CN109609403B

CN109609403B - Biocontrol bacterium and application thereof in prevention and control of downy mildew of crops

Info

Publication number: CN109609403B
Application number: CN201811583608.6A
Authority: CN
Inventors: 郭坚华; 张丽娜; 蒋春号
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2018-12-24
Filing date: 2018-12-24
Publication date: 2021-12-31
Anticipated expiration: 2038-12-24
Also published as: CN109609403A

Abstract

The invention relates to a biocontrol bacterium and application thereof in preventing and controlling downy mildew of crops, belonging to the technical field of biological prevention and control of crops. The biocontrol bacterium CGMCC No.16732 can effectively prevent and treat the downy mildew of crops, particularly the downy mildew of cucumbers, soybeans and grapes in the aspect of planting application of the cucumbers, the soybeans and the grapes; in addition, the inventor also finds that the biocontrol bacterium CGMCC No.16732 can also reduce the occurrence of powdery mildew of crops, has the growth promoting effect on the crops and improves the crop yield; moreover, compared with the chemical agents in the prior art, the biocontrol bacterium is nontoxic to human and livestock, does not pollute the environment, has no residue, can particularly improve the micro-ecological environment for crop planting, and is beneficial to the sustainable development of the ecological environment.

Description

Biocontrol bacterium and application thereof in prevention and control of downy mildew of crops

Technical Field

The invention relates to a biocontrol bacterium and application thereof in preventing and controlling downy mildew of crops, belonging to the technical field of biological prevention and control of crops.

Background

Downy mildew refers to plant diseases caused by downy mildew in fungi, is widely distributed all over the world and also widely occurs in China, is a main disease of various crops, seriously affects the quality and the yield of the crops and causes serious economic loss.

Peronospora is an obligate parasitic bacterium, and there are Pseudoperonospora cubensis (Pseudoperonospora cubensis) which causes downy mildew of cucumber, Peronospora man-schurica (Peronospora) which causes downy mildew of soybean, and Plasmopara viticola (Plasmopara viticola) which causes downy mildew of grape.

Downy mildew generally starts to attack from lower leaves of plants, light green water stain-shaped small spots are generated at the early stage of attack, the edges of disease spots are not obvious, yellow irregular disease spots develop at the later stage, and grey-white mildew layers are generated on leaf backs when the humidity is high and gradually become dark grey. The diseased leaves gradually turn yellow and dry when the drought occurs in the greenhouse, and the diseased leaves are mildewed when the air humidity is high.

At present, the breeding work of downy mildew resistance has not been developed in a breakthrough manner, the main control measures of downy mildew in agricultural production are still means of spraying chemical agents and the like, the main control measures of downy mildew are still means of spraying chemical agents and the like, common pesticides comprise metalaxyl (Mefenoxam), strobilurin (QoIs, such as azoxystrobin) and the like, but long-term high-frequency use of a bactericide with a single action site causes a large amount of drug-resistant physiological races of downy mildew, and meanwhile, the problems of serious food safety and environmental pollution are brought, and the health of people is seriously threatened. Therefore, the search for new safe and effective alternative products for controlling downy mildew is becoming a major part of our work.

The utilization of beneficial environmental microorganisms to control the occurrence of diseases and induce improvement of crop stress tolerance is a hot point of research in recent years, and especially, the microorganisms are pollution-free to the environment, which has led to the interest of scientists in controlling diseases and coping with adverse environments by biological control means.

The biological control has the characteristics of abundant natural resources, convenient in-situ production and application, low production cost, wide application range and the like, and can protect and improve the ecological environment of farmlands, does not pollute the environment and is safe to people and livestock compared with chemical control. The biocontrol product has multiple factors and components, is beneficial to delaying the occurrence and development of drug resistance of pathogenic microorganisms, has continuous and lasting inhibition effect on plant diseases and insect pests, can reduce the application times and quantity of the medicament, and the production raw materials and the effective components belong to natural products, can return to the nature and ensure sustainable development.

Thus, biological control of downy mildew has become a focus of research, and it is desired in the art to screen for microorganisms capable of controlling downy mildew.

Disclosure of Invention

In view of the above problems and/or other problems of the related art, an aspect of the present invention provides a biocontrol bacterium, which is Bacillus subtilis, deposited in the china general microbiological culture collection center of the culture collection management committee of microorganisms with a collection number of CGMCC No. 16732.

The invention also provides a fungus medicine composition adopting the biocontrol bacterium, which comprises a bactericide and the fungus powder of the biocontrol bacterium; the bactericide is any one or a mixture of more of a copper preparation, a dyson bactericide, a thiram bactericide, pyraclostrobin and trifloxystrobin bactericide.

The invention also provides a bacterial preparation, wherein the bacterial preparation comprises the biocontrol bacteria and an agriculturally and pharmaceutically acceptable auxiliary agent, or the bacterial preparation comprises the bacterial composition and the agriculturally and pharmaceutically acceptable auxiliary agent.

In still another aspect, the invention provides the application of the biocontrol bacterium in preventing and treating downy mildew of crops.

