CN111778173B

CN111778173B - Bacillus subtilis Pro1A2, microbial inoculum and preparation method thereof, and application of bacillus subtilis Pro1A2 in cultivation of melons

Info

Publication number: CN111778173B
Application number: CN202010244458.7A
Authority: CN
Inventors: 梁慎; 赵卫星; 李晓慧; 常高正; 康利允; 高宁宁; 徐小利; 王龙飞; 杨作运
Original assignee: INSTITUTE OF HORTICULTURE HENAN ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: Lankao County Boyang Agricultural Development Co.,Ltd.
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2022-04-15
Anticipated expiration: 2040-03-31
Also published as: CN111778173A

Abstract

The invention discloses a bacillus subtilis Pro1A2, a microbial inoculum thereof, a preparation method and application thereof in melon cultivation, and aims to solve the technical problems of poor disease control effect and difficult overcoming of continuous cropping obstacles in melon cultivation. The invention screens out a strain of bacillus subtilis Pro1A2 for the first time, and the strain is preserved in China center for type culture collection with the preservation number of CCTCC number M2017194. The microbial agent prepared from the bacillus subtilis Pro1A2 has good control effects on plant fungal diseases such as gummy stem blight of melon, wheat take-all, rice blast and the like, has good control effects on bacterial diseases, and has strong inhibition effects on bacterial fruit blotch of melon, bacterial piercing germ of peach, bacterial wilt of tobacco, soft rot of cabbage, bacterial canker of tomato and the like; can also overcome the continuous cropping obstacle of premature senility of melons; high control efficiency, simple use and no environmental pollution.

Description

Bacillus subtilis Pro1A2, microbial inoculum and preparation method thereof, and application of bacillus subtilis Pro1A2 in cultivation of melons

Technical Field

The invention relates to the technical field of biological control, in particular to bacillus subtilis (Bacillus subtilis)Bacillus subtilis) Pro1A2, microbial inoculum thereof, a preparation method and application in melon cultivation.

Background

Common diseases of the melons comprise fruit blotch, gummy stem blight, fusarium wilt, powdery mildew, downy mildew, sclerotinia, angular leaf spot, anthracnose and the like, and continuous cropping aggravate the diseases and continuous cropping obstacles, and if the diseases and the continuous cropping obstacles are not effectively prevented and controlled in time, the yield and the quality of the melons are seriously influenced.

In the prior art, various measures can be taken to prevent and control diseases, such as plant quarantine, utilization of disease-resistant varieties, crop-changing rotation, agricultural prevention and control, chemical prevention and control, physical prevention and control, biological prevention and control and the like.

The disease-resistant variety is the simplest, most convenient, most economical and most effective prevention and control way, enhances seed quarantine, well performs the work of detecting the germ-carrying of the seeds, and should be introduced from a disease-free area, and should avoid the germ-carrying seeds from being mixed into qualified seeds during seed production. But is limited by factors such as narrow genetic background of germplasm resources, limited disease-resistant materials and the like; the most common, fastest and most direct prevention and treatment way is chemical drug prevention and treatment, which reduces the occurrence of diseases to a certain extent, but limits the application of chemical pesticides due to the problems of pesticide residue, food safety, environmental pollution, ecological balance damage, drug resistance of pathogenic substances and the like caused by excessive and frequent use of chemical bactericides; agricultural prevention and control and physical prevention and control only reduce the occurrence of diseases to a certain extent.

Along with the development and progress of social civilization, people have increasingly improved the call for pollution-free and pollution-free green foods, biological control becomes a research hotspot for plant disease control, and the biological control has good compatibility with the future development direction of human beings in terms of production mode, food safety, nutritional health, ecological balance and biological diversity.

The crop rotation is also an effective way for preventing and controlling or reducing plant diseases and insect pests, but is difficult to popularize and implement under the conditions that the per capita cultivated land area of China is extremely limited and geographically distributed unevenly.

At present, biocontrol bacteria are taken as an important part of biological control, and the research on biological control of plant diseases is hot. Due to the wide variety of biocontrol bacteria, various biocontrol bacteria such as fungi, bacteria, actinomycetes and the like are widely applied in production.

The development of new biocontrol strains is urgently needed to play an important role in harmonious coexistence of people and nature, maintenance of ecological balance and promotion of agricultural sustainable development process.

