CN111548951A

CN111548951A - Bacillus subtilis Pro6A5, microbial inoculum and preparation method thereof, and application of bacillus subtilis Pro6A5 in cultivation of melons

Info

Publication number: CN111548951A
Application number: CN202010246791.1A
Authority: CN
Inventors: 赵卫星; 李晓慧; 梁慎; 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: INSTITUTE OF HORTICULTURE HENAN ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-08-18
Anticipated expiration: 2040-03-31
Also published as: CN111548951B

Abstract

The invention discloses a bacillus subtilis Pro6A5, a microbial inoculum thereof, a preparation method and application thereof in melon cultivation, and aims to solve the technical problem of limited disease control in melon cultivation. The invention screens out a strain of bacillus subtilis Pro6A5 for the first time, and the strain is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2017196. The microbial agent prepared from the bacillus subtilis Pro6A5 has good effect of preventing and treating bacterial diseases, and has strong inhibiting effect on melon bacterial fruit blotch, peach bacterial punch, tobacco ralstonia solanacearum, Chinese cabbage soft rot, tomato canker and the like; the continuous cropping obstacle of the melons in the saline-alkali soil can be overcome; high control efficiency, simple use and no environmental pollution.

Description

Bacillus subtilis Pro6A5, microbial inoculum and preparation method thereof, and application of bacillus subtilis Pro6A5 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) Pro6A5, a microbial inoculum thereof, a preparation method and application in melon cultivation.

Background

Common diseases of the melons comprise blight, powdery mildew, downy mildew, sclerotinia, angular leaf spot, anthracnose and the like, and continuous cropping of the melons further 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, agricultural control, chemical control, physical control, biological 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.

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.

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.

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 that the bacillus subtilis Pro6A5, the microbial inoculum thereof and the preparation method are provided and applied to melon cultivation so as to solve the technical problems that the disease control effect is poor and the salt-alkali adversity stress or/and the continuous cropping obstacle are difficult to overcome in the melon cultivation

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

screening to obtain bacillus subtilisBacillus subtilisPro6A5, 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, wherein the preservation number is CCTCC NO: m2017196.

Preparing a microbial agent comprising said bacillus subtilis Pro6a5 and/or a metabolite of said bacillus subtilis Pro6a 5.

Provides a preparation method of a microbial agent, which comprises the following steps:

(1) inoculating the bacillus subtilis Pro6A5 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 2-3 × 10⁹cfu/ml。

The bacillus subtilis Pro6A5 or the microbial agent is applied to prevention and control of melon bacterial fruit blotch, tomato bacterial canker, Chinese cabbage soft rot, tobacco bacterial wilt or peach bacterial puncture germs and the like, and can also be used for inhibiting or overcoming saline-alkali adversity stress or/and melon continuous cropping and continuous cropping obstacles.

Providing a method of administering said bacillus subtilis Pro6a5 or said microbial agent comprising: 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 seed dressing agent can be applied to the muskmelon in the whole growth period, such as before sowing, the seed dressing agent and the seeds are adopted for seed dressing; during seedling raising, the microbial inoculum and a matrix are mixed in proportion to prepare a specific matrix, and the specific matrix is applied to the rhizosphere of crops by root irrigation or sprayed on the surfaces of leaf surfaces, flower buds and fruits in the initial stage of diseases.

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

the bacillus subtilis Pro6A5 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 NO: m2017196. The microbial agent prepared from the bacillus subtilis Pro6A5 has good effect of preventing and treating bacterial diseases, and has strong inhibiting effect on melon bacterial fruit blotch, peach bacterial punch, tobacco ralstonia solanacearum, Chinese cabbage soft rot, tomato canker and the like; the ecological environment of the melon rhizosphere soil can be adjusted and improved so as to overcome the saline-alkali soil stress obstacle or/and the melon continuous cropping obstacle; high control efficiency, simple use and environmental protection.

Drawings

FIG. 1 is a phylogenetic tree analysis of B.subtilis Pro6A5 with reference model strains.

FIG. 2 is a comparative plot of the emergence status of melons tested in example 7.

FIG. 3 is a comparative plot of the root system of the melon tested in example 7 after 20 days of sowing.

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 melon field (collected by Liuqian in grape trellis village in Lankai county, Lancey, county, etc. at 20 days 9 and 2016), wetting soil layer of 5-20 cm, pulverizing, weighing 10g, placing in a triangular flask containing 100ml of sterile water, shaking for 3-5 min, and heating in 80 deg.C 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^-5Spreading 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, sterilizing at 121 deg.C 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) Bacterial disease antagonistic bacteria screening

Inoculating the 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 bacterial fruit blotch of melonAcidovorax citrulli) And (3) taking the strain as a target, and separating and screening antagonistic bacteria by adopting a flat plate strain injection method.

