CN113755408B

CN113755408B - Multifunctional bacillus subtilis, composite microbial inoculum and application thereof

Info

Publication number: CN113755408B
Application number: CN202111222649.4A
Authority: CN
Inventors: 郭兴龙; 康耀卫; 黄燕菲; 苑莹
Original assignee: Hebei Mengbang Biotechnology Co ltd
Current assignee: Hebei Mengbang Biotechnology Co ltd
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2022-09-02
Anticipated expiration: 2041-10-20
Also published as: CN113755408A

Abstract

The invention provides multifunctional bacillus subtilis, a composite microbial inoculum and application thereof, and relates to the technical field of microorganisms. The bacillus subtilis is bacillus subtilis strain KY374 which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 23455; the strain KY374 has good colonization capability on plant roots. The strain has high affinity to plant roots, extremely strong bactericidal activity to important pathogenic microorganisms of various crops, and a function of promoting the growth of the crop roots, particularly has high plant root colonization capability under natural conditions, can promote the growth of the plants, and has extremely high commercial value and wide market prospect.

Description

Multifunctional bacillus subtilis, composite microbial inoculum and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to multifunctional bacillus subtilis, a composite microbial inoculum and application thereof.

Background

Plant diseases are one of the important factors affecting crop yield reduction. Plant diseases include species such as root rot, blight, damping off, blight, verticillium wilt, and the like. The life history of pathogens of such diseases is partly or mostly present in the soil, and when conditions are appropriate, the pathogens germinate and infect the leaves, stems or roots of the plants causing the plants to develop diseases (Shi-Dong LI et al, 2011). Fusarium oxysporum f.sp.cubense, Botrytis cinerea, Fusarium solani, Verticillium dahlia, Helminthosporium umbilicifolium and Fusarium oxysporum have great harm to crops, and once the plant pathogenic fungi occur, the yield is greatly reduced, thus bringing great economic loss to agricultural production. Botrytis cinerea can infect stems, leaves, flowers and fruits of various vegetables, fruits and ornamental plants, has a wide host range and is harmful (Bouchra C et al, 2003); fusarium solani causes Root Rot, blight, Root Rot, etc. in various plants (Roy K W, 1997; company of Fusarium solani and F.oxysporum as domestic Agents of Fruit Rot and Root Rot of Muskmelon [ J ], 1993), causing severe disease in various crops and cash crops; fusarium oxysporum infection-induced blight is a destructive soil-borne disease, which causes serious crop yield reduction and even crop failure.

At present, physical, chemical and biological methods are generally adopted to prevent and treat plant diseases at home and abroad. Physical prevention and control are time-consuming and labor-consuming, and the prevention and control effect is limited and greatly influenced by environmental conditions, so the application is limited; the chemical method is the most frequently used method for preventing and treating plant diseases at home and abroad. For half a century, the application of chemical agents to control plant soil-borne diseases plays an important role in agricultural production, but long-term unreasonable use of chemical pesticides has created many problems. The high toxicity and high residue of chemical pesticide not only causes harm to the health of people and livestock, but also causes serious pollution to soil, water and atmosphere; meanwhile, as the drug resistance of germs and pests is continuously enhanced, the use amount and the use frequency of pesticides are continuously increased, so that the soil fertility is seriously reduced, the soil microflora is disordered, non-target organisms are injured, the ecological balance is damaged, a vicious circle that the use amount and the diseases are mutually increased is generated, the pesticide residue in agricultural products is increased, and great threat is formed on the safety of people and livestock. Various problems caused by using a large amount of chemical agents do not meet the requirements of sustainable development of agricultural health, so that people are prompted to search a new plant disease and insect pest control strategy which is friendly to human and environment and has good control effect.

The biological control has the characteristics of environmental friendliness, effectiveness, durability, no drug residue and the like, particularly avoids a series of problems caused by chemical control (Xumeina, 2005; xu Zhi, 2000), becomes a research hotspot for controlling plant soil-borne diseases at home and abroad at present, and has wider application prospect. Biological control refers to a method for inhibiting the vitality and reproductive capacity of pathogenic bacteria by using one or more microorganisms, and the common mechanisms are as follows: improving the physical and chemical properties and the nutritional status of soil, promoting the growth of plants, improving the health level of the plants and enhancing the disease resistance of host plants; the parasitic and antibiotic effects of antagonistic microbes such as biocontrol bacteria, fungi and actinomycetes and the competitive effects of the antagonistic microbes and the nutrients and the ecological niches of pathogenic bacteria are utilized to inhibit and eliminate the pathogenic bacteria; inducing a host plant to develop systemic resistance to pathogenic bacteria (Current status and maintenance for biological control of plant diseases in China [ J ], 2010; Liu Ming et al, 2004). Control of plant diseases by beneficial microorganisms starting with Hartely in 1921 for damping-off by fungi (Tika B Adhikari et al, 2001); in 1973 Kerr successfully controlled plant root tumor diseases with Agrobacterium radiobacter K84.

Plant disease-controlling microorganisms have been involved for many years in fungal, actinomycete, bacterial and even viral (phage) populations. Bacteria play an important role in the activities of naturally occurring biological control and human applied biological control due to the huge population, high fertility, complex metabolic activity, diverse action modes on pathogens and easy artificial culture. Currently, the most widely used biocontrol bacteria include Bacillus (Bacillus spp.), Pseudomonas (Pseudomonas spp.), and actinobacillus (Agrobacterium radiobacter) (Chengli et al, 2003).

