource for early blight of potatoes and applied to actual agricultural production practice.

According to a specific embodiment of the invention, the Bacillus belgii has the 16S rDNA sequence shown in SEQ ID NO. 1. According to a specific embodiment of the present invention, the Bacillus belgii has the gyrB gene sequence shown in SEQ ID NO. 2.

The invention also provides a microbial agent, which comprises the bacillus belgii. In at least some embodiments of the present invention, the microbial inoculant is in the form of a dry powder comprising at least 100 hundred million CFU of live bacillus belgii per gram of microbial inoculant. According to the specific embodiment of the invention, the provided microbial agent has obvious disease-resistant and growth-promoting effects on potato early blight, and the specific effects are as follows: compared with a blank control group CK and a market microbial inoculum, the incidence rate of the potato early blight pot culture is respectively reduced by 50.65 percent and 7.99 percent, and the relative prevention effect reaches 60.93 percent.

According to embodiments of the present invention, the provided microbial agents may be obtained by fermentation. For example, the bacterial powder is prepared by fermenting, freezing and drying a WB-2 strain of Bacillus belgii to obtain bacterial powder, wherein the viable count content of the Bacillus belgii bacterial powder preparation is not less than 1.3 multiplied by 10 ¹¹ Ten thousand per gram. According to a particular embodiment of the invention, the microbial inoculum is obtained by: for said Bacillus beilesensisPerforming fermentation culture to obtain a fermentation product; and carrying out spray drying and crushing treatment on the basis of the fermentation product so as to obtain the microbial agent. According to a particular embodiment of the invention, the fermentation culture comprises: performing activated fermentation culture on the bacillus beleisis so as to obtain a zymocyte liquid; and carrying out amplification fermentation culture on the zymophyte liquid so as to obtain a fermentation product.

According to a specific embodiment of the present invention, the medium for the amplified fermentation culture includes corn starch, soybean meal, yeast powder, sucrose, peptone, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, calcium carbonate, and an antifoaming agent. For example, in at least some embodiments, the medium used for the scale-up fermentation culture comprises, in parts by weight: 2-5 parts of corn starch, 3-5 parts of soybean meal, 0.1-0.3 part of yeast powder, 0.5-2 parts of cane sugar, 0.1-0.3 part of peptone, 0.1-0.3 part of dipotassium hydrogen phosphate, 0.2-0.5 part of potassium dihydrogen phosphate, 0.1-0.3 part of sodium chloride, 0.05-0.2 part of calcium carbonate and 0.2-0.5 part of defoaming agent. The pH of the amplified fermentation medium can be controlled to be about 7. The size of the fermentation tank for the amplified fermentation culture can be 10L, 50L or 500L; the fermentation tanks with different volumes are used in a step-by-step enlarged mode. Drying and crushing the fermentation product to obtain the corresponding microbial agent. In the case of liquid fermentation culture, the medium used may be LB medium. For example, the fermentation broth can be obtained by culturing at 37 ℃ for 4 to 8 hours.

The Bacillus belgii can be prepared into a microbial agent, can be used as a single microbial agent, and can be compounded with other microbial agents to prepare a composite microbial agent. Therefore, the invention also provides a complex microbial inoculant which comprises a first microbial inoculant and a second microbial inoculant, wherein the first microbial inoculant is the microbial inoculant. The second microbial agent is not particularly required, and the second microbial agent can be used for preventing and treating plant diseases and insect pests or diseases. The second microbial agent comprises at least one selected from a bacillus amyloliquefaciens agent and other bacillus belgii agents. According to a specific embodiment of the invention, the complex microbial inoculant comprises 30-35 parts by weight of the first microbial inoculant and at least one of the following components: 30-35 parts by weight of a bacillus amyloliquefaciens microbial agent; 30-35 parts of other Bacillus belgii inoculants. The mentioned bacillus amyloliquefaciens microbial inoculum refers to a microbial inoculum containing bacillus amyloliquefaciens, and the mentioned other bacillus belgii microbial inoculants refer to microbial inoculants containing other bacillus belgii. The mentioned Bacillus amyloliquefaciens or other Bacillus belgii can be obtained by self-screening or purchase, and can be obtained by fermentation culture, drying and crushing. Of course, the bacillus amyloliquefaciens or other bacillus belgii agents can also be directly purchased and obtained.