In another aspect, the invention provides the application of the biocontrol strain in preventing and treating powdery mildew of crops.

The invention further provides application of the biocontrol bacterium in cucumber, soybean or grape planting.

The invention also provides application of the fungus medicine composition in preventing and treating downy mildew of crops or powdery mildew of crops.

The invention also provides the application of the fungus medicine composition in the aspect of planting cucumbers, soybeans or grapes.

The invention also provides application of the fungus medicine preparation in preventing and treating downy mildew of crops or in preventing and treating powdery mildew of crops.

The invention also provides the application of the bacterial preparation in the aspect of planting cucumber, soybean or grape.

The biocontrol bacterium CGMCC No.16732 provided by the invention can effectively prevent and treat the downy mildew of crops, particularly the downy mildew of cucumbers, soybeans and grapes in the aspect of planting application of the cucumbers, the soybeans and the grapes; in addition, the inventor also unexpectedly finds that the biocontrol bacterium CGMCC No.16732 can also relieve the occurrence of powdery mildew of crops, has the growth promoting effect on the crops and improves the crop yield; moreover, compared with the chemical agents in the prior art, the biocontrol bacterium is nontoxic to human and livestock, does not pollute the environment, has no residue, can particularly improve the micro-ecological environment for crop planting, and is beneficial to the sustainable development of the ecological environment.

Detailed Description

The present invention will be further described with reference to specific embodiments, but the present invention is not limited to these specific embodiments.

Materials, reagents and the like used in the following embodiments are commercially available unless otherwise specified. The specific techniques or conditions are not indicated, and the procedures or conditions are described in the literature in the art (for example, refer to J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, science Press, translated by Huang Petang et al) or in accordance with the product instructions.

The following describes the operation process of the present inventors separating and screening the biocontrol bacterium AG11 (Bacillus subtilis) CGMCC No.16732 from natural environment.

(1) Isolation of potential biocontrol bacteria

Isolation of potential biocontrol bacteria present in vitro of plants:

weighing 3g of root-surrounding soil of cucumbers (collected in a cucumber greenhouse in Huaiyun area in Huai' an city) as samples, and respectively adding the samples into 27mL of sterilized water (containing sterilized glass beads);

placing the obtained sample in 180rmp shaking table, oscillating for 0.5h, standing for 5min, respectively sucking supernatant for gradient dilution, and taking dilution 10⁴、10⁵And 10⁶Double dilution; respectively taking 0.1mL of LB-coated plate from three gradient dilutions, culturing at 28 ℃ for 24-48 h, selecting the plate with the colony number between 50-300, counting and selecting all colonies, purifying, inoculating into a test tube containing 5mL of LB liquid culture medium, and performing 28 ℃ CShaking at 200rpm for 24-48 h, mixing the bacterial suspension with an equal volume of 80% glycerol solution, and storing at-70 deg.C for later use (Berg et al, 2000).

Isolation of potential biocontrol bacteria present on the surface of plants:

cutting 3g of root, stem and leaf of cucumber (collected from cucumber greenhouse in Huaiyin area of Huai-An city) respectively, placing in 30mL sterile phosphate buffer (pH 7.0-7.2), shaking by 180rmp shaking table for 0.5h, taking out plant sample, standing for 5min, respectively sucking supernatant, performing gradient dilution, and diluting by 10⁴、10⁵And 10⁶Double dilution; respectively taking 0.1mL of LB-coated plate from three gradient dilutions, culturing for 24-48 h at 28 ℃, selecting a plate with the colony number between 50-300, counting and selecting all colonies, purifying, inoculating into a test tube containing 5mL of LB liquid culture medium, shaking at 28 ℃ and 200rpm for culturing for 24-48 h, taking a bacterial suspension, uniformly mixing with an equal volume of 80% glycerol solution, and storing at-70 ℃ for later use (Krechelet al, 2002).

(2) Enzyme production and metabolite activity determination primary screening biocontrol strain

The substances such as protein, chitin, glucan, cellulose and the like are components of the cell wall of the plant pathogenic fungi, can generate strains with the activities of the degrading enzymes of the substances, can degrade the cell wall of the plant pathogenic fungi preliminarily presumed, and have antagonistic activity on the plant pathogenic fungi, so the strains can be used as the standard for primarily screening the biocontrol fungi. Selecting a strain in a vigorous growth period, placing the strain on a protease enzyme activity determination culture medium, a culture medium (Chi-adsorbents) taking colloidal chitin as a unique carbon source, a beta-1, 3-glucanase enzyme activity determination culture medium and a cellulase enzyme activity determination culture medium, culturing for 3 days at 28-30 ℃ after inoculation, observing the existence of a transparent ring, and respectively recording the inner diameter and the outer diameter of the transparent ring. Strains with one or more of the above enzyme producing and secondary metabolite producing activities are retained for further screening.

Finally, a strain is separated from the cucumber leaf part, and is confirmed to be a potential biocontrol strain through detecting the enzyme activity and carrying out a plate antagonism test, and the strain is named as biocontrol bacterium AG 11.