Disclosure of Invention

The invention aims to solve the technical problems of providing the bacillus subtilis Pro1A2, a microbial inoculum thereof and a preparation method thereof, and applying the bacillus subtilis Pro1A2 in melon cultivation so as to solve the technical problems of poor disease control effect and/or difficult overcoming of continuous cropping obstacles in melon cultivation

In order to solve the technical problems, the invention adopts the following technical scheme:

screening to obtain bacillus subtilisBacillus subtilisPro1A2, and is preserved in the China center for type culture Collection (address: Wuhan university, Wuhan city, Hubei province, China, zip code 430072) in 2017, 4 months and 14 days, and the preservation number is CCTCC number M2017194.

Preparing a microbial agent comprising said bacillus subtilis Pro1a2 and/or a metabolite of said bacillus subtilis Pro1a 2.

Data a method for preparing a microbial inoculant comprising the steps of:

(1) inoculating the bacillus subtilis Pro1A2 to a solid culture medium, and culturing at 28-35 ℃ for 24-36 hours;

(2) inoculating the strain cultured in the step (1) into a liquid culture medium, and culturing at 28-35 ℃ for 12-16 hours to obtain a seed solution;

(3) inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 3-5%, fermenting and culturing for 36-48 hours at the temperature of 28-35 ℃, the rotation speed of 180-200 rpm and the pH value of 7.5-8.0, and collecting fermentation liquor to obtain the microbial agent.

Preferably, the viable bacteria concentration of the microbial agent is 2-4 multiplied by 10⁹cfu/ml。

The bacillus subtilis Pro1A2 or the microbial agent can be used for preventing and treating melon fusarium oxysporum, watermelon anthracnose pathogen, melon vine blight pathogen, tomato gray mold pathogen, wheat take-all pathogen, apple brown spot pathogen, wheat sharp eyespot pathogen, cucumber phytophthora, melon bacterial fruit blotch pathogen, tomato bacterial canker pathogen, cabbage soft rot pathogen, tobacco ralstonia solanacearum and peach bacterial puncture pathogen, and can also be used for overcoming or inhibiting continuous cropping and continuous cropping obstacles of melons, saline-alkali stress obstacles and the like.

The administration method of the bacillus subtilis Pro1A2 or the microbial agent comprises the following steps:

the fertilizer is applied in at least one mode of root irrigation, seed soaking and plant spraying; or

After being compounded with a bactericide or a plant regulator, the plant bactericide is applied in at least one mode of root irrigation, seed soaking and plant spraying. The method can be used in the whole melon growth period, and has obvious use effect in the melon seedling period and before pollination, for example, the method adopts the mixed application of a fermentation preparation and a seedling substrate before melon seedling; or the water-soluble fertilizer is used as a field planting and flushing microbial inoculum and is flushed and applied to the rhizosphere of the muskmelon through root irrigation and water-medicine integration or sprayed and applied through plant overground parts such as leaves, stems and the like of the muskmelon.

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

the bacillus subtilis Pro1A2 with excellent comprehensive performance is obtained by screening for the first time and is preserved in China center for type culture collection with the preservation number of CCTCC number M2017194. The microbial agent prepared from the bacillus subtilis Pro1A2 has good control effects on plant fungal diseases such as gummy stem blight of melon, wheat take-all, rice blast and the like, has good control effects on bacterial diseases, and has strong inhibition effects on bacterial fruit blotch of melon, bacterial piercing germ of peach, bacterial wilt of tobacco, soft rot of cabbage, bacterial canker of tomato and the like; the ecological environment of the soil at the root zone of the melon can be adjusted and improved, so that continuous cropping obstacles (premature senility, wilting and the like) of the melon can be overcome; high control efficiency, simple use and environmental protection.

Drawings

FIG. 1 is a phylogenetic tree analysis diagram between Bacillus subtilis Pro1A2 and a reference model strain;

FIG. 2 is a comparison of melon plants 15 days after colonization in example 7.

FIG. 3 is a comparison of melon plants at fruit set-up in example 7; wherein A is a test group, B is a control group showing mild premature senility, and C is a control group showing severe premature senility.

Detailed Description

The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.

The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.