According to the specific embodiment of the invention, the weight ratio of the bacillus belgii to the bacillus amyloliquefaciens to the other bacillus belgii in the composite microbial agent is 1-3: 1 to 3. In at least one specific embodiment, the weight ratio of the bacillus belgii, the bacillus amyloliquefaciens and other bacillus belgii in the composite microbial agent is 1: 1. other Bacillus belgii species mentioned herein may be different from the Bacillus belgii species provided by the present invention.

According to the specific embodiment of the invention, the other mentioned Bacillus belgii inoculum can be obtained by fermenting and culturing LJ (the strain preservation number is CGMCC No.21827, the strain is described in Chinese patent application text with the application number of 202111115791.9) or obtained by fermenting Bacillus belgii 45 #.

According to the specific embodiment of the invention, the effective viable count of the bacillus belvesii in each gram of the composite microbial inoculum is at least 100 hundred million CFU, and at least one of the following bacteria is selected from the group consisting of: the effective viable count of the bacillus amyloliquefaciens is at least 100 hundred million CFU; the number of viable bacteria of other Bacillus belgii bacteria is at least 100 hundred million CFU. CFU is generally explained in the art as a colony forming unit.

The provided strain, or microbial agent or composite microbial agent can be used alone or added into a fertilizer for use. The invention also provides a microbial fertilizer which comprises the bacillus beilesensis, the microbial agent or the composite microbial agent. According to the specific embodiment of the invention, the provided microbial fertilizer has the effects of preventing diseases and promoting growth, and has the following specific effects: compared with the conventional fertilization and the market microbial inoculum, the incidence rates of the potato early blight are respectively reduced by 53.33 and 19.44; the relative control effect is respectively improved by 76.19 percent and 27.78 percent; the yield is respectively increased by 18.69 percent and 12.19 percent, and the soluble starch is respectively increased by 3.5 percent and 26 percent. The microbial fertilizer compounded by the bacterial strain has the functions of promoting the growth of crops, improving the yield of the crops and preventing and treating the potato early blight.

According to an embodiment of the invention, the microbial fertilizer contains at least 0.5 hundred million CFU of effective viable microorganism per gram of microbial fertilizer.

According to a specific embodiment of the invention, the microbial fertilizer contains one to five thousandths of the bacillus belgii, or one to five thousandths of the microbial agent, or one to five thousandths of the complex microbial agent. For example, it may be one-thousandth, two-thousandth, three-thousandth, four-thousandth, or five-thousandth.

According to a specific embodiment of the present invention, the microbial fertilizer further comprises a base fertilizer selected from at least one of a compound fertilizer and an organic-inorganic fertilizer. According to a specific embodiment of the invention, the mentioned compound fertilizer is 12-18-15/S.

The invention also provides application of the bacillus beiLeisi in preparation of microbial agents, complex microbial agents or microbial fertilizers.

The invention also provides a method for preventing and treating diseases, which comprises the following steps: the method comprises the step of applying an effective amount of Bacillus belgii, a microbial agent, a composite microbial agent or a microbial fertilizer to plants, wherein the Bacillus belgii is the Bacillus belgii, the microbial agent is the microbial agent, the composite microbial agent is the composite microbial agent, and the microbial fertilizer is the microbial fertilizer. According to an embodiment of the present invention, the mentioned diseases include, but are not limited to, diseases caused by potato early blight, rice bakanae disease, wheat scab, rice sheath blight, potato black nevus or rice blotch. The mentioned plants include but are not limited to potato, rice, wheat, etc.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 isolation and selection of Bacillus belgii

The Bacillus belgii is collected from soil around the rhizosphere of a potato early blight disease strain and is obtained by separation through a dilution coating flat plate method and a flat plate marking method, and the method comprises the following steps:

(1) and (3) separating soil flora: selecting rhizosphere soil of the early blight disease strain of the potato in a potato planting area of Mei Port street in Shandong Linyi City of Shandong province for screening. The method comprises the following specific steps: and collecting 10 parts of soil sample at the position of 0-10 cm away from the ground surface of the rhizosphere of the early blight disease strain by adopting a five-point sampling method. Each part of soil sample weighs 20g, and is put into a sampling bag, numbered and stored at 4 ℃. Respectively weighing 10g of soil sample, placing into 90mL (250mL) of sterile water, and performing shaking culture at 28 ℃ and 180r/min for 30min to obtain soil suspension. It is diluted to 1 × 10 by gradient dilution coating plate method ^-3 、1×10 ^-4 、1×10 ^-5 Each gradient is provided with 3 parallels, the parallels are placed at 28 ℃ for cultivation for 2d, and then single colonies of 7 strains with different forms, sizes and colors are selected to be subjected to streak purification cultivation on an LB solid culture medium, and the numbers are as follows: WB-1, WB-2, WB-3, WK-1, WK-2, WK-3 and WK-4, the growth of colonies is observed regularly, and the colonies are stored at 4 ℃ for later use.

(2) Screening of potato early blight antagonistic strain

Primary screening: using the five-point confrontation method, bacterial cakes with a length of d of 0.5cm were respectively punched on the edges of the plates of potato early blight (Alternaria solani), rice bakanae disease (Fusarium moniliforme), wheat scab disease (Fusarium graminearum), rice sheath blight disease (Rhizoctonia solani), potato black mole disease (Rhizoctonia solanikuhn) and maize northern leaf blight (Exserohilum turcicum) by a sterilization punch, and then transferred to the center of a new PDA plate. Meanwhile, the 7 strains screened in the step 1 are respectively and equidistantly point-connected on the periphery of the panel by using a sterilizing toothpick, a PDA panel only inoculated with corresponding pathogenic bacteria cakes is taken as a control group (CK), the panel is placed in a constant temperature incubator at 25 ℃ for culture, and the inhibition process and degree of the strains on the pathogenic bacteria are observed and recorded until the pathogenic bacteria in the CK grow over the whole PDA panel.

Re-screening: and repeating the steps for re-screening to obtain the strains with better antagonistic effect on the 6 agricultural fungal pathogens, and determining the antagonistic rate by adopting a cross method.

Finally, the number WB-2 is determined as the target antagonistic strain, the antagonistic effect graph is shown in figure 1, and the bacteriostatic rate is shown in Table 1. Wherein the antagonism rate is calculated by the following formula:

antagonism (%) - (control group pathogen colony diameter-treatment group pathogen colony diameter)/control group pathogen colony diameter × 100

Wherein the control colony radius in the formula refers to the colony radius of corresponding pathogenic bacteria treated by the following PDA culture medium.

Antagonistic ratio of functional strains on pathogenic bacteria of agricultural fungi in Table 17

In addition, PCR amplification was performed on the genes of the WB-2 strain metabolites, and the results showed that synthetic genes of fenD (293bp), ituC (575bp), srfAA (273bp), and srfAB (308bp) could be amplified from the metabolites of the strain, as shown in FIG. 2.

Example 2 identification of Bacillus belgii

Morphological, physiological, biochemical and molecular biological identification are respectively carried out on the Bacillus belgii WB-2. The results are as follows:

(1) morphological characteristics:

antagonistic bacteria WB-2 are pale yellow, regular-edged, nearly round, slightly convex, moist in surface, opaque, gram-positive, and capable of producing spores, as shown in FIG. 3.

(2) Physiological and biochemical characteristics:

the physiological and biochemical characteristics of the antagonistic strain WB-2 were identified by using a bacterial trace biochemical identification tube, which was purchased from Qingdao Haibobo biology Co., Ltd, and the specific operation method was examined by referring to the instruction manual and combining with the method described in Berger's Manual of bacteria identification (eighth edition).