Secondly, the identification process of the biocontrol bacterium AG11 of the invention is as follows:

molecular biological identification

The biocontrol bacterium AG11 obtained by screening is cultured in LB culture medium under the environment of 28 ℃ to logarithmic phase, and the bacterium is collected by centrifugation at 5000 r/min for 5 min.

The genome DNA of the collected thallus is extracted by adopting a genome DNA rapid extraction kit of Shanghai Saibaoshi Gene technology Limited.

Then adopting a PCR amplification kit of Tiangen Biochemical technology Co., Ltd to amplify the gyrB gene segment of the biocontrol bacteria, and taking the extracted genome DNA product as a template; the sequence of the forward primer used for amplification is as follows: 5 '-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3'; the reverse primer sequence adopted by amplification is as follows: 5' -AGCAGGATACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3 "; in the two primer sequences, "N" represents any base in A/G/C/T, "R" represents any base in A/G, and Y represents any base in C/T; the primer synthesis company randomly combines primers when synthesizing the primers, and the synthesized primers obtained are a mixture of these random combinations. The specific PCR amplification procedure was performed according to the kit instructions.

After the PCR product was subjected to 1% agarose gel electrophoresis, the PCR product with the target band was observed under an ultraviolet lamp, and the PCR product was subjected to sequencing by Biotechnology engineering (Shanghai) Co., Ltd. And (3) carrying out homology comparison on the sequencing result through NCBI BLAST software (carrying out similarity comparison with gyrB gene fragments of known bacteria), and identifying the biocontrol bacterium AG11 of the invention as bacillus subtilis according to the comparison result.

Third, preservation of strains

The biocontrol bacterium AG11 for preventing and treating downy mildew of crops belongs to bacillus subtilis, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: No. 3 of West Lu No.1 of Su Xingzhou, Chaoyang, Beijing city, institute of microbiology, China academy of sciences), wherein the preservation date is 11 months 09 days in 2018, and the preservation number is CGMCC No. 16732.

Fourthly, the culture method and the application of the biocontrol bacteria

The inventionThe biocontrol bacterium 5YN8 is streaked on an LB plate, cultured for 24 hours at 28 ℃, picked into a test tube containing 5ml of LB culture solution after a single bacterium grows out, and cultured for 12 hours at 200rpm at 28 ℃ to be used as seed solution. Inoculating the seed solution to 500mL LB culture solution at a ratio of 1:100, culturing at 28 deg.C and 200rpm for 24 hr until the total viable bacteria concentration is 5 × 10⁹～1×10¹⁰CFU/ml。

The biocontrol bacterium AG11 of the invention is streaked on an LB plate, cultured for 24h at 28 ℃, picked into a test tube containing 5ml of LB culture solution after a single bacterium grows out, cultured for 12h at 200rpm at 28 ℃ and used as seed solution. Inoculating the seed solution to 500mL LB culture solution at a ratio of 1:100, culturing at 28 deg.C and 200rpm for 24 hr until the total viable bacteria concentration is 5 × 10⁹～1×10¹⁰CFU/ml。

In general, before the disease of crops, the biocontrol bacteria AG11 of the present invention is diluted 500 to 1000 times to be sprayed on the leaf surface of the crops at intervals of 10 to 20 days after transplanting, and the transplanted crops can be irrigated with roots after the culture obtained by diluting 100 to 200 times to be transplanted into the soil immediately after the crops are transplanted into the soil.

Fifth, the result of the greenhouse disease prevention test of the biocontrol bacterium AG11 of the invention on cucumber downy mildew and soybean downy mildew

Taking cucumber seed (Jinyou No. 35, purchased from Tianjin Kerun agriculture science and technology Co., Ltd.) and using 10% H₂O₂Sterilizing the surface of the solution for 10min, cleaning with sterile water, and air drying. Each nutrition pot is used for sowing 10 seeds. After normal watering management for 4 weeks, transplanting the seedlings with consistent growth vigor into a 330ml plastic cup, and carrying out spray treatment after 10 true leaves grow out.

Similarly, the tested soybean seeds (source: Jiangsu province academy of agricultural sciences) were treated with 10% H₂O₂Sterilizing the surface of the solution for 10min, cleaning with sterile water, and air drying. Each nutrition pot is used for sowing 10 seeds. After normal watering management for 4 weeks, transplanting the seedlings with consistent growth vigor into a 330ml plastic cup, and carrying out spray treatment after 10 true leaves grow out.

The specific spray treatment operation is as follows: each seedling of the treatment group is sprayed with 30ml of biocontrol agentSuspension of AG11 (viable cell concentration 1X 10)⁷CFU/ml, and containing 0.01% wt of surfactant Tween-20), and 30ml of sterilized water (containing 0.01% wt of surfactant Tween-20) was sprayed on each seedling in the control group. The treatment group and the control group were each provided with 4 replicates, each replicate for 48 seedlings.