The bacillus is a biocontrol bacterium widely distributed in the nature, has huge population, stronger fecundity and stable physicochemical properties, is applied to more scientific researches and production, and is an ideal biocontrol bacterium screening object. Examples of reported biocontrol bacillus include bacillus subtilis (b.subtilis), bacillus polymyxa (b.polymyxa), bacillus thuringiensis (b.thuringiensis), bacillus amyloliquefaciens (b.amyloliquefaciens) (Jamil Shafi et al, 2017), bacillus cereus (b.cereus), bacillus megaterium (b.megaterium), and bacillus pumilus (b.brevis). The bacillus subtilis has the characteristics of no environmental pollution, no harm to human and livestock, strong stress resistance, endogenetic spores, high propagation speed, simple nutritional requirement and safety to crops, can generate various antibacterial substances (Mengmli and the like, 2018) such as bacteriocin, enzymes, active proteins, lipopeptides, polypeptides and the like, has a wide antibacterial spectrum and has a good control effect on various plant diseases, is listed as a microorganism safe to human and livestock by the U.S. food and drug administration and the Ministry of agriculture in China, and has been widely applied to the fields of medicine, livestock raising, aquatic products, crop planting and the like (Chen Z and the like, 2010, Mishra S and the like, 2012) as a probiotic and biological control agent product, and is particularly developed into a product for biological control of various plant diseases.

In recent years, research reports about bacillus subtilis biocontrol agents at home and abroad mainly focus on strain laboratory screening and antibacterial efficacy evaluation (leaf cloud peaks, 2011; leaf cloud peaks and the like, 2011) on a culture dish, the growth and activity conditions of different bacillus subtilis strains around soil or crop roots are rarely researched, and large-scale practical application research in fields is more scarce, so that the biological control at present faces a huge challenge: the control effect of field biological control products is unstable, and the same problem is faced by the bacillus subtilis agent as the biological control product in the market. The main factors influencing the field control effect of the biological control product are whether the product has plant affinity and whether the product survives and can be stably propagated in the soil. If the affinity of the product and the plant is not high, the activity of the product in the soil after application is weak, the product is easily replaced by the competition of bacteria or harmful microorganisms existing in the soil, the number of the microorganisms in the applied product is not superior, the secretion of effective bactericidal substances is easily reduced due to the fact that the microorganisms cannot stably exist and propagate in the soil on the field ground in the application process, the prevention and control effect is unstable, the maximum effect cannot be exerted, the biocontrol function of the product is influenced, the large-area popularization and application of the product is further influenced, the problem of the farmer finally chooses to use chemical pesticides to solve is solved, the national weight-losing and pesticide-reducing policy cannot be responded, the environment-friendly policy is adopted, and a closed loop of 'chemical pesticides-drug resistance diseases-increasing the chemical pesticide dosage' is finally formed. Therefore, there is a need for further screening of Bacillus subtilis strains having plant affinity, stable existence and reproduction in field soil, and stable biocontrol effect, suitable for large-scale application.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide multifunctional bacillus subtilis, a composite microbial inoculum and application thereof, and aims to solve the technical problems that a biological control product in the prior art is unstable in field control effect, does not have plant affinity and is easily influenced by soil environment.

The technical scheme provided by the invention is as follows:

in one aspect, the invention provides a multifunctional Bacillus subtilis which is a Bacillus subtilis strain KY374, is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation number of CGMCC No. 23455; the strain KY374 has good colonization capability on plant roots.

The method comprises the steps of collecting 30 soil samples of different provinces and cities, screening bacillus subtilis by adopting a method combining heating-enrichment with high flux, screening 3724 high-temperature-resistant spore-producing microbial strains from about 2.5 ten thousand microbial strains, and further screening the 3724 high-temperature-resistant spore-producing microbial strains by adopting specific primers of the bacillus subtilis on the basis to obtain 35 bacillus subtilis with positive reaction with the specific primers. By respectively inoculating 35 strains of bacillus subtilis to the root system of the corn, 5 strains of bacillus subtilis are found to have good colonization capacity in the root system of the corn, and meanwhile, further tests are carried out on biological functions of the bacillus subtilis, such as fungicidal biological activity, capability of promoting germination of crop seeds and the like. As a result, the bacillus subtilis strain which has high plant affinity and stable bactericidal activity is named as KY 374. Through further research, the sequence of the 16s DNA gene of the bacillus subtilis strain is shown as SEQ ID NO: 5, belonging to Bacillus subtilis strain.

The bacillus subtilis KY374 strain obtained by screening has good affinity with monocotyledons and dicotyledons, has obvious antagonistic action on soil-borne pathogenic microorganisms, and also has the functions of stress resistance (salt resistance, acid and alkali resistance) and promotion of crop germination.

In another aspect, the present invention provides a complex microbial inoculant comprising the aforementioned Bacillus subtilis strain KY374 or a fermentation product of the Bacillus subtilis strain. The composite microbial inoculum comprises effective amount of the bacillus subtilis, for example, the viable bacteria amount is 10 ⁵ cfu/g or more. The strain KY374 can be mixed with agriculturally acceptable carriers, excipients and/or adjuvants. The complex microbial inoculum can be a liquid microbial inoculum or a solid microbial inoculum.

In another aspect, the invention also provides application of the Bacillus subtilis strain KY374 or the composite microbial inoculum in inhibiting or controlling plant diseases. In particular, the bacterial strain or the composite microbial inoculum can be used for preventing and treating plant soil-borne diseases.

In one embodiment, the plant disease comprises one or more of apple alternaria leaf spot, pear black spot, wheat sharp eyespot, wheat root rot, cotton wilt, watermelon wilt, tomato early blight and banana wilt specialization type 3.

In another aspect, the invention also provides application of the Bacillus subtilis strain or the composite microbial inoculum in promoting plant growth.

In one embodiment, the promoting plant growth comprises promoting plant root growth and/or promoting plant seed germination.

In one embodiment, the plant comprises a monocot or a dicot; preferably, the plant is a crop plant of the class monocotyledonae or dicotyledonae.