The detection results are shown in the following table 2, and the results show that the test strain WB-2 can utilize various saccharides, alcohols and amino acids, so that the metabolic spectrum of the test strain WB-2 on a carbon source is wide, and the carbon source provides a carbon skeleton for biosynthesis, so that the test strain is beneficial to colonization of the test strain in soil.

TABLE 2 physio-biochemical Properties of antagonistic Strain WB-2

features/Characteristics	WB-2	features/Characteristics	WB-2
				Gram stain (Gram stain)	+	Sorbitol (Sorbitol)	+
Methyl red (Methyl red)	-	Nitrate reduction (Nitrate reduction)	-
				Fructose (Fructose)	+	D-arabinose (D-arabinosine)	+
Glucose (Glucose)	+	Lysozyme (Lysozyme)	+
				Maltose (Maltose)	+	Mannitol (Mannitol)	+
Lactose (Lactose)	+	Starch hydrolysis (Amylohydrolysis)	+

Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

(3) Molecular biological characteristics:

1)16S rDNA gene sequence (shown as SEQ ID NO:1, 1044bp) and phylogenetic analysis

WB-2(MW365328)

GATGCGGGTGCTATAATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTA GCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGG TGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAA CGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGG ACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGG ACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGC TCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCT AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAA GCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGT GAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAG AAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAAC ACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTG GGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAG TGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGG GAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT GGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACTCCTCT GACATCCTAAAGATAGGACTCCCCTTCGGGGGAAAGTGACAGGTGTGCATGGTTGCC TCAACTCCGGGCCTGAG(SEQ ID NO:1)

Through analysis of a strain 16S rDNA evolutionary tree, the similarity of the strain and Bacillus velezensis strain C1(MK788353) reaches 97 percent, and the genetic relationship is the most similar, as shown in FIG. 4.

2) gyrB gene sequence (shown as SEQ ID NO:2, 1193bp) and phylogenetic analysis

WB-2(MW884255)

ATCGTCGCGGAGCGGATATAAGTATCCGGCGGTCTTCACGGTGTAGGGGCGTCTGTCG TAAACGCCTTGTCGACCACTCTTGACGTTACGGTTCATCGTGACGGAAAAATCCACTA TCAGGCGTACGAGCGCGGTGTACCTGTGGCCGATCTTGAAGTGATCGGTGATACTGAT AAGACCGGAACGATTACGCACTTCGTTCCGGATCCGGAAATTTTCAAAGAAACAACC GTATACGACTATGATCTGCTTTCAAACCGTGTCCGGGAATTGGCCTTCCTGACAAAAG GCGTAAACATCACGATTGAAGACAAACGTGAAGGACAAGAACGGAAAAACGAGTAC CACTACGAAGGCGGAATCAAAAGCTATGTTGAGTACTTAAACCGTTCCAAAGAAGTC GTTCATGAAGAGCCGATTTATATCGAAGGCGAGAAAGACGGCATAACGGTTGAAGTTG CATTGCAATACAACGACAGCTATACAAGCAATATTTATTCTTTCACGAATAATATCAACA CATACGAAGGCGGCACGCACGAGGCCGGATTTAAAACCGGTCTGACCCGTGTCATAA ACGACTATGCAAGAAGAAAAGGGATTTTCAAAGAAAATGATCCGAATTTAAGCGGGG ATGATGTGAGAGAAGGGCTGACTGCCATTATTTCAATTAAGCACCCTGATCCGCAATTC GAAGGTCAGACGAAAACGAAGCTCGGCAACTCCGAAGCGAGAACGATCACTGATAC GCTGTTTTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCGGACTCAGCCCGCAAA ATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGCAGCGAAAAAAGCGCG GGAATTGACCCGCCGCAAAAGTGCGCTTGAGATTTCCAATCTGCCGGGCAAACTGGC GGACTGTTCTTCTAAAGATCCGAGCATTTCCGAGCTGTATATCGTAGAGGGTGACTCTG CGGGCGGATCAGCGAAACAGGGACGGGACCGTCATTTCCAAGCCATTCTGCCGCTGC GCGGTAAGATTCTGAACGTTGAGAAAGCCAGACTTGATAAGATTCTCTCAAACAATGA GGTCAGATCAATGATCACGGCCCTCGGAACAGGAATCGGAGAAGATTTTATCTTGAAA AACGCGTTTCTTAAGAGTCTTTGACGAAGCGATGAGGCGTT(SEQ ID NO:2)