Example 1: isolation and selection of Bacillus subtilis

(1) Isolation and purification of Agrobacterium

Collecting 300g of wet tillage layer soil (collected by Liang cautios in Sanyizi village Digitge village of Lanceu county) 5-20 cm away from the surface layer in 2016 (9/20 days) of 300g, crushing, weighing 10g, placing in a triangular flask containing 100ml of sterile water, shaking for 3-5 min, and heating in 80 ℃ water bath for 10 min. The soil suspension was measured and diluted with sterile water to prepare 10^-2、10^-3、10^-4And 10^-5A soil suspension; respectively suck the above 10^-3、10^-4、10^-5Coating 50 μ L of the soil suspension on NA culture medium (beef extract 3g, yeast powder 1g, peptone 5g, glucose 10g, agar 12g, water 1L, pH 6.8-7.0,sterilized at 121 ℃ for 20 min), plates were coated on a clean bench and air dried. The mixture was inverted and cultured in a biochemical incubator at 37 ℃ for 48 hours. After the single spore colonies grow out, selecting the single colony, purifying by adopting a continuous scribing method, inoculating the single spore colony strain obtained by purification to NA inclined plane culture, and preserving for later use.

The strain preservation method comprises the following steps: preserving the slant culture at 4 ℃; or preparing into 20% glycerol bacterial suspension, and storing at-80 deg.C.

2) Screening of antagonistic bacteria against fungal diseases

Inoculating separated and purified Bacillus into LB culture medium (tryptone 10g, yeast powder 5g, NaCl 10g, water 1L, pH7.0, sterilizing at 121 deg.C for 20 min) to culture for 48 hr, and testing melon Fusarium oxysporumFusarium oxysporium) As a target, a plate confrontation culture method is adopted to determine the bacteriostasis effect.

The specific method comprises the following steps: after activation of melon fusarium wilt germs, inoculating the melon fusarium wilt germs to the center of a PDA (personal digital assistant) plate, culturing for 48h at 28 ℃, inoculating bacillus strains to be tested around the pathogens, observing the bacteriostatic effect after 48h, measuring the size of a bacteriostatic zone, and selecting 6 strains with good antagonism to enter a re-screening process.

3) Bacterial disease antagonistic bacteria screening

Inoculating separated and purified bacillus into LB liquid for 48h, and culturing to obtain melon bacterial fruit blotch bacteriaAcidovorax citrulli) And (3) taking the plate as a target, and measuring the bacteriostatic effect by adopting a plate bacterium injection method.

The specific method comprises the following steps: preparing the activated muskmelon fruit blotch into 10⁸And (3) uniformly coating 50 muL of CFU suspension on an LB (LB) flat plate, then uniformly punching a hole on the LB flat plate by using a sterile oxford cup, taking out a culture medium in the hole, then injecting 100 muL of bacillus liquid, culturing at 30 ℃ for 36-48 h, measuring the size of an antibacterial zone, and selecting 6 strains with good antagonism to enter a re-screening process.

(3) Antagonistic strain rescreening

1) Preparing sterile fermentation filtrate: inoculating 6 antagonistic strains into LB culture solution, performing shake culture at 30 deg.C for 48h, collecting the culture solution in 50ml centrifuge tube, centrifuging at 5000 rpm for 5min, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain sterile fermentation filtrate.

2) Antagonists against pathogenic fungi: and adding 27ml of PDA culture medium melted at 50 ℃ and 3ml of sterile fermentation filtrate into a 50ml sterile centrifuge tube, quickly and fully mixing uniformly, pouring into a sterile culture dish, preparing a double-screen plate, inoculating target pathogenic bacteria in the center of the plate after the plate is solidified, respectively taking the PDA plate of sterile water and LB culture solution as a reference, culturing at constant temperature of 28 ℃ for 3-5 days, measuring the diameter of a bacterial colony, and calculating the inhibition rate.

。

3) Disease pathogenic bacteria are used for preparing an LB flat plate, and the pathogenic fungi to be tested are as follows: muskmelon fusarium wilt bacterium (Fusarium oxysporium) Watermelon anthracnose: (Colletotrid umgloeosporiodes) Gummy stem blight of melon: (Mycosphaerella melonis) Botrytis cinerea (Botrytis cinerea)Botrytis cirerea) Wheat take all over (Gaeumannomyces grami) Brown spot of apple bacterium: (Marssonina coronaria) Rhizoctonia cerealis (Rhizoctonia cerealis) Phytophthora cucumis (A) and (B)Phytophthora melonis）。

The test pathogenic bacteria were: muskmelon bacterial fruit blotch (Acidovorax citrulli) Bacterial canker of tomato: (Clavibater michiganensis) Chinese cabbage Soft rot fungus: (Erwinia carotovora pv.carotovora) Bacterial wilt of tobacco (Ralstonia solanacearum) Peach bacterial fruit of the species Perforaria (Xanthomonas campestris ）。

4) After the prepared LB flat plate is inverted, the center of the bottom of the dish is taken as the center of a circle, the LB flat plate is evenly divided into a plurality of fan shapes by a marking pen, then the LB flat plate is placed in an upright mode, the center of a sterile oxford cup is used for punching, culture medium in the hole is taken out, 100 mu L of sterile fermentation filtrate is injected into different fan-shaped areas, and the concentration of the sterile fermentation filtrate is 10⁸And uniformly coating 10 muL of CFU pathogenic bacteria suspension on a sector area, culturing at 30 ℃ for 24h, observing the bacteriostatic effect, and measuring the bacteriostatic width.