In one embodiment, the plant comprises one or more of soybean, corn, mung bean and wheat; preferably, the plant is maize. The strain KY374 and the plant have better effectsAnd (4) affinity. In the present study, only 5 out of 35 different Bacillus subtilis strains showed good affinity for the corn root system. In the invention, the method for judging the affinity is to irrigate the crops by using the bacterial suspension of the bacillus subtilis after the crops (such as corns) germinate, and the bacterial amount separated from the root systems of the crops can reach 10 after the crops grow for 1 week ⁵ cfu/g or more is considered to have affinity for the crop.

In another aspect, the invention also provides application of the Bacillus subtilis strain KY374 or the composite microbial inoculum in preparation of plant growth promoters or microbial organic fertilizers. The use of the bacterial strain KY374 or the composite microbial inoculum can obviously reduce the fertilizer consumption. The Bacillus subtilis strain or the composite microbial inoculum can be used for preparing field biological control products (such as biological control microbial inoculum, seed coating agent and the like) so as to promote the growth and development of plants under the field natural condition.

In another aspect, the present invention also provides a method for promoting plant growth under natural soil conditions, which comprises applying an effective amount of said Bacillus subtilis strain KY374 or said complex inoculant to said plant or to the environment around the plant roots. In particular, the strain may be applied to the plant (e.g., by root watering, root dipping, etc.) or to the plant seed (e.g., dressing, coating), as a bacterial suspension, or to the soil surrounding the plant or plant seed. The plant includes crops of the monocotyledonous or dicotyledonous class, including, but not limited to, soybean, corn, mung bean, wheat, or the like, for example.

Bacillus subtilis strain KY374 is collected from Qingshan lake region of Nanchang city in Jiangxi province; the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 22 days 9 months 2021, the address is the microorganism research institute of China academy of sciences No.3, West Lu 1, Beijing, Chaoyang, and the strain preservation number is CGMCC No. 23455. Survival was detected by the depository at 2021, 9 months and 22 days.

Has the advantages that:

the bacillus subtilis KY374 and the composite microbial inoculum containing the same have high affinity to plants, can promote the germination of plant seeds and the growth of root systems, and can promote the increase of crop yield; meanwhile, the bacillus subtilis KY374 and the compound microbial inoculum containing the same can comprehensively prevent and treat various common plant diseases (wide antibacterial spectrum to plant pathogenic bacteria), and are favorable for improving the quality of crops. Particularly, the strain can be fast colonized in a large amount at the plant rhizosphere under the natural condition, colonize and survive for a long time, has ecological niche advantage, and can promote the growth and nutrient absorption of plants under the natural soil condition.

The bacillus subtilis KY374 disclosed by the invention has good salt resistance, alkali resistance and acid resistance and outstanding comprehensive performance, and is a multifunctional microbial strain which has high plant affinity, can stably prevent and treat plant diseases, has stress resistance and promotes crop seed germination. The invention has important significance for reducing the using amount of the fertilizer and maintaining the healthy and sustainable development of agriculture.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an agarose electrophoresis result of a Bacillus subtilis-specific PCR verification experiment performed on 50 randomly selected Bacillus strains according to the present invention (wherein

lanes

8, 11, and 30 are positive for Bacillus subtilis, and

lanes

25 and 26 are respectively positive control Bacillus subtilis 92068 and blank control);

FIG. 2 shows the results of antagonistic experiments of 5 Bacillus subtilis strains and various phytopathogens obtained by screening;

FIG. 3 shows that 5 Bacillus subtilis strains screened by the invention have 5%, 10%, 15% and 20% KNO ₃ The growth results of (a);

FIG. 4 shows the results of the growth of 5 Bacillus subtilis strains selected according to the present invention on R2A medium at pH11, pH12 and pH 4.5;

FIG. 5 shows the growth promoting effect of 5 screened Bacillus subtilis strains on corn seeds;

FIG. 6 shows the growth promoting results of 5 Bacillus subtilis strains screened by the present invention on wheat seeds;

FIG. 7 shows the growth promoting effect of 5 Bacillus subtilis strains on mung bean seeds;

FIG. 8 shows the growth promoting effect of 5 Bacillus subtilis strains on soybean seeds;

FIG. 9 shows the observation result of the strain KY374 of the present invention under a microscope;

FIG. 10 shows the results of plant affinity assay of the strains of example 7;

FIG. 11 shows the results of colony specific PCR verification in example 7 of the present invention (wherein the gel is PCR results of randomly selected strains from coated Rif-R2A medium: 92068 and 92068-1 in

lanes

1 and 2, respectively, and blank electrophoresis in lane 26; and the gel is PCR results of randomly selected strains from coated Sm-R2A medium: KY374 and KY374-2 in

lanes

1 and 2, respectively, and blank electrophoresis in lane 26).

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 selection of Bacillus subtilis

The method for screening the bacillus subtilis by using a high-temperature heating-high-flux method comprises the following specific steps of:

1.1. collection of soil samples

Representative soil is selected from 30 different provinces across the country for collection, such as sand soil, clay, black soil and the like, samples can be from different areas such as farmlands, pastures, forest soil and the like, particularly, soil for planting corn, sugarcane and the like is sampled, each point collects 15-20 g of samples, source areas (provinces and counties), year and month of collection and soil sources (plants, sand soil or the like) are marked, the samples are stored in a refrigerator at minus 80 ℃, a soil resource library is established, and 230 soil samples from all provinces and cities across the country are collected at present.