Through the analysis of a gyrB gene evolutionary tree of the strain, the similarity of the strain and Bacillus velezensis strain NN95(MT119763.1) reaches 94 percent, and the genetic relationship is the most similar, as shown in FIG. 5.

In conclusion, the antagonistic strain WB-2 was identified as Bacillus velezensis (Bacillus velezensis) by the above analysis of morphological characteristics, physiological and biochemical characteristics and molecular biological identification of the antagonistic strain WB-2. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is the microorganism research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation number is CGMCC No. 23508.

Example 3 verification of prevention and treatment effects of a single disease-preventing microbial agent on potato early blight

Firstly, a microbial agent is prepared according to the following method:

step 1: inoculating the purified and cultured Bacillus belgii WB-2 into a triangular flask containing 100ml (250ml) of LB culture solution, and performing shake culture at 37 ℃ for 6h at 180r/min to obtain a zymocyte solution; meanwhile, purifying, inoculating and culturing industrialized functional strains: LJ (Bacillus belgii), 45# (Bacillus belgii) and 5# (Bacillus subtilis) (LJ, 45#, 5# are all functional strains independently screened by the Linyi research and development center of chemical fertilizer Co., Ltd.).

Step 2: inoculating WB-2 zymocyte liquid into a pilot-scale fermentation tank (10L-50L-500L), performing fermentation culture at 37 ℃, and then sequentially performing spraying, drying and subpackaging, wherein the viable count is measured to be 130 hundred million/g. Mixing with diatomite and other materials, and compounding to obtain the Bacillus beiLeisi WB-2 powder. The formula of the pilot culture medium is as follows: 3.0% of corn starch, 4.0% of soybean meal, 0.15% of yeast powder, 1.0% of sucrose, 0.2% of peptone, 0.2% of dipotassium hydrogen phosphate, 0.3% of potassium dihydrogen phosphate, 0.15% of sodium chloride, 0.1% of calcium carbonate and 0.3% of defoaming agent, and the pH is adjusted to 7.0. The single bacteria and the rest strains in the compound bacteria are operated according to the preparation flow of WB-2 bacteria powder.

The single strain pot culture test is characterized in that 4 treatments are set according to the results of pathogenic bacteria antagonistic data, namely, a control group (CK-1) is not added with a microbial inoculum, a treatment group 1(WB-3), a treatment group T2-1(WB-2) and a treatment group T3-1(WK-3) are respectively added with 4 treatments, each treatment group is 5 treatments in parallel, and the treatments are applied to the potato pot culture test by a spraying method. The results of the single-strain potting are shown in Table 3.

TABLE 3 Single Strain pot effect validation results

Numbering	Index of disease condition	Relative control effect (%)
			Control group (CK)	83.36	-
Treatment group 1(WB-3)	50.23	39.74
			Treatment group 2(WB-2)	40.82	51.03
Treatment group 3(WK-3)	49.27	40.89

As can be seen from Table 3, Bacillus belgii WB-2 showed the best effect. The concrete effects are as follows: compared with CK-1, the disease indexes of the potato plant early blight are respectively reduced by 42.54, and the relative control effect reaches 51.03%.

The microbial fertilizer is prepared by coating and mixing the bacterial powder with better single-strain pot culture verification effect and the special fertilizer for the potatoes. The compound fertilizer acts on potato plants in a base fertilizer mode, and field disease prevention effect verification is carried out. The specific implementation case is as follows: the WB-2 and WK-3 combinations of the single-bacterium formula are respectively coupled with the special fertilizer for potatoes uniformly, and then added according to 2 per mill, so that the microbial fertilizer is obtained through stable production, and the effective viable count is more than or equal to 0.5 hundred million/g. The special fertilizer for the potatoes is 12-18-15/S. The field experiment was conducted in the potato plantation base of Mei Tou street in Hedong region of Linyi city, Shandong province.