The above experiment was repeated three times, each with an LB plate of 100 μ L sterile water as a control, and after statistics, the inhibitory effect of the sterile fermentation filtrate on each pathogenic bacterium was calculated, with the results shown in tables 1 and 2. And (4) re-screening and screening the Pro1A2 strain with good control effect.

TABLE 1 bacteriostatic effect of Pro1A2 strain on pathogenic fungi

。

TABLE 2 bacteriostatic effect of Pro1A2 strain on pathogenic bacteria

Note: , + ++ represents a band greater than 10 mm; + represents an antibacterial band of 5-10 mm; + the zone of inhibition is less than 5 mm.

Example 2: identification of Bacillus subtilis Pro1A2

(1) Analysis of physicochemical Properties

1) Colony characteristics and morphology observation

Inoculating Bacillus Pro1A2 on NA culture medium, and culturing in biochemical incubator at 37 deg.C for 48 h. And observing the colony morphology and the growth condition of the strain on the culture medium. Bacillus colonies cultured for 48h were picked and gram-stained on glass slides to observe the size and morphology of the cells.

2) Strain physiological and biochemical test determination Pro1A2 Strain physiological and biochemical test determination adopts culture medium and experimental method recommended by 'Manual of common bacteria System identification' and 'Bacillus'. The physiological and biochemical tests mainly comprise: methyl red reaction, catalase test, starch hydrolysis test, hydrogen peroxide test, gelatin liquefaction test, casein decomposition test, anaerobic growth test, indole reaction, cellulose degradation test, growth temperature test, V-P test and carbon source utilization test.

The results of the physical and chemical property analyses are shown in Table 3.

TABLE 3 culture characteristics, morphology and physio-biochemical characteristics of Pro1A2 Strain

Note: + is positive; negative.

(3) The molecular biology and the evolutionary relationship analysis of the strain are as follows:

pro1A2 strain molecular biological analysis mainly adopts 16SrDNA gene fragment, 16SrDNA sequence PCR amplification upstream and downstream primers are synthesized by Nanjing Kingsry bioengineering technology service company, respectively:

16SF：5’-ACGGTTACCTTGTTACGACTT-3’；

16SR：5’-AGAGTTGATCATGGCTCAG-3’。

LB liquid culture of Pro1A2 strain was used as a template, and 16SR and 16SF were used as PCR amplification primers.

The PCR reaction system is as follows: 10 XPCR Buffer (containing Mg)²⁺) 5 muL, 0.5 muL of a template, 2 muL of dNTP (10 mmol/L), 2 muL of a primer 16SR (10 mumol/L), 2 muL of 16SF (10 mumol/L), 1 muL of TagDNA polymerase (5U/muL) and 37.5 muL of deionized water.

The PCR amplification conditions were: 5min at 94 ℃; 1 min at 94 ℃; 45 s at 60 ℃; 1.2 min at 72 ℃; 35 cycles; 10 min at 72 ℃.

The PCR reaction products were detected by 1% agarose gel electrophoresis to obtain 1 expected fragment of about 1.4 kb in length. The target band is purified by an agarose DNA recovery kit (Beijing Baitach Biotechnology, Inc.), and then sent to Shanghai Biotechnology, Inc. for sequencing to obtain a sequence with the size of 1417bp, which is shown as SEQ NO. 1.

BLAST analysis (http:// www.ncbi.nlm.nih.gov/BLAST) is carried out on the sequencing result, DNAMAN software is used for carrying out sequence comparison and calculating the sequence similarity between the test strain and the reference strain, and a phylogenetic tree between the test strain and the reference model strain is constructed, as shown in figure 1.