After 5 days after the spray treatment of the treatment group and the control group, the spray inoculation (inoculation concentration of 10) was performed on the experimental group and the control group⁵Peronosporangia Peronospora suspension/ml), the amount sprayed is such that the leaf surface is completely wetted through and drips off.

Moisturizing at 20-25 deg.C, counting diseases after the control group is attacked (about 15 days), and calculating biocontrol effect.

For statistics of the incidence, the classification criteria for cucumber downy mildew disease are presented below:

level 0: no disease spots;

level 1: the inoculation point has slight scab, and the diameter of the inoculation point is less than 0.5 cm;

and 3, level: the diameter of the lesion is 0.5-1.3 cm;

and 5, stage: the yellowing area of the inoculation point is less than the cotyledon area 1/2, and the necrosis area is less than 1/3;

and 7, stage: necrotic plaque area is 1/3-2/3;

and 9, stage: the area of necrotic spots is above 2/3 or the whole plant dies.

For statistics of the incidence, the classification criteria of soybean downy mildew disease are described below:

level 0: no disease spots;

level 1: the disease area accounts for less than 10% of the area of the blade;

and 2, stage: the disease area accounts for 11 to 20 percent of the area of the leaf;

and 3, level: the disease area accounts for 21-50% of the leaf area;

4, level: the disease area accounts for 51 to 70 percent of the area of the leaf;

and 5, stage: the area of the affected part is more than 71 percent of the area of the blade.

Calculation formulas for "disease severity" and "biocontrol effect":

disease severity { ∑ (disease grade number × number of plants with the disease grade) }/(total number of plants × highest disease grade);

the biocontrol effect is (control group disease severity-treatment group disease severity)/control group disease severity;

the results of the cucumber downy mildew test are shown in table 1 below.

TABLE 1

	Disease severity (%)	Biocontrol effect (%)
			Treatment group	2.75±1.42	70.43
Control group	9.30±0.02	-

The results of disease severity were mean. + -. standard error (the same applies hereinafter).

The results of the soybean downy mildew test are shown in table 2 below.

TABLE 2

	Disease severity (%)	Biocontrol effect (%)
			Treatment group	58.75±1.31	72.25
Control group	16.30±0.16	-

The results of the greenhouse disease prevention tests in tables 1 and 2 show that the biocontrol bacterium AG11 of the present invention has significant control effect on cucumber and soybean downy mildew.

Sixthly, the result of the greenhouse disease prevention test of the biocontrol bacterium AG11 of the invention on grape downy mildew

And (4) collecting third to fifth unfolded leaves (source: Shanghai African garden) of disease-free grape branches of 1 year, immediately storing in an ice box, and transporting back. And (4) washing the cut leaves with sterile water, and airing at normal temperature. Leaf disks (diameter 11mm) were punched out with a punch and placed in a petri dish containing 0.8% water agar.

Each leaf disc was inoculated with 35. mu.L of a downy mildew spore suspension (concentration 10)⁵Sporangia/ml), the droplets were removed 24h after inoculation and cultured under alternating light and dark conditions at 20 deg.C/18 deg.C for 16/8 h.

After 12h of culture, 50. mu.L of a suspension of biocontrol bacterium AG11 (viable cell concentration 1X 10)⁷CFU/ml) is point-connected on a grape leaf disc of the treatment group, and then cultured under the condition of alternating light and dark at 20 ℃/18 ℃ for 16/8 h; the control group was spotted on a grape leaf disk using 50. mu.L of sterilized water and cultured in the same manner.

Moisturizing at 20-25 deg.C, counting diseases (number of affected discs) after the control group has suffered from diseases (about 7 days), and calculating biocontrol effect.

The statistics of the disease occurrence are that the disease classification is carried out by grading the spore production by inoculating a leaf disc, and the classification standard of the grape downy mildew disease is described as follows:

level 0: no obvious spore production;

level 1: 1-10% of the area of the leaf disc produces spores;

and 2, stage: 10% -25% of the area of the leaf disc produces spores;

and 3, level: producing spores in 25-50% of the area of the leaf disc;

4, level: spore production is carried out on 50-75% of the area of the leaf disc;

and 5, stage: 75-100% of the area of the leaf disc produces spores.

The sporulation index was calculated according to the following formula:

sporulation index ═ Σ (leaf disc number of each stage × relative stage value)/total leaf disc number

The results of the grape downy mildew test are shown in Table 3 below.

TABLE 3

	Spore production index (%)	Biocontrol effect (%)
			Treatment group	0.91±0.03	73.08
Control group	3.38±0.21	-

From the results of the greenhouse disease prevention test for grape downy mildew in table 3 above, it was shown that the biocontrol bacterium AG11 of the present invention has significant control effect on grape downy mildew.

Seventhly, the result of the greenhouse disease prevention test of the biocontrol bacterium AG11 on cucumber powdery mildew and soybean powdery mildew

The process of growing seedlings and transplanting cucumber seeds and soybean seeds is described in the fifth section above.