The field disease prevention effect verification of single bacterium treatment sets 4 groups of treatments in total, which are respectively as follows: CK1 is conventional planting, T1 is a self-developed transformation production microbial inoculum No. 45 (Bacillus belgii), the addition amount is 1 kg/mu, and the test group (WB-2) and the test group (WK-3) are adopted. The specific test results are as follows:

TABLE 4 functional strains effective viable count of single bacteria shelf life tracking data

TABLE 5 Single Strain field Effect test results

As shown in Table 4, the coupling data tracking of the single bacterium powder and the fertilizer 12-18-15/S shows that the WB-2 effect is the best, and the viable bacteria retention rate at 180 days is 94%. The data result of the field disease prevention effect of single bacterium treatment shows that the WB-2 treatment effect is the best, and compared with CK1 and T1, the incidence of potato early blight of a T2 test group is respectively reduced by 31.73 and 2.01; the relative control effect is respectively improved by 45.33 percent and 2.87 percent; the yield is respectively increased by 7.7 percent and 3.7 percent, and the soluble starch is respectively increased by 2.1 percent and 4.6 percent.

Example 4 verification of prevention and treatment effects of the composite disease-prevention microbial agent on potato early blight

According to the potted plant test effect, strain compounding is carried out, the formula of the compound microbial inoculum is that each bacterium powder is compounded and combined into triple bacteria randomly, the proportion is 1:1:1, and the theoretical living bacteria addition amount of the compound bacteria is 0.5 hundred million/g. The prepared compound microbial agent is used for verifying early blight potato potted plants and field disease prevention effects.

6 groups of treatment are set in the composite microbial inoculum pot culture test, namely a control group (CK) without microbial inoculum, a market microbial inoculum T1 (Zhongnong green kang-potato special microbial inoculum), a test group T3(LJ, WB-2 and 45# compounded) and a test group T5(LJ, WB-2 and 5# compounded), and 5 parallel test groups are set in each group. The test results are shown in Table 6.

Table 6 verification results of potting effect of compound microbial inoculum

Numbering	Treatment of	Index of disease condition	Relative control effect (%)
				CK	-	83.12	-
T1	Market microbial inoculum	40.46	51.35
				T3	LJ+WB-2+45#	32.47	60.93
T5	LJ+WB-2+5#	38.89	53.24

As can be seen from Table 6, test group T3(LJ, WB-2 and 45# formulations) exhibited the best results. The concrete effects are as follows: compared with CK and a test group T1, the disease indexes of the potato plant early blight are respectively reduced by 50.65 and 7.99, and the relative prevention effect reaches 60.93 percent.

4 groups of treatment are set in the field test of the compound microbial inoculum, namely control groups CK1 and T1-1; test groups T3-1, T5-1, each group handling 3 cells, 20m each ² The fertilizer application amount is 50 kg/mu. Wherein CK1 is for conventional planting, T1-1 is a special microbial inoculum of agricultural green kang-potato in the market microbial inoculum, and the addition amount is 1 kg/mu; test groups T3-1 and T5-1 are complex microbial inoculum prepared in complex strain pot experiment. The used bacterial powder is coupled with potato special fertilizer (12-18-15/S) to be used as base fertilizer for treatment. The results of the field experiment data of the compound microbial inoculum treatment are shown in Table 7.

Table 7 field effect verification results of complex microbial inoculum

As can be seen from Table 7, test group T3-1(LJ, WB-2 and 45# combinations) exhibited the best effect compared to CK1 and test group T1-1. The concrete expression is as follows: the incidence rates of the potato early blight of the T3-1 test group are respectively reduced by 53.33 and 19.44; the relative control effect is respectively improved by 76.19 percent and 27.78 percent; the yield is respectively increased by 18.69 percent and 12.19 percent, and the soluble starch is respectively increased by 3.5 percent and 26 percent.