1.2. Screening of bacillus subtilis by high-temperature heating-high-flux method

(1) Taking 0.5g of soil sample, shaking uniformly in 15mL of purified water, absorbing 1mL of mixed solution into two 1.5mL centrifuge tubes, taking one centrifuge tube, heating in a metal bath at 80 ℃ for 30min, shaking uniformly after cooling to room temperature, taking 100 mu L of the mixed solution of the two centrifuge tubes, diluting in a gradient manner, respectively taking 100 mu L of the mixed solution, and respectively coating the diluted mixed solution on an R2A solid culture medium (0.5 g of yeast powder, 0.5g of peptone, 0.5g of tryptone, 0.5g of glucose, 0.5g of soluble starch, 0.3g of dipotassium hydrogen phosphate, 0.3g of sodium pyruvate, 0.05g of magnesium sulfate heptahydrate, 15g of agar powder and 1000mL of water), respectively coating 3R 2A solid culture media in each degree, culturing the strain at 37 ℃ in an incubator for growth, and observing the difference of the number and the types of the mixed solution of the soil sample after heating treatment and the normal soil sample strain;

(2) and (3) selecting the strain from the soil sample dilution culture medium after the heating treatment, carrying out streak culture and purification on a solid R2A culture medium (an incubator at 37 ℃), and carrying out primary verification of the bacillus subtilis specific PCR on the obtained strain after the strain grows.

1.3. Bacillus subtilis specificity PCR verification

And (3) carrying out bacillus subtilis specific PCR verification on the strain obtained in the step (2) and judging whether the obtained strain is bacillus subtilis (positive control is bacillus subtilis 92068).

1.3.1CTAB method for extracting bacterial DNA

(1) Inoculating a single colony in 5mL of R2A, and culturing overnight at 30 ℃; (2) inoculating 1mL of seed culture solution into 100mL of R2A liquid, and culturing at 37 ℃ and 220R/min for 16 hours; (3) centrifuging at 5000r/min for 10 min, and discarding the supernatant; (4) adding 10mL of TE, centrifuging, washing, dissolving the thalli by using 10mL of TE, uniformly mixing, and storing at-20 ℃ for later use; (5) taking 3.5mL of bacterial suspension, adding 184 mu L of 10% SDS, mixing uniformly, adding 37 mu L of 10mg/mL proteinase K, mixing uniformly, and incubating for 1 hour at 37 ℃; (6) adding 740 mu L of 5mol/L NaCl, then adding 512 mu L of CTAB/NaCl, uniformly mixing, and incubating for 10 minutes at 65 ℃; (7) adding chloroform/isoamyl alcohol with the same volume, mixing uniformly, centrifuging for 5 minutes at 10000r/min, and keeping the supernatant; (8) equal volume of phenol was added to the supernatant: chloroform: isoamyl alcohol (25: 24: 1), mixing uniformly, centrifuging for 5 minutes at 10000r/min, and reserving supernatant; (9) adding isopropanol with the volume of 0.6 time, uniformly mixing, centrifuging for 5 minutes at 10000r/min, collecting DNA precipitate, and centrifuging and washing the DNA precipitate by using 70% ethanol; (10) DNA was dissolved in 1mL of TE, RNaseA was added to a final concentration of 20. mu.g/mL, and the mixture was stored at 4 ℃.

1.3.2 amplification and sequencing

PCR amplification of 16S rDNA was performed using Bacillus subtilis specific primers EN1F (SEQ ID No. 1: 5'-CCAGTAGCCAAGAATGGCCAGC-3') and EN1R (SEQ ID No. 2: 5'-GGAATAATCGCCGCTTTG TGC-3'). And (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90s for 30 cycles. And (3) carrying out 1.5% agarose gel electrophoresis on the PCR product, carrying out electrophoresis imaging on the PCR product after the agarose gel electrophoresis, comparing the obtained strain with a control according to the gel running result of the control bacillus subtilis 92068, and preliminarily judging whether the strain is bacillus subtilis.

1.4. Analysis of results

The soil samples collected from 30 different provinces and cities are screened by a high-temperature heating-high-throughput method through the method, and about 3724 strains of the bacillus are obtained through screening, wherein 35 strains of the bacillus have positive reactions (the proportion is about 0.9 percent) with specific primers of the bacillus subtilis, and the bacillus subtilis belongs to the bacillus subtilis.

FIG. 1 shows the results of agarose electrophoresis after random selection of 50 strains from 3724 obtained Bacillus strains, and specific PCR verification of Bacillus subtilis using Bacillus subtilis 92068 given by the Chinese academy of agricultural sciences as a control (lane 25 in FIG. 1), in which 3 strains (

lanes

8, 11, and 30) among the 50 strains reacted positively with primers specific to Bacillus subtilis. In addition, the research results also show that the number of the bacillus subtilis is very different in different soil samples, and the bacillus subtilis is not separated in a plurality of soil samples collected by us.

Example 2 screening of the resulting Bacillus subtilis strains for affinity testing with maize plants

2.1. Preparation of a bacterial suspension

Inoculating 35 different Bacillus subtilis strains into 250mL triangular flask containing 50mL R2A liquid culture medium, culturing at 30 deg.C for 48 hr at 200R/min, and respectively preparing into OD _600nm The bacterial solution (0.1) is ready for use.

2.2. Affinity test of different Bacillus subtilis for maize plants

(1) Taking a proper amount of vermiculite, carrying out autoclaving at 121 ℃ for 20min, then subpackaging into 35 parts by using a proper cup, controlling the weight error of each part of vermiculite within 5g, and carrying out numbering and marking according to different bacillus subtilis strains;

(2) selecting full, uniform-sized and unbroken corn seeds, inoculating the corn seeds into the vermiculite packaged in the step (1), planting about 5 corn grains in each cup, and watering at proper time until the corn sprouts;

(3) after the corns germinate, respectively pouring 30mL of the corresponding prepared bacillus subtilis liquid, and respectively collecting and pouring corn root systems of different bacillus subtilis liquid after the corns grow for 1 week (timely watering is carried out during the period to ensure the normal growth of the corns), and recording the corn root systems;

(4) respectively collecting root samples from corn roots irrigated by different bacillus subtilis liquid, and separating and metering bacteria from the root samples by adopting a gradient dilution separation method. Shearing corn roots, respectively carrying out bacterial separation from the corn roots, adding a small amount of water, fully grinding, collecting grinding liquid, gradient diluting and coating the grinding liquid on an R2A solid culture medium, culturing in an incubator at 30 ℃, and carrying out strain quantity statistics when strains grow;

note: the number of the strains is more than or equal to 10 ⁵ cfu/g, which is judged to have affinity with the plant; the larger the number of the bacteria is, the better the affinity of the strain to the plant is;<10 ⁵ cfu/g, which is judged to have no good affinity with the plant;>10 ⁵ cfu/g, determined as the same plantHas high affinity.