The DNA sequence is compared and analyzed with a plurality of databases through BLASTB. subtilisThe strains are highly homologous, and the consistency is more than 99%. From the 16SrDNA sequence of Pro1A2 strain with known Bacillus species16SrDNA sequence comparison and construction of phylogenetic tree visible Pro1A2 strains andBacillus subtilis MF767441.1 gathered into one group, and the homology reached 99.65%. Deducing that the Pro1A2 strain is bacillus subtilis by combining physiological and biochemical characteristicsBacillus subtilis。

Example 3: preparation of microbial agent

Pro1A2 microbial inoculum is prepared by the following steps:

(1) strain culture and preparation

Inoculating bacillus subtilis Pro1A2 to a test tube slant soybean casein solid culture medium (tryptone 15g, soybean protease hydrolysate 5g, sodium chloride 5g, glucose 2.5g, agar powder 12g, water added to a constant volume of 1000ml, pH 7.0-7.5, subpackaging, and sterilizing at 121 ℃ for 20 min), and culturing at 28-35 ℃ for 24-36 hours to obtain thalli;

(2) seed liquid preparation

Inoculating the test tube strain prepared in the step (1) into a soybean casein liquid culture medium (15 g of tryptone, 5g of soybean protease hydrolysate, 5g of sodium chloride and 2.5g of glucose, adding water to a constant volume of water to 1000ml, adjusting the pH to 7.0-7.5, subpackaging, and sterilizing at 121 ℃ for 20 min), and performing shake culture at 28-35 ℃ for 12-16 hours to prepare a seed solution;

(3) preparation of fermentation Medium

Respectively taking glucose, corn flour, soybean cake powder and dipotassium hydrogen phosphate, adding water to prepare culture solution containing 1.2% of glucose, 1.8% of corn flour, 3% of soybean cake powder and 0.2% of dipotassium hydrogen phosphate;

(4) liquid fermentation production

After sterilization of fermentation equipment and a fermentation culture medium, inoculating the seed solution to the culture solution prepared in the step (3) according to the inoculation amount of 5%, performing fermentation culture for at least 36-48 hours at the temperature of 28-35 ℃, the rotating speed of 180rpm and the pH value of 8.0, and collecting fermentation liquor to obtain a liquid microbial inoculum with the bacterial concentration of no 2.8 multiplied by 10⁹cfu/ml。

Example 4: test verification of Pro1A2 microbial inoculum for preventing and treating blight at seedling stage of melons

In 2019 in the greenhouse of agricultural academy of sciences, Henan province, 4 months.

Melon fusarium wilt bacteria (preserved in watermelon and melon research laboratory of gardening research institute of academy of agricultural sciences, Henan province) are used as pathogenic bacteria to be tested, and 50% carbendazim (Fuda agricultural chemical Co., Ltd. of Jiangyun city) and clear water are used as controls.

The specific operation steps are as follows:

(1) soaking 20 muskmelon seeds of Zhongyun in 0.1% potassium permanganate solution for 15min, cleaning with sterile water for 3 times, accelerating germination at 28 ℃, sowing the seeds into seedling pots filled with substrates when radicles are exposed to white, performing damp-heat sterilization on the seedling substrates, namely grass carbon, vermiculite and cultivated soil (2: 1: 1), at 121 ℃ for 1.0h, and performing seedling culture in a sunlight greenhouse at 20-25 ℃.

(2) Taking out 10d PDA culture plate for melon wilt pathogen, adding anhydrous 20ml, scraping colony surface with spreader, releasing conidium in water, filtering suspension with 2 layers of sterile lens wiping paper, and making into 1 × 10⁶CFU/ml conidia suspension for pathogenicity determination.

(3) When the melon grows to 2 leaves and one heart, the seedlings with roots are gently pulled up, the roots are washed by water and then soaked in the conidium suspension for 15min, and then the melon seedlings which are inoculated with the bacteria are planted again and planted in a seedling pot filled with a seeding matrix.

(4) And placing the inoculated plants in a light culture room, performing moisture preservation culture for 48h, then respectively inoculating the liquid microbial inoculum diluted by 10 times and the liquid microbial inoculum diluted by 20 times, using clear water and the 800 times solution of 50 percent carbendazim as contrast, performing light irradiation for 14h every day, and controlling the temperature to be 25-28 ℃.

(5) Investigating 1 time every 2 days after inoculation for 10 days, observing the disease condition, recording the disease level, and finally calculating the disease index and the prevention and control effect after 25 days.

The above experiments were performed 3 times in 3 replicates with 20 melon seedlings each.

The disease level of the sweet melon fusarium wilt is 5, namely:

level 0: no symptom, no difference between the growth vigor and the growth vigor of the plants compared with clear water;

level 1: cotyledon yellowing or slight stem base browning;

and 2, stage: leaf wilting or stem base 1/2 browning;

and 3, level: cotyledon and true leaf wilting or plant dwarfing, or stem base 2/3 browning;

4, level: all vascular bundles at the base of the stem to the apical end brown and the root necrosed.