The operation of the spraying treatment is also substantially the same as in the sixth section, specifically as follows:

spraying 30ml of suspension bacterial liquid of biocontrol bacterium AG11 (viable bacteria concentration is 1 × 10) to each seedling of the treatment group⁷CFU/ml, and containing 0.01% of surfactant Tween-20), and 30ml of sterilized water (containing 0.01% of surfactant Tween-20) was sprayed on each seedling in the control group. The treatment group and the control group were each provided with 4 replicates, each replicate for 48 seedlings.

After 5 days after the spray treatment of the treatment group and the control group, the spray inoculation (inoculation concentration of 10) was performed on the experimental group and the control group⁵Spore suspension of powdery mildew of individual sporangia/ml), the amount sprayed is preferably such that the leaf surface is completely wetted through and drips off.

Regarding the statistics of the onset, the following sets forth the grading criteria for powdery mildew (both cucumber seedlings and soybean seedlings apply):

level 0: no disease spots;

level 1: the disease area accounts for less than 5% of the leaf area;

and 3, level: the disease area accounts for 6 to 10 percent of the area of the leaf;

and 5, stage: the disease area accounts for 11 to 20 percent of the area of the leaf;

and 7, stage: the disease area accounts for 21-40% of the leaf area;

and 9, stage: the area of the affected part is more than 40 percent of the area of the blade.

Calculation formulas for "disease severity" and "biocontrol effect":

the cucumber powdery mildew test results are shown in table 4 below.

TABLE 4

	Disease severity (%)	Biocontrol effect (%)
			Treatment group	9.30±0.28	65.05
Control group	3.25±0.42	-

The soybean powdery mildew test results are seen in table 5 below.

TABLE 5

	Disease severity (%)	Biocontrol effect (%)
			Treatment group	12.3±0.04	57.89
Control group	5.18±0.03	-

The results of the greenhouse disease prevention tests in tables 4 and 5 show that the biocontrol bacterium AG11 has a remarkable control effect on powdery mildew of cucumbers and soybeans.

Eighthly, the result of the greenhouse disease prevention test of the biocontrol bacterium AG11 on grape powdery mildew

Grape leaf discs were obtained in the same manner as in the sixth section above.

each leaf disc was inoculated with 35. mu.L of powdery mildew spore suspension (concentration 10)⁵Sporangia/ml), the droplets were removed 24h after inoculation and cultured under alternating light and dark conditions at 20 deg.C/18 deg.C for 16/8 h.

The statistics of the disease occurrence are that the disease classification is carried out by the spore yield classification of leaf disc inoculation, and the classification standard of grape powdery mildew is introduced as follows:

level 0: no obvious spore production;

level 1: 1-10% of the area of the leaf disc produces spores;

and 2, stage: 10% -25% of the area of the leaf disc produces spores;

and 3, level: producing spores in 25-50% of the area of the leaf disc;

and 5, stage: 75-100% of the area of the leaf disc produces spores.

The sporulation index was calculated according to the following formula:

The results of the grape downy mildew test are shown in Table 6 below.

TABLE 6

	Spore production index (%)	Biocontrol effect (%)
			Treatment group	3.2±0.12	62.18
Control group	1.21±0.09	-

The results of the greenhouse disease prevention test for grape powdery mildew in table 6 show that the biocontrol bacterium AG11 has a remarkable control effect on grape powdery mildew.

Ninth, the results of the field test of the biocontrol bacterium AG11 of the present invention on cucumber planting (controlling downy mildew and powdery mildew, and promoting growth)

The cucumber seeds (jin you 35, purchased from Tianjin Ke Run agriculture technology Co., Ltd.) after germination acceleration are sown in the basin bowl filled with sterile soil, one seed is planted in each basin, and the soil is kept dry before sowing.

Transplanting the cucumber into the field to perform plot experiment when the cucumber grows to 2-leaf stage, wherein the experiment adopts completely random design, each treatment is repeated for 4 times, and the area of each repeated plot is 30m²(3m multiplied by 7.5m) and protection rows are arranged among the cells.

Spraying the cucumber for the 1 st time after 10 days of transplanting (5-6 true leaves are grown), and spraying 30ml of the biocontrol bacterium AG11 bacterial liquid (viable bacteria concentration is 1.0 multiplied by 10) on each seedling of the treated group⁷CFU/mL, containing 0.01% wt surfactant Tween-20), and 30mL sterile water (containing 0.01% wt surfactant Tween-20) was sprayed to each seedling of the control group.

The field experiments all used standard field management modes and no other bactericide was used. Spraying the cucumber fertilizer once every 10 days, and spraying the cucumber fertilizer 4 times in the whole growth period of the cucumber; after 60 days, the cucumber disease control (the disease control of downy mildew and powdery mildew) and the growth promotion result (observing the change of crop seedlings, counting the biomass of each plant, namely the plant height, the stem thickness, the chlorophyll content, the connective number and the single fruit weight, and counting the yield increase rate) are counted.

For the criteria for grading cucumber downy mildew diseases, as well as the calculation of the severity of the disease and the biocontrol effect see the fifth section above.