(5) And (5) respectively selecting 25 colonies from the R2A solid culture medium in the step (4) to carry out verification of bacillus subtilis specific PCR, and verifying whether the colonies are bacillus subtilis. If most of the colonies did not react positively with the primers specific for Bacillus subtilis, it was suggested that the Bacillus subtilis inoculated with the maize did not have good affinity for the maize root system (i.e., good colonization ability), or that the bacteria isolated on R2A medium were not derived from the inoculated Bacillus subtilis (i.e., not the 35 strains of Bacillus subtilis obtained by screening), but were derived from background microbial strains in the environment.

2.3. Results of affinity tests of different Bacillus subtilis for maize plants

Carry out the fungus liquid watering to the maize through carrying out 35 different bacillus subtilis, carry out the bacterial separation respectively from corresponding maize root system and carry out the bacterial strain quantity statistics, according to the affinity standard of judging: after the corn grows for 1 week, the bacterial quantity of 5 bacillus subtilis in 35 bacillus subtilis separated from the corn root system can reach 10 ⁵ cfu/g or more, these strains were identified as having affinity for corn (see Table 1), and the number of other tested strains could not reach 10 ⁵ The amount of bacteria is 4.45 × 10 (e.g. KY417 (ZZ-1)) cfu/g or more ⁴ cfu/g); the strains meeting the affinity standard are KY231, KY374, KY384, KY423(LTZ-3) and KY474(ALE-2), wherein the strains with the most bacteria are KY374 and KY423(LTZ-3), and the bacteria amount of the two strains is 4.77 x 10 ⁶ cfu/g and 1.58 x 10 ⁶ cfu/g, has high affinity with corn.

TABLE 1.5 Strain number results for Bacillus subtilis with affinity for maize plants

Bacterial strains	Amount of bacteria isolated from root (cfu/g)
		KY231	1.27*10 ⁵
KY384	3.41*10 ⁵
		KY374	4.77*10 ⁶
KY423(LTZ-3)	1.58*10 ⁶
		KY474(ALE-2)	5.36*10 ⁵
KY417(ZZ-1)	4.45*10 ⁴

Example 3 test of the Activity of Bacillus subtilis to kill pathogenic microorganisms

3.1. Experiment for testing bactericidal activity of functional strains on plant pathogenic bacteria

Respectively inoculating 5 screened bacillus subtilis strains (KY231, KY374, KY384, KY423(LTZ-3) and KY474(ALE-2)) and plant pathogenic bacteria of apple alternaria leaf spot, pear black spot, wheat sharp eyespot, wheat root rot, cotton fusarium wilt, watermelon fusarium wilt, tomato early blight and banana fusarium wilt specialization type 3 on a PDA culture medium at the same time, and observing whether the bacillus subtilis strains have antagonistic action on the plant pathogenic bacteria. Bacillus subtilis 92068 strain was set as a control.

3.2. Test result of bacterial strain against phytopathogen

The result determination (figure 2 and table 2) shows that the 5 screened bacillus subtilis strains (KY231, KY374, KY384, KY423(LTZ-3) and KY474(ALE-2)) have stronger or similar antagonistic action on wheat root rot, pear black spot, apple alternaria leaf spot and tomato early blight compared with the control strain 92068, and the antagonistic strength of different bacillus subtilis strains on the same plant pathogen has individual difference.

TABLE 2 antagonistic experiment results of Bacillus subtilis strain and various plant pathogenic bacteria

(note: "+" represents that the strain has a strong antagonistic action against plant pathogenic bacteria; "+" represents that the strain has an antagonistic action against plant pathogenic bacteria; "-" represents that the strain has a weak antagonistic action against plant pathogenic bacteria or hardly has)

The results of the combination of FIG. 2 and Table 2 show that the strains which antagonize phytopathogens most in the 5 screened Bacillus subtilis strains are KY374, KY384, KY423 and KY474 times compared with the control strain 92068.

Example 4 test of Bacillus subtilis Strain for stress resistance

4.1. Strain stress resistance determination

Inoculating 5 screened Bacillus subtilis strains (KY231, KY374, KY384, KY423(LTZ-3) and KY474(ALE-2)) with 5% KNO ₃ 、10％KNO ₃ 、15％KNO ₃ 、20％KNO ₃ And the growth, inhibition and growth speed of the strain are observed on R2A culture media of pH4.5, pH11 and pH 12. Bacillus subtilis 92068 strain was set as a control.

4.2. Strain stress resistance test results

Combining FIG. 3, FIG. 4 and Table 3, the results show that 5 screened strains of Bacillus subtilis (KY231, KY374, KY384, KY423(LTZ-3) and KY474(ALE-2)) have good salt tolerance compared with the control strain 92068, and have alkali tolerance similar to that of the control strain 92068 on a culture medium with pH11 and pH 12; wherein, the strains KY231 and KY423 and the control strain 92068 can grow on a culture medium with the pH value of 12, and the strains KY374 and KY474 have weaker growth vigor; on the culture medium with pH4.5, the strains KY374 and KY384 can normally grow, and the growth of other strains is inhibited.

TABLE 3 growth results of Bacillus subtilis strains on R2A medium at pH4.5 and pH12

5％KNO ₃

10％KNO ₃

15％KNO ₃

20％KNO ₃

pH4.5

pH11

pH12

92068

++

Weak (weak)

++

KY231

++

-

++

KY374

++

+

Weak (weak)

KY384

++

+

-

KY423

++

-

++

KY474

++

-

++

+

(note: "+" represents that the strain can grow and is not inhibited under these conditions; "+" represents that the strain can grow and begins to be inhibited under these conditions; "weak" represents that the strain grows weakly under these conditions; "-" represents that the strain cannot grow under these conditions.)