Control effect% = (control disease index-treatment disease index)/control disease index x 100

The results of the prevention and control experiments are shown in table 4:

TABLE 4 prevention and treatment effects of Pro1A2 on blight at seedling stage of melon

。

The control effect of the liquid microbial inoculum diluted by 10 times and 20 times of the microbial inoculum in the embodiment 3 is higher than 50 percent of carbendazim, and the control effect is more than 85 percent.

Example 5: application test of Pro1A2 microbial inoculum as seed treatment agent for preventing and treating melon fruit blotch

In 2019, 10 months in a greenhouse of agricultural academy of sciences in Henan province, and the melon seeds (containing bacterial fruit blotch) and the healthy seeds to be tested are Zhongyun 20 (provided by watermelon and melon research laboratory of horticultural research institute of agricultural academy of Henan province); the test agent was Garinon (manufactured by Beixing chemical industry Co., Ltd.).

The control was 600 times garinong liquid treatment and clear water. Each group of experiments comprises that different medicaments are used for treating healthy seeds and bacteria-carrying seeds for 6 hours, and each group of experiments comprises 6 treatments: CK1 (healthy seed); CK2 (seed with bacteria) + CK3 (seed with bacteria 600 times gaminolone); carrying bacteria seeds + the liquid microbial inoculum of example 3 diluted by 10 times; carrying bacteria seeds + the liquid microbial inoculum of example 3 diluted by 20 times; carrying bacteria seeds + the liquid microbial inoculum of example 3 diluted by 50 times; the bacteria-carrying seeds + the liquid microbial inoculum of example 3 diluted 100 times. And (3) obtaining seeds treated by different medicaments, wherein 30 seeds are taken for each treatment, each seed is planted at four corners of the same small flowerpot, and each treated seed is planted in 6 small pots. Normally culturing for 2 weeks after planting seeds, investigating the incidence of cotyledon 2 weeks after emergence of seedlings, repeating for 3 times, and calculating the prevention and treatment effect.

Control effect after seed treatment = [ 1-incidence rate of treated cotyledon/incidence rate of bacteria-carrying seed control ] × 100%

The results are shown in Table 5.

TABLE 5 prevention and control Effect of Pro1A2 microbial inoculum on bacteria-carrying seeds

。

From the above, it can be seen that: compared with healthy seeds, bacteria-carrying seeds and medicament treatment thereof, the liquid microbial inoculum prepared in the embodiment 3 can be diluted by 10 times and 20 times to improve the seedling rate, the microbial inoculum diluted by 10 times has better control effect, and the seedling rate is improved by 20 times. This indicates that the liquid microbial inoculum of example 3 has control ability on bacterial fruit blotch and growth promoting ability on melon seeds.

Example 6: experimental study on Pro1A2 microbial inoculum for preventing and treating greenhouse melon fruit blotch

The method is carried out in a multi-year continuous cropping greenhouse for muskmelon Saizidakojicun in Lankou county, Henan province in 2019 for 3-6 months, wherein soil of the greenhouse is sandy loam, soil fertilizers are poor, and the muskmelon is cultivated by mulching.

The tested melon variety is Zhongyun 20 (provided by watermelon and melon research laboratory of gardening research institute of academy of agricultural sciences in Henan province); the test agent was 20% sodium dichloroisocyanurate wettable powder (produced by Sanfeng division of industries, Ltd., Yanshan, Shandong, Jinan, Japan). For the test pathogen melon bacterial fruit blotch (preserved in muskmelon research laboratory of gardening research institute of academy of agricultural sciences of Henan province), 750 times of solution treatment of 20% sodium dichloroisocyanurate wettable powder and clear water are used as comparison.

Each set of experiments was divided into 5 treatments, which were: the treatment is carried out by treating with clear water, treating with 750 times of liquid of sodium chloroisocyanurate wettable powder, diluting with 10 times of bacterial liquid of the liquid bacterial agent in example 3, diluting with 20 times of bacterial liquid of the liquid bacterial agent in example 3 and diluting with 50 times of bacterial liquid of the liquid bacterial agent in example 3, wherein 30 strains are treated, and the treatment is randomly arranged and repeated for 3 times.