For the grading criteria of cucumber powdery mildew disease, as well as the calculation of disease severity and biocontrol effect see the seventh section above.

See table 7 below for the field test results for control of cucumber downy mildew.

TABLE 7

	Disease severity (%)	Control effect (%)
			Treatment group	38.9±0.09	70.43
Control group	11.5±1.71	-

See table 8 below for field test results for cucumber powdery mildew control.

TABLE 8

	Disease severity (%)	Control effect (%)
			Treatment group	12.3±0.09	55.28
Control group	5.5±1.71	-

From the results of the above tables 7 and 8, it can be seen that the biocontrol bacterium AG11 of the present invention can significantly reduce the disease severity of cucumber downy mildew, and the average biocontrol effect thereof reaches 70.43%, and also can significantly reduce the disease severity of cucumber powdery mildew, and the average biocontrol effect thereof reaches 55.28%.

The results of the field test on the effect of the biocontrol bacterium AG11 of the present invention on the growth promotion of cucumber plants are shown in Table 9 below.

TABLE 9

As can be seen from the results in table 9, the biocontrol bacterium AG11 of the present invention has significant growth promoting effect on cucumber cultivation, significant increase in biomass (plant height, stem thickness, chlorophyll, connective number and fruit weight), and yield increase of 41.89% compared with the control group.

Ten, the field test result of the biocontrol bacterium AG11 of the invention on soybean planting (preventing and treating downy mildew and powdery mildew and promoting growth)

For field planting of soybeans, an equidistant hill-drop cultivation method is adopted, the row spacing is 70 cm, the hill spacing is 15-20 cm, 3-4 plants are planted in each hole, the experiment adopts completely random design, each treatment is repeated for 4, and the area of each repeated cell is 30m²(3m multiplied by 7.5m) and protection rows are arranged among the cells.

Spraying the soybean for the 1 st time when the fifth true leaf grows out, spraying every 20 days for 4-5 times in the whole growth period, and spraying 30ml of the biocontrol bacterium AG11 bacterial liquid (viable bacteria concentration is 1.0 × 10) to each seedling of the treated group⁷CFU/mL, containing 0.01% wt surfactant Tween-20), and the same amount of sterile water (containing 0.01% wt surfactant Tween-20) was sprayed and treated for each seedling of the control group. The field experiments all used standard field management modes and no other bactericide was used.

At the initial stage of soybean pod setting, statistics is carried out on soybean disease control (disease control of downy mildew and powdery mildew) and growth promotion results (observation of changes of crop seedlings, statistics of biomass of each plant, namely plant height, branch number and pod setting number, and statistics of yield increase rate).

For the criteria for grading soybean downy mildew disease, and the calculation of disease severity and biocontrol effect see section five above.

For soybean powdery mildew disease grading criteria, as well as calculation of disease severity and biocontrol effect see section seven above.

See table 10 below for the results of field trials for control of soybean downy mildew.

Watch 10

	Disease severity (%)	Control effect (%)
			Treatment group	17.3±0.09	70.43
Control group	58.5±4.31	-

See table 11 below for field test results for soybean powdery mildew control.

TABLE 11

	Disease severity (%)	Control effect (%)
			Treatment group	10.5±1.33	52.9
Control group	22.3±0.09	-

From the results of the above tables 10 and 11, it can be seen that the biocontrol bacterium AG11 of the present invention can significantly reduce the disease severity of soybean downy mildew, and the average biocontrol effect thereof reaches 70.43%, and simultaneously can significantly reduce the disease severity of soybean powdery mildew, and the average biocontrol effect thereof reaches 52.9%.

The results of field trials of the effect of the biocontrol bacterium AG11 of the present invention on the growth promotion aspect of soybean plants are seen in table 12 below.

TABLE 12

As can be seen from the results in table 12, the biocontrol bacterium AG11 of the present invention has a significant growth promoting effect on soybean planting, and each biomass (plant height, branch number and pod number) is significantly increased, and the yield is increased by 35.64% compared with the control group.

Eleventh, the field test result of the biocontrol bacterium AG11 of the invention for grape planting (preventing downy mildew and powdery mildew, and promoting growth effect)

Selecting 2-year-old organic vineyards (places: Shanghai African garden) in field experiments, adopting completely random design, treating 4 times each time, wherein the area of each repeated cell is 25m²(5m is multiplied by 5m), and protection rows are arranged among cells.

Spraying the 1 st time when grape in field has sporadic disease, and spraying the bacteria solution of the biocontrol bacteria AG11 (viable bacteria concentration is 1.0 × 10)⁷CFU/mL, containing 0.01% wt surfactant Tween-20), spraying sterile water (containing 0.01% wt surfactant Tween-20) to control group; the application amount is preferably that the medicine is uniformly applied to the front and back surfaces of the leaves and slightly drips.