Example 5 Bacillus subtilis Strain experiments on plant growth promoting action on the rhizosphere (PGPR)

5.1 preparation of the bacterial suspension

Control

92068 and 5 Bacillus subtilis strains (KY231, KY374, KY384, KY423(LTZ-3) and KY474(ALE-2)) were inoculated into 250mL Erlenmeyer flask containing 50mLR2A liquid culture medium, and cultured at 30 deg.C and 200r/min for 48 hr to prepare OD _600nm The bacterial solution was 0.1.

5.2 seed coating

Coating soybean seeds, corn seeds, mung bean seeds and wheat seeds respectively, spraying 300 mu L of bacterial liquid on each 100 g of seeds, selecting the seeds which are full and uniform in size and are not crushed and coated, inoculating the seeds into a culture medium (5 g of agar and 1000L of water) with the agar concentration of 0.5% for germination test, repeating each treatment for 4 times in 10 grains in each dish, and culturing in a dark place.

Seeds coated with the bacillus subtilis 92068 strain were set as positive controls, and seeds coated with water, respectively, were set as blank controls. The root growth of the seeds was observed from day 2 onward, and the germination index of each seed was calculated.

Note: the germination index G of the soybean and mung bean seeds is 0N + 1N + 2N + 3N + 4N (N represents the number of the grades); the germination index G of the corn and the wheat seeds is N (0+ X) + N (1+ X) + N (2+ X) + N (3+ X) + N (4+ X) (N represents the number of the grades, and X represents the total number of lateral buds and lateral roots of the seeds of the grades);

0 represents no germination; 1 represents a bud length of 0-0.5 cm; 2 represents a sprout length of 0.5-1.0 cm; 3 represents a bud length of 1.0-1.5 cm; 4 represents a shoot length of more than 3 cm.

5.3 seed Germination test results

Fig. 5 to 8 show the growth promotion results of the bacillus subtilis strain on corn seeds, wheat seeds, mung bean seeds and soybean seeds, respectively, and table 4 shows the influence of different bacterial liquids on the germination indexes of the corn seeds, the wheat seeds, the mung bean seeds and the soybean seeds.

TABLE 4 influence of different bacterial solutions on germination index of corn seed, wheat seed, mung bean seed and soybean seed

The results of the combination of the fig. 5-fig. 8 and the table 4 show that the 5 bacillus subtilis strains (KY231, KY374, KY384, KY423(LTZ-3) and KY474(ALE-2)) have the effect of promoting germination of soybean seeds, corn seeds, mung bean seeds and wheat seeds, and the germination index is obviously higher than that of the control bacillus subtilis strain 92068. Compared with a control bacillus subtilis strain 92068 and a blank control (water), the accelerating effect of the strains KY231, KY374 and KY384 on wheat seed germination is very significant and different on the level P <0.01 by SPSS analysis; the promotion effect of the strains KY374, KY384 and KY423(LTZ-3) on the mung bean seed germination is very obvious and different on the P <0.01 level through SPSS analysis; the promotion effect of the strains KY231, KY374, KY384 and KY474(ALE-2) on soybean seed germination is very significantly different on the level that P is less than 0.01 through SPSS analysis; the promoting effect of KY374 strain on corn seed germination is very different at the P <0.01 level by SPSS analysis.

The synthesis can be concluded: the strain KY374 has the function of promoting germination of soybean seeds, corn seeds, mung bean seeds and wheat seeds, and has very obvious difference (P is less than 0.01). Therefore, the strain KY374 has good plant affinity, and simultaneously, the strain KY374 has the characteristics of good salt resistance, alkali resistance, acid resistance and wide bacteriostatic spectrum on plant pathogenic bacteria.

Example 6 sequencing of 16sDNA of Strain KY374 and analysis of physiological morphology of Strain

6.1 determination of 16sDNA of KY374 Strain

6.1.1CTAB method for extracting bacterial DNA (the specific steps are the same as in example 1.3.1).

6.1.2 amplification and sequencing

PCR amplification of 16S rDNA was performed using 16S rDNA universal primer 27f (SEQ ID No. 3: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (SEQ ID No. 4: 5'-GGTTACCTTGTTACGACTT-3').

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 60s, for 35 cycles. And (3) performing 1.5% agarose gel electrophoresis on the PCR product, recovering, purifying and sequencing the PCR product after the agarose gel electrophoresis, performing Blast search on homologous sequences in GenBank according to the obtained 16S rDNA sequence, analyzing and comparing the homologous sequences, and establishing a phylogenetic tree.

6.2 sequencing results of 16s DNA of Strain KY374

The sequencing result shows that the 16s DNA sequence of the strain KY374 is shown as the following SEQ ID No. 5: TTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGC are provided.

The 16sDNA sequence of the strain KY374 and the known Bacillus subtilis are subjected to homologous alignment analysis (by adopting a Fast Minimum Evolution method), and the homology of 100% with the known Bacillus subtilis (such as Bacillus subtilis strain JCL-16, IB-22) is proved.

6.3 morphological Observation of the Strain

The selected strains were inoculated on an R2A plate, cultured at room temperature for 2d, and the size, shape, color, gloss, viscosity, bulge shape, transparency, edge characteristics, and the like of the colonies were observed.

The observed strain is cultured and grown on an R2A culture medium for 2d, the strain is observed to be rod-shaped under a microscope, the bacterial colony is in a circular sheet shape on the culture medium, the bacterial colony is white, rough and wrinkled, the edge is irregular, large, opaque and non-viscous, and the diameter of the bacterial body is about 2-3 mu m and the diameter of the spore is about 1-2 mu m as measured by the microscope, as shown in figure 9.