In the description of the specification, references to the description of "one embodiment," "some embodiments," "an example," "a specific example," or "an implementation" or the like mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> research and development center of Linyi agriculture of Zhonghua chemical fertilizer Co

<120> Bacillus belgii and application thereof in preventing and treating crop diseases

<130> BI3212159

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1044

<212> DNA

<213> Bacillus velezensis

<220>

<223> 16S rDNA

<400> 1

gatgcgggtg ctataatgca agtcgagcgg acagatggga gcttgctccc tgatgttagc 60

ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 120

ccggggctaa taccggatgg ttgtctgaac cgcatggttc agacataaaa ggtggcttcg 180

gctaccactt acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca 240

aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300

ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360

gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac 420

aagtgccgtt caaatagggc ggcaccttga cggtacctaa ccagaaagcc acggctaact 480

acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta 540

aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg 600

tcattggaaa ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt 660

gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg 720

acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780

taaacgatga gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt 840

aagcactccg cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc 900

cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct 960

tgactcctct gacatcctaa agataggact ccccttcggg ggaaagtgac aggtgtgcat 1020

ggttgcctca actccgggcc tgag 1044

<210> 2

<211> 1193

<212> DNA

<213> Bacillus velezensis

<220>

<223> gyrB

<400> 2

atcgtcgcgg agcggatata agtatccggc ggtcttcacg gtgtaggggc gtctgtcgta 60

aacgccttgt cgaccactct tgacgttacg gttcatcgtg acggaaaaat ccactatcag 120

gcgtacgagc gcggtgtacc tgtggccgat cttgaagtga tcggtgatac tgataagacc 180

ggaacgatta cgcacttcgt tccggatccg gaaattttca aagaaacaac cgtatacgac 240

tatgatctgc tttcaaaccg tgtccgggaa ttggccttcc tgacaaaagg cgtaaacatc 300

acgattgaag acaaacgtga aggacaagaa cggaaaaacg agtaccacta cgaaggcgga 360

atcaaaagct atgttgagta cttaaaccgt tccaaagaag tcgttcatga agagccgatt 420

tatatcgaag gcgagaaaga cggcataacg gttgaagttg cattgcaata caacgacagc 480

tatacaagca atatttattc tttcacgaat aatatcaaca catacgaagg cggcacgcac 540

gaggccggat ttaaaaccgg tctgacccgt gtcataaacg actatgcaag aagaaaaggg 600

attttcaaag aaaatgatcc gaatttaagc ggggatgatg tgagagaagg gctgactgcc 660

attatttcaa ttaagcaccc tgatccgcaa ttcgaaggtc agacgaaaac gaagctcggc 720

aactccgaag cgagaacgat cactgatacg ctgttttctt ctgcgctgga aacattcctt 780

cttgaaaatc cggactcagc ccgcaaaatc gttgaaaaag gtttaatggc cgcaagagcg 840

cggatggcag cgaaaaaagc gcgggaattg acccgccgca aaagtgcgct tgagatttcc 900

aatctgccgg gcaaactggc ggactgttct tctaaagatc cgagcatttc cgagctgtat 960

atcgtagagg gtgactctgc gggcggatca gcgaaacagg gacgggaccg tcatttccaa 1020

gccattctgc cgctgcgcgg taagattctg aacgttgaga aagccagact tgataagatt 1080

ctctcaaaca atgaggtcag atcaatgatc acggccctcg gaacaggaat cggagaagat 1140

tttatcttga aaaacgcgtt tcttaagagt ctttgacgaa gcgatgaggc gtt 1193

Claims

1. The Bacillus velezensis is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 23508.

2. A microbial agent, comprising the bacillus belgii of claim 1.

3. The microbial inoculant according to claim 2, wherein the microbial inoculant is in the form of a dry powder comprising at least 100 million CFU of live bacillus beijerinckii per gram of microbial inoculant.