The field experiments all used standard field management modes and no other bactericide was used. The bacterial liquid of the biocontrol bacterium AG11 of the invention is sprayed every 10 days, and after 4 times of spraying, the grape disease control (the disease control of downy mildew and powdery mildew) and the growth promotion result (the change of crop seedlings is observed, the biomass of each plant is counted, namely the stem thickness, the chlorophyll content, the fruit setting number and the single fruit weight, and the yield is counted) are counted.

For the grading criteria of the grape downy mildew disease, and the calculation of the severity of the disease and the biocontrol effect see section six above.

For the grading criteria of grape powdery mildew disease, and the calculation of disease severity and biocontrol effect see section eight above.

See table 13 below for the field test results for control of grape downy mildew.

Watch 13

	Disease severity (%)	Control effect (%)
			Treatment group	19.48±1.79	71.05
Control group	67.31±5.48	-

See table 14 below for field test results for grape powdery mildew control.

TABLE 14

	Disease severity (%)	Control effect (%)
			Treatment group	5.48±1.79	64.20
Control group	15.31±5.48	-

From the results in tables 13 and 14, it can be seen that the biocontrol bacterium AG11 of the present invention can significantly reduce the disease severity of grape downy mildew, and the average biocontrol effect thereof reaches 71.05%, and also can significantly reduce the disease severity of grape powdery mildew, and the average biocontrol effect thereof reaches 64.20%.

The results of field trials of the effect of the biocontrol bacterium AG11 of the present invention on the growth promoting aspect of grapes are shown in Table 15 below.

Watch 15

From the results in table 15, it can be seen that the biocontrol bacterium AG11 of the present invention has significant growth promoting effect on grape cultivation, significant increase in biomass (stem thickness, chlorophyll content, fruit set number and fruit weight) and yield increase of 54.94% compared with the control group.

Twelfth, test of the Effect of the biocontrol bacterium AG11 of the present invention on improving the micro-ecological environment for crop cultivation

In the following, the effect of the biocontrol bacterium AG11 of the present invention on the microecological environment of crop planting is examined by taking cucumber planting as an example.

Subjecting cucumber seed to 10 wt% concentration of H₂O₂Sterilizing the surface of the solution for 10min, cleaning with sterile water, and air drying.

Sowing the cucumber seeds after germination acceleration into a basin bowl filled with sterile soil, wherein each basin is provided with one seed, and the soil is kept dry before sowing. When the seedlings grow to 2-leaf stage, transplanting the seedlings into the field, treating 48 seedlings in each group, randomly placing the seedlings, and repeating the steps for 3 times.

After waiting for about 30 days (10 true leaves were grown), it was sprayed. The specific spray treatment operation is as follows: spraying suspension of biocontrol bacterium AG11 (viable bacteria concentration of 1 × 10) on leaves of the treated plants⁷CFU/ml, and containing 0.01% wt of surfactant Tween-20), and 30ml of sterilized water (containing 0.01% wt of surfactant Tween-20) was sprayed on each seedling in the control group. The treatment group and the control group were each provided with 3 replicates of 48 seedlings each.

After 30 days, the leaves were sampled (marked in advance to prevent the influence of newly grown leaves), and the biocontrol bacteria-applied leaves were cut with sterilizing scissors. The leaf samples taken back were weighed 10g each, transferred under aseptic conditions to 100ml of an eluent (0.1mol/L potassium phosphate buffer, pH7.0), shaken at 15 ℃ at 200r/min for 30min, and sonicated in an ultrasonic cleaner for 5 min. Collecting eluate, centrifuging at 4 deg.C 10000r/min for 20min, collecting precipitate, and extracting microorganism DNA.

The improved chemical cracking process is adopted to extract plant phyllospheric genome DNA directly, and the method comprises the following steps: suspending 0.2g of the collected plant phyllospheric bacteria thallus in 800 μ L of lysate, adding 5 μ L of 20mg/ml proteinase K, and performing warm bath at 37 deg.C for 30 min; adding 20% SDS solution to make the final concentration 2%, mixing, warm-bathing at 60 deg.C for 1h, taking out, and shaking for 4-5 times; adding equal volume of chloroform, mixing, and centrifuging at 1000r/min for 8 min; adding isopropanol with volume 0.6 times of that of the supernatant, standing overnight at normal temperature, and centrifuging at 12000r/min for 20 min; the supernatant was discarded, dried and resuspended in 30. mu.L of TE buffer. 16S high throughput sequencing was performed on the leaf periphery genome.

The inventors examined the bacteria with a large change in the periphyton microbial community of the leaves of the treated group and the control group before the treatment and 30 days after the treatment. Through comparison, the following results are found: the bacteria of pseudomonas, streptomyces, sphingomonas and serratia which are treated by the biocontrol bacterium AG11 bacterial liquid spray (treatment group) have higher population numbers which are higher than those of the bacteria of a control group, which shows that: the biocontrol bacterium AG11 bacterium liquid spray treatment can obviously improve the population quantity of the beneficial microbes around the leaves.

The population quantity of the methylobacterium, the microbacter, the acinetobacter and the agrobacterium is lower than that of the control group, which shows that the spray treatment of the biocontrol bacterium AG11 bacterial liquid can obviously reduce the population quantity of potential pathogenic microorganisms around leaves.