Example 7 experiment verification of affinity of strain KY374 to corn plants in natural soil

In the embodiment, the method for screening the bacillus subtilis strain with good affinity with the plant adopts the planting environment system which is vermiculite material sterilized by high pressure, has the advantages of avoiding the interference of natural microorganisms in natural soil or existing bacillus subtilis strain to the test result of screening the bacillus subtilis with good affinity with the plant, and simultaneously facilitating the normal operation of strain screening; but cannot show that the bacillus subtilis strain screened by the system has the same affinity and root system colonization capacity to corn crops under natural conditions.

In order to further test the affinity of the strain KY374 to plants or the colonization ability of the root system, a widely-used commercial strain 92068 is used as a control to compare the affinity of the strain KY374 to corn under natural soil conditions and the colonization ability of the root system. Since there are a large number of natural microbial populations in natural soil, which also include natural Bacillus subtilis, it is desirable to biomarker strain KY374 and commercial strain 92068 to facilitate the differentiation of target strains from Bacillus subtilis in soil in order to avoid the inability to distinguish the target strains.

The strain KY374 and the commercial strain 92068 are subjected to biological labeling by two different antibiotics, spontaneous mutation methods are utilized to respectively obtain spontaneous mutants of the commercial strain 92068 for rifampicin, namely a mutant strain 92068-1 and a spontaneous streptomycin-resistant mutant strain KY374-2 of the strain KY374, mutant bacterial liquids of the two antibiotics are adopted to irrigate plants planted in natural soil, and specific PCR is carried out by combining with specific PCR primers of bacillus subtilis for further confirmation, so that the colonization ability of a target strain to roots of the plants under natural soil conditions, namely the affinity with the plants, can be researched. The following are the specific procedures and results of the experiments.

7.1 Strain KY374 and 92068 Strain Single resistance mutation experiment

(1) 92068 and KY374 strains are inoculated into a 250mL conical flask containing 50mL R2A liquid culture medium, and cultured for 48 hours at the temperature of 30 ℃ at the speed of 200R/min;

(2) spreading 200. mu.L, 400. mu.L and 600. mu.L bacterial suspensions of strain 92068 on R2A (Rif-R2A) solid culture medium containing 100. mu.g/mL rifampicin antibiotic respectively; 200 mu L, 400 mu L and 600 mu L of KY374 bacterial suspension is taken and coated on R2A (Sm-R2A) solid culture medium containing 50 mu g/mL streptomycin antibiotic, and the strain to be cultured is cultured at 30 ℃ in an incubator;

and (3) selecting 5 strains of bacteria growing in Rif-R2A and Sm-R2A solid culture media, carrying out streak purification on R2A, carrying out Bacillus subtilis specific PCR verification on the purified strains, verifying whether bacterial colonies are Bacillus subtilis or not, judging whether the strains are greatly different from wild strains or not according to electrophoresis band results, wherein the difference is large and indicates that the strains are not Bacillus subtilis, and timely eliminating the strains.

7.2 Strain KY374 and 92068 Single resistance mutation results

The purified and agarose-electrophoretically confirmed strains are cultured again in solid medium of Rif-R2A and 50 ug/mLSm-R2A at 100 ug/mL, antibiotic false-positive strains are excluded, and finally strain 92068-1 and strain KY374-2 can stably grow on Rif-R2A and Sm-R2A at 100 ug/mL respectively, so that the subsequent plant affinity tests are carried out by using the two strains.

7.3 Experimental verification of plant affinity of strain KY374

(1) Sowing proper amount of corn in a pot plant filled with natural soil (corn land soil in Yonganzhen town of Zhang lake in Zhaoqing, Guangdong), observing the moistening condition of the soil in the pot, and irrigating at proper time (generally irrigating once every 2 days) until the corn germinates;

(2) inoculating the variant strain 92068-1 and variant strain KY374-2 into 250mL conical flask containing 100mLR2A liquid culture medium, culturing at 30 deg.C at 200r/min for 48 hr, and respectively preparing into OD _600nm 0.8 of bacterial suspension;

(3) taking OD _600nm Mixing 100mL bacterial suspensions of 0.8 strain 92068-1 and KY374-2, pouring the mixture into a germinated corn pot, after 2 hours, after the soil completely absorbs bacterial liquid, pulling out a proper amount of corn seedlings, setting the corn seedlings as a zero point, shearing the root of the corn seedlings to be about 1g, adding a small amount of water, fully grinding, collecting grinding liquid, diluting and coating the grinding liquid after gradient, then coating the grinding liquid on 100 mu g/mL Rif-R2A and 50 mu g/mL Sm-R2A solid culture media, culturing in an incubator at 30 ℃, counting the number of the strains when the strains grow, and calculating the proportion of the strains;

(4) observing the growth condition of corn every day, watering the corn in proper amount, pulling out a proper amount of corn seedlings after two days, setting the corn seedlings as 2 days points (the corn seedlings are taken once every two days for counting the number of strains, each point is respectively marked as 2 days, 4 days and 6 days), cutting off about 1g of the root parts of the corn seedlings, adding a small amount of water for fully grinding, coating the corn seedlings in a gradient dilution manner on 100 mu g/mL Rif-R2A and 50 mu g/mL Sm-R2A solid culture media, culturing the corn seedlings in an incubator at 30 ℃, counting the number of the strains after the strains grow, and calculating the proportion of the corn seedlings and the strain.

(5) After the last gradient dilution (6 days), 25 colonies were randomly selected from 100. mu.g/mL Rif-R2A and 50. mu.g/mL Sm-R2A solid medium cultured in an incubator for 24 hours, respectively, and verified by Bacillus subtilis-specific PCR to determine whether the colonies were Bacillus subtilis.