4. The microbial agent according to claim 2, wherein the microbial agent is obtained by:

carrying out fermentation culture on the Bacillus belgii so as to obtain a fermentation product;

and carrying out spray drying and crushing treatment on the basis of the fermentation product so as to obtain the microbial agent.

5. The microbial inoculant according to claim 4, wherein said fermentation culture comprises:

carrying out activated fermentation culture on the Bacillus belgii so as to obtain a zymocyte liquid;

and carrying out amplification fermentation culture on the zymophyte liquid so as to obtain a fermentation product.

6. The microbial inoculant according to claim 5, wherein the medium used for the amplified fermentation culture comprises corn starch, soybean meal, yeast powder, sucrose, peptone, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, calcium carbonate and antifoaming agents.

7. The microbial inoculum according to claim 6, wherein the medium for the scale-up fermentation culture comprises, in parts by weight:

2-5 parts of corn starch,

3-5 parts of soybean meal,

0.1 to 0.3 portion of yeast powder,

0.5 to 2 parts of cane sugar,

0.1 to 0.3 part of peptone,

0.1 to 0.3 part of dipotassium hydrogen phosphate,

0.2 to 0.5 portion of monopotassium phosphate,

0.1 to 0.3 portion of sodium chloride,

0.05 to 0.2 portion of calcium carbonate,

and

0.2-0.5 part of defoaming agent.

8. A complex microbial inoculant comprising the microbial inoculant of any one of claims 2-7.

9. The complex microbial inoculant according to claim 8, wherein the complex microbial inoculant comprises a first microbial inoculant and a second microbial inoculant, the first microbial inoculant is the microbial inoculant of any one of claims 2 to 7, and the second microbial inoculant comprises at least one selected from bacillus amyloliquefaciens and other bacillus belgii.

10. The complex microbial inoculant according to claim 9, wherein the complex microbial inoculant comprises 30-35 parts by weight of the first microbial inoculant and at least one of the following components:

30-35 parts by weight of bacillus amyloliquefaciens;

30-35 parts of other Bacillus belgii.

11. The complex microbial inoculum of claim 9, wherein the effective viable count of bacillus belgii per gram of complex microbial inoculum is at least 100 hundred million CFU, and at least one selected from the following:

the effective viable count of the bacillus amyloliquefaciens is at least 100 hundred million CFU;

the number of viable bacteria of other Bacillus belgii bacteria is at least 100 hundred million CFU.

12. A microbial fertilizer, which comprises the Bacillus belgii of claim 1, or the microbial agent of any one of claims 2 to 7, or the composite microbial agent of any one of claims 8 to 11.

13. A microbial fertilizer according to claim 12, comprising at least 0.5 hundred million CFU of microbially effective viable bacteria per gram of microbial fertilizer.

14. The microbial fertilizer according to claim 12, wherein the microbial fertilizer contains one to five per thousand of the bacillus belericus, or one to five per thousand of the microbial agent, or one to five per thousand of the composite microbial inoculum.

15. The microbial fertilizer according to claim 12, further comprising a base fertilizer selected from at least one of a compound fertilizer and an organic-inorganic fertilizer.

16. Use of the bacillus beleisi of claim 1 for the control of diseases caused by early blight of potato, rice bakanae disease, wheat scab, rice sheath blight, potato black nevus or rice blotch.

17. A method of preventing a disease, comprising:

applying an effective amount of Bacillus belgii, a microbial agent, a complex microbial agent or a microbial fertilizer to a crop, wherein the Bacillus belgii is the Bacillus belgii of claim 1, the microbial agent is the microbial agent of any one of claims 2 to 7, the complex microbial agent is the complex microbial agent of any one of claims 8 to 11, and the microbial fertilizer is the microbial fertilizer of any one of claims 12 to 15;

the diseases are selected from diseases caused by potato early blight, rice bakanae disease, wheat scab, rice sheath blight, potato black nevus or rice blotch.

18. The method of claim 17, wherein the crop is potato, rice, wheat.