In particular, the population numbers of erwinia and carnobacterium were high on the uninfected new leaves before treatment, and after the spray treatment with the biocontrol bacterium AG11 of the present invention, the population numbers of both bacteria were significantly reduced even with the invasion of pathogenic bacteria, indicating that the invasion of pathogenic fungi and the reduction in the population numbers of both bacteria were correlated.

Therefore, it can be seen that the biocontrol bacterium AG11 of the invention has a significant improvement effect on the micro-ecological environment for crop planting.

In conclusion, the biocontrol bacterium AG11(CGMCC No.16732) provided by the invention can effectively control the downy mildew of crops, and particularly can effectively control the downy mildew of cucumbers, soybeans and grapes in the aspect of planting application of the cucumbers, the soybeans and the grapes; in addition, the inventor also unexpectedly finds that the biocontrol bacterium CGMCC No.16732 can also relieve the occurrence of powdery mildew of crops, has the growth promoting effect on the crops and improves the crop yield; moreover, compared with the chemical agents in the prior art, the biocontrol bacterium is nontoxic to human and livestock, does not pollute the environment, has no residue, can particularly improve the micro-ecological environment for crop planting, and is beneficial to the sustainable development of the ecological environment.

After the biocontrol bacterium AG11 has the control effect on the downy mildew of crops, a conventional bactericide (chemical agent) for controlling the downy mildew can be compounded with the bacterium powder of the biocontrol bacterium AG11 to obtain a bacterium-medicine composition.

The conventional bactericides for preventing and treating downy mildew include copper preparations, dyson bactericides, thiram bactericides, pyraclostrobin and trifloxystrobin bactericides.

Therefore, any one or more of the conventional bactericides can be compounded with the bacterial powder of the biocontrol bacterium AG11 to obtain the bacterial-medicine composition, and the skilled person can mix the proportions according to the specific needs and the drug effects and can verify the composition through limited tests.

In addition, after the biocontrol bacterium AG11 has the control effect on the downy mildew and powdery mildew of crops, the biocontrol bacterium AG11 can be combined with a pesticide acceptable auxiliary agent to obtain a bactericide preparation for controlling the downy mildew and the powdery mildew of crops.

After the control effect of the biocontrol bacterium AG11 on the downy mildew and powdery mildew of cucumbers, soybeans and grapes and the growth promotion effect of the cucumbers, the soybeans and the grapes are obtained, the biocontrol bacterium AG11 can be combined with a pesticide acceptable auxiliary agent to obtain a bactericide preparation applied to the planting of the cucumbers, the soybeans and the grapes.

Similarly, the above-mentioned fungicide composition (composition of fungicide and fungus powder of the biocontrol fungus AG11 of the present invention) can be combined with agrochemically acceptable adjuvant to obtain fungicide preparations for controlling downy mildew and powdery mildew of crops, and fungicide preparations for planting cucumber, soybean and grape.

It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.

The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims

1. a bio-control bacterium, is characterized in that: described bio-control bacterium is Bacillus subtilis ( Bacillus subtilis ), is preserved in China Microorganism Culture Collection General Microorganism Center, and its deposit number is CGMCC No.16732.

2. a kind of bacterial medicine composition adopting the biocontrol bacteria as claimed in claim 1, is characterized in that: described bacterial medicine composition comprises the bacteria powder of bactericide and the described biocontrol bacteria of claim 1; Said bactericide It is any one or a mixture of copper preparations, dysen fungicides, fume fungicides, pyraclostrobin and tristrobin fungicides.

3. a bacterial medicine preparation, it is characterized in that: described bacterial medicine preparation comprises biocontrol bacteria as claimed in claim 1 and pesticide acceptable auxiliary agent, or described bacterial medicine preparation comprises as claimed in claim 2 The bacterial pharmaceutical composition and the agrochemically acceptable adjuvant.

4. the application of biocontrol bacteria as claimed in claim 1 in preventing and treating cucumber downy mildew, soybean downy mildew or grape downy mildew.

5. the application of the biocontrol bacteria as claimed in claim 1 in preventing and treating cucumber powdery mildew, soybean powdery mildew or grape powdery mildew.

6. The application of biocontrol bacteria as claimed in claim 1 in promoting the growth of cucumber, soybean or grape.

7. The application of the fungal composition as claimed in claim 2 in preventing and treating cucumber downy mildew, soybean downy mildew or grape downy mildew or in preventing and treating cucumber powdery mildew, soybean powdery mildew or grape powdery mildew.

8. The application of the bacterial medicine composition as claimed in claim 2 in promoting the growth of cucumber, soybean or grape.

9. The application of the fungus preparation as claimed in claim 3 in preventing and treating cucumber downy mildew, soybean downy mildew or grape downy mildew or in preventing and treating cucumber powdery mildew, soybean powdery mildew or grape powdery mildew.

10. The application of the bacterial medicinal preparation according to claim 3 in promoting the growth of cucumber, soybean or grape.