The specific operation steps are as follows: the application is carried out at the early stage of the onset of the bacterial fruit blotch of the muskmelon, 1 time is carried out at intervals of 5 days, the application time is carried out at the evening with clear weather, the spraying treatment of the stem leaves is carried out, the spraying degree is that the front and the back of the plant leaves are full of medicine drops, and the water drops start to slightly drop at the leaf tips. The investigation is that 5 plants are randomly selected for each treatment, the whole plant leaves are investigated, the percentage of the lesion area of each leaf to the whole leaf area is graded, the disease base number is investigated before the first pesticide application, and the control effect is investigated 7 days after the last pesticide application. Calculating the disease index, the growth rate of the disease index and the correction control effect.

Calculating the formula:

disease index =100 × [ Σ (number of diseased leaves at each stage × relative stage value) ]/(survey total leaf number × 9);

control effect (%) = [1- (CK)₀Disease refers to x PT₁Diseased finger)/(CK₁Disease refers to x P_T0Disease finger)]×100%

CK₀Is the index of the pre-administration condition, CK, of the control area₁As a control zone post-dose disease index, PT₀For pre-drug index in the treatment area, PT₁Is the index of the disease condition after the treatment area is applied.

Grading standard of diseased leaves:

level 0: no disease spots;

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

and 3, level: the lesion area accounts for 6 to 15 percent of the whole leaf area;

and 5, stage: the lesion area accounts for 16 to 25 percent of the whole leaf area;

and 7, stage: the lesion area accounts for 26-50% of the whole leaf area;

and 9, stage: the lesion area accounts for more than 50% of the whole area.

The results are shown in Table 6.

TABLE 6 Experimental results of prevention and treatment of bacterial fruit blotch of muskmelon in greenhouse by Pro1A2 microbial inoculum

。

The results show that: the liquid microbial inoculum of the embodiment 3 has control effect on bacterial fruit blotches from 10 times to 100 times of dilution, the control effect of the microbial inoculum diluted 100 times is very poor and only 15.66 after 2 times of spraying, the control effect of the microbial inoculum diluted 10 times on the bacterial fruit blotches of greenhouse melons is good, and the control effect reaches 92.18%.

Example 7: experimental study on Pro1A2 microbial inoculum for overcoming continuous cropping obstacle of melons

In autumn of 2018, in the county of Lankao, the county of grape trellis, Zhao buttress building village, melon continuous cropping greenhouse test. The soil belongs to sandy loam, the content of organic matters is less, due to high temperature in autumn, the plant grows vigorously, the photosynthesis and transpiration of the plant are strong, the demand of overground parts of the melon on moisture and nutrients is high, the overground parts of the melon grow vigorously, the underground root system grows slowly, particularly, the fruit expansion period begins, the overground parts of the melon need a large amount of nutrition and moisture, the conveying capacity of the root system nutrient and moisture is low, the growth and development demand of the melon cannot be met, and the phenomena of withering and premature senility of the plant are shown.

Experimental treatment:

the method comprises the steps of contrasting 1000kg of decomposed cow dung and 40kg of compound fertilizer (N: P: K =15:15: 15) as base fertilizer, treating 1000kg of decomposed cow dung and 40kg of compound fertilizer (N: P: K =15:15: 15) as base fertilizer, spraying 100 times diluted liquid microbial inoculum prepared in example 3 for one time 7d before seedlings are out of a nursery, spraying 200 times diluted liquid microbial inoculum prepared in example 3 for root irrigation after 7d after field planting, irrigating 100ml of roots in each hole, diluting 200 times diluted liquid microbial inoculum prepared in example 3 for 3d before pollination, irrigating 200ml of roots in each hole, and investigating seedling growth and later plant aging conditions.

The results of the experiment are shown in Table 7.

TABLE 7 analysis of the effect of Pro1A2 on the premature senility of autumn melon

。

After seedlings are planted in a week, the seedlings are treated by root irrigation with the liquid microbial inoculum prepared in the embodiment 3 diluted by 200 times, the survival rate of the seedlings is improved after treatment, the survival rate reaches more than 99 percent, the seedlings grow neatly, the seedlings recover quickly, grow robustly and have developed root systems; as shown in fig. 2, the leaves of the melon plants are thick and green, the stems are thick and strong, the growth of the plants is very uniform and consistent, the leaves of the control plants are thin, the stems are thin, the growth of the plants is not uniform, and the growth potential is general 15 days after the planting; as shown in fig. 3, at the beginning of the fruit expansion period, the treated plants grow strongly, the leaves rarely yellow, the fruits grow normally, the yield of the first-grade fruits after ripening is higher, and the consistency of the yield and the quality after harvesting and the seeds of the product is higher; and after the contrast fruit is expanded, the plants gradually have a yellowing phenomenon, and in a high-temperature sunny day, the withering of individual plants begins to occur, the yellowing and withering phenomena are more serious as the fruit grows and develops, a large-area death phenomenon occurs for some plants, the center sugar of the mature fruit is low, the center sugar of most fruits is below 15 ℃, the first-level fruit rate is very low, and some plants are premature senility, die and stop harvesting in the later stage.