7.4 Strain KY374 plant affinity experiment verification result

FIG. 10 and the results in Table 5 show that the numbers of the strain 92068-1 and the strain KY374-2 at zero point were 0.58 hundred million/g and 0.54 hundred million/g, respectively, at a ratio of 1:1, and the numbers of the strain were 0.03 hundred million/g and 0.96 hundred million/g with time, at a ratio of 1: 20, the amounts differ by a factor of approximately 20; as can be seen from the results in FIG. 11, the Bacillus subtilis-specific PCR of the selected strain from the solid medium of Rif-R2A at 100. mu.g/mL showed no or no bands at different positions from the original strains 92068 and 92068-1, indicating that the number of the strain 92068-1 was decreasing or even approaching 0 over time; and the strain selected from the Sm-R2A solid culture medium is subjected to Bacillus subtilis specific PCR, and the position of the strain band is the same as that of the original strain KY374 and KY374-2 bands, so that the strain KY374-2 has high affinity with corn, can be colonized on the root of the corn and cannot be reduced along with the increase of time.

TABLE 5 Experimental validation results of plant affinity of the strains

	92068-Rif-R2A (Yi/g)	KY374-Sm-R2A (hundred million/g)
			Day 0	0.58	0.54
2 days	0.43	0.52
			4 days	0.21	0.89
6 days	0.027	0.96

According to the research, a Bacillus subtilis KY374 which has high plant affinity and can stably prevent and treat plant diseases is separated from soil collected in a Qingshan lake region of Nanchang city in Jiangxi by adopting a high-temperature heating-high-flux screening method, and the strain has high homology with Bacillus subtilis through sequence determination and analysis of 16sDNA of the strain. Compared with a commercial strain bacillus subtilis 92068, the strain KY374 has higher affinity with plants, has the function of promoting germination of soybean seeds, corn seeds, mung bean seeds and wheat seeds in a seed germination experiment, has obvious difference (P is less than 0.01), and has the characteristics of better salt resistance, alkali resistance and acid resistance and wider bacteriostasis spectrum on plant pathogenic bacteria. Therefore, KY374 is a bacillus subtilis strain which has high plant affinity, can stably prevent and treat plant diseases, and is a functional microbial strain which has stress resistance and can promote crop seed germination.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Hebei Germing-Hebang Biotech Co., Ltd

<120> multifunctional bacillus subtilis, composite microbial inoculum and application thereof

<130> PA21028443

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence

<400> 1

ccagtagcca agaatggcca gc 22

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<212> DNA

<213> Artificial sequence

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ggaataatcg ccgctttgtg c 21

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

agagtttgat cctggctcag 20

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<212> DNA

<213> Artificial sequence

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ggttaccttg ttacgactt 19

<210> 5

<211> 1421

<212> DNA

<213> 16s DNA

<400> 5

ttcggcggct ggctcctaaa aggttacctc accgacttcg ggtgttacaa actctcgtgg 60

tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120

ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180

atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc attgtagcac 240

gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300

ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360

gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420

acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga ggatgtcaag 480

acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540

cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg gagtgcttaa 600

tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc atcgtttacg 660

gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc ctcagcgtca 720

gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta cgcatttcac 780

cgctacacgt ggaattccac tctcctcttc tgcactcaag ttccccagtt tccaatgacc 840

ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc gagcccttta 900

cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag 960

ttagccgtgg ctttctggtt aggtaccgtc aaggtaccgc cctattcgaa cggtacttgt 1020

tcttccctaa caacagagct ttacgatccg aaaaccttca tcactcacgc ggcgttgctc 1080

cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc 1140

cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgttgccttg 1200

gtgagccgtt acctcaccaa ctagctaatg cgccgcgggt ccatctgtaa gtggtagccg 1260

aagccacctt ttatgtttga accatgcggt tcaaacaacc atccggtatt agccccggtt 1320

tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc cgtccgccgc 1380

taacatcagg gagcaagctc ccatctgtcc gctcgacttg c 1421

Claims

1. The multifunctional bacillus subtilis is characterized in that the bacillus subtilis is bacillus subtilis (Bacillus subtilis: (A) (B))Bacillus subtilis) The strain KY374 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC number 23455; the strain KY374 has good colonization ability on plant roots and affinity with plants.

2. A complex bacterial agent comprising the Bacillus subtilis (Bacillus subtilis) of claim 1Bacillus subtilis) A strain or a fermentation product of said bacillus subtilis strain.

3. The Bacillus subtilis of claim 1 (b), (c), (d) and d)Bacillus subtilis) The application of the strain or the composite microbial inoculum of claim 2 in inhibiting or controlling plant diseases.

4. The use according to claim 3, wherein the plant disease comprises one or more of apple alternaria leaf spot, pear black spot, wheat sharp eyespot, wheat root rot, cotton wilt, watermelon wilt and tomato early blight.

5. The Bacillus subtilis of claim 1 (b), (c), (d) and d)Bacillus subtilis) The application of the strain or the composite microbial inoculum of claim 2 in promoting plant growth.

6. The use of claim 5, wherein the promotion of plant growth comprises promotion of plant root growth and/or promotion of plant seed germination.

7. Use according to claim 5 or 6, wherein the plant comprises a monocotyledonous or dicotyledonous plant.

8. Use according to claim 7, wherein the plant is a crop plant of the class Monocotyledoneae or Dicotyledoneae.

9. Use according to claim 7, wherein the plant comprises one or more of soybean, corn, mung bean and wheat.

10. Use according to claim 7, wherein the plant is maize.

11. The Bacillus subtilis of claim 1 (b), (c), (d) and d)Bacillus subtilis) The application of the strain or the composite microbial inoculum of claim 2 in preparing plant growth promoters or microbial organic fertilizers.

12. A method for promoting plant growth under natural soil conditions, comprising administering the Bacillus subtilis of claim 1 (a)Bacillus subtilis) The strain or the complex microbial inoculant of claim 2 is applied to the plant or the environment around the plant.