In conclusion, the experiment treatment effectively solves the continuous cropping obstacle caused by early withering and withering of the melons in autumn.

While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.

SEQUENCE LISTING

<110> horticultural research institute of academy of agricultural sciences of Henan province

<120> bacillus subtilis Pro1A2, microbial inoculum and preparation method thereof, and application of bacillus subtilis Pro1A2 in cultivation of melons

<130> 2020

<160> 1

<170> PatentIn version 3.2

<210> 1

<211> 1417

<212> DNA

<213> Bacillus subtilis

<400> 1

tggcgggtct ataatgcagt cgagcgaatg gattaagagc ttgctcttat gaagttagcg 60

gcggacgggt gagtaacacg tgggtaacct gcccataaga ctgggataac tccgggaaac 120

cggggctaat accggataac attttgaacc gcatggttcg aaattgaaag gcggcttcgg 180

ctgtcattta tggatggacc cgcgtcgcat tagctagttg gtgaggtaac ggctcaccaa 240

ggcaacgatg cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc 300

cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag 360

caacgccgcg tgagtgatga aggctttcgg gtcgtaaaac tctgttgtta gggaagaaca 420

agtgctagtt gaataagctg gcaccttgac ggtacctaac cagaaagcca cggctaacta 480

cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa 540

agcgcgcgca ggtggtttct taagtctgat gtgaaagccc acggctcaac cgtggagggt 600

cattggaaac tgggagactt gagtgcagaa gaggaaagtg gaattccatg tgtagcggtg 660

aaatgcggag agatatggag gaacaccagt ggcgaaggcg actttctggt ctgtaactga 720

cactgaggcg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 780

aaacgatgag tgctaagtgt tagagggttt ccgcccttta gtgctgaagt taacgcatta 840

agcactccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc 900

gcacaagcgg tggagcatgt gatttaattc gaagcaacgc gaagaacctt accaggtcgt 960

gacatcctct gaaaacccta gaagatagag cttcctcctt cgggaccaga agtgacaggt 1020

ggtgcatgct tgtcgtcagc tcgtgtcgtg aaatgttgga ttaagtcccg caacgagcgc 1080

aacccttgat cttagtggcc atcattaagt tgagcactct aaggtgaact gccggtgaca 1140

aaccggagga aggtgaggat gacgtcaaat catcatgtcc ccttatgacc tgggctacac 1200

acgtgctaca atggaacggt acaaagagct cgccaagaac acgcgaaggt ggagctaatc 1260

tcatcaaacc gttctcagtt cggattgtag gctgcaaccc gcctacatga agctggaatc 1320

gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380

ccgtcacacc acgagagttt gtaacacccg aagtcgg 1417

Claims

1. A Bacillus subtilis Pro1A2 with preservation number of CCTCC number M2017194.

2. A microbial inoculant comprising bacillus subtilis Pro1a2 of claim 1.

3. The preparation method of the microbial agent is characterized by comprising the following steps of:

(1) inoculating the Bacillus subtilis Pro1A2 of claim 1 onto a solid medium, and culturing at 28-35 deg.C for 24-36 hr;

4. The method for preparing a microbial agent according to claim 3, wherein the viable bacteria concentration of the microbial agent is 2-4 x 10⁹cfu/ml。

5. Use of the bacillus subtilis Pro1a2 of claim 1 or the microbial inoculant of claim 2 for controlling any one of fusarium oxysporum f.melo, colletotrichum citrulli, fusarium oxysporum f.melo, botrytis cinerea, fusarium graminearum, alternaria mali, rhizoctonia cerealis, phytophthora cucumerinum, cucumis melo, bacterial canker of tomato, fusarium solani schott, ralstonia solanacearum, and peach bacterial peronospora persica.

6. Use of the bacillus subtilis Pro1a2 of claim 1 or the microbial inoculant of claim 2 for overcoming or inhibiting continuous cropping obstacles of melons.

7. A method of administering the bacillus subtilis Pro1a2 of claim 1 or the microbial inoculant of claim 2, comprising:

the fertilizer is applied by at least one of root irrigation, seed soaking and plant spraying.