The specific method comprises the following steps: taking activated melon fruit blotch germs, preparing 108CFU suspension, taking 50 muL to be evenly coated on an LB flat plate, then evenly punching the LB flat plate by using an aseptic oxford cup, taking out a culture medium in the hole, then injecting 100 muL of bacillus liquid, culturing for 36-48 h at 30 ℃, observing the antibacterial effect, measuring the size of an antibacterial zone, and primarily screening to obtain 29 antagonistic strains of bacillus.

(3) Antagonistic strain rescreening

1) Preparing sterile fermentation filtrate: inoculating 29 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) The LB plate was prepared with pathogenic bacteria of bacterial disease, 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 pv.pruni）。

3) The prepared LB plate is placed upside down and then placed in a dishEvenly dividing the LB flat plate into a plurality of fan-shaped areas by using a marking pen with the center of the bottom as the circle center, then placing the LB flat plate, punching the center of a sterile oxford cup, taking out a culture medium in a hole, then injecting 100 mu L sterile fermentation filtrate, and simultaneously dripping 10 of sterile fermentation filtrate into different fan-shaped areas⁸And 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, 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, and the results are shown in table 1. And (4) re-screening and screening the Pro6A5 strain with good control effect.

TABLE 1 bacteriostatic effect of Pro6A5 strain on various 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 Pro6A5

(1) Analysis of physicochemical Properties

1) Colony characteristics and morphology observation

Inoculating Bacillus Pro6A5 on NA culture medium, and culturing in biochemical incubator at 30 deg.C for 48 hr. Observing the colony morphology, color and 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) Physiological and biochemical test determination of strain

The Pro6A5 strain physiological and biochemical experiment determination adopts the culture medium and experiment method recommended by 'Manual of identification of common bacteria system' 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 2.

TABLE 2 culture characteristics, morphology and physio-biochemical characteristics of Pro6A5 Strain

Note: + is positive; negative.

(3) Molecular biology and evolutionary relationship analysis of strains

Pro6A5 strain molecular biological analysis mainly adopts 6SrDNA 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 Pro6A5 strain was used as a template, and 16SR and 16SF were used as PCR amplification primers.

The PCR reaction system is 10 × PCR 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 1.5 kb in length. The target band was purified using agarose DNA recovery kit (Beijing Baitach Biotechnology, Inc.), and then sent to Shanghai bioengineering, Inc. for sequencing to obtain 1456 bp sequence as shown in 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.

DNA sequence ofBLAST comparative analysis with multiple ones in the databaseB. subtilisThe strains are highly homologous, and the consistency is more than 99%. Comparison of the 16SrDNA sequence of Pro6A5 strain with the known multiple Bacillus 16SrDNA sequences and construction of phylogenetic trees revealed that Pro6A5 strain was associated withB.subtilisKF278709.1 gathered into a group, in the same length of high homology sequence comparison, homology to 99.45%. Deducing that the Pro6A5 strain is the bacillus subtilis by combining physiological and biochemical characteristicsBacillus subtilis。

Example 3: preparation of microbial agent

The preparation steps of the Pro6A5 microbial inoculum are as follows:

(1) strain culture and preparation

Inoculating Bacillus subtilis Pro6A5 into test tube slant soybean casein solid medium (tryptone 17g, soybean peptone 3g, NaCl 5g, KH)₂PO₄2.5g, 2.5g of glucose and 12g of agar powder, adding water to a constant volume of 1000ml, carrying out sterilization at the temperature of 121 ℃ for 20 min), and culturing at the temperature of 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 (tryptone 17g, soybean peptone 3g, NaCl 5g, KH)₂PO₄2.5g of glucose, adding water to a constant volume of 1000ml, sterilizing at the temperature of 121 ℃ for 20min at the pH of 7.0), and performing shake culture at the temperature of 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 2.4 × 10⁹cfu/ml。

Example 4: antagonists of Pro6A5 bacterial agents against various pathogenic bacteria

(1) Preparation of sterile fermentation filtrate

Putting the liquid microbial inoculum obtained in the embodiment 3 into a 50ml centrifuge tube, centrifuging for 5min at 5000 rpm, taking supernatant, and filtering by a 0.22 mu m microporous filter membrane to obtain sterile fermentation filtrate.

(2) Antagonists of sterile fermentation filtrates against various pathogenic bacteria

Adding the sterile fermentation filtrate into a 50 ℃ melting LB culture medium, diluting the filtrate according to the proportion of 5 times, 10 times, 20 times and 50 times to prepare bacteriostatic flat plates with the total volume of 25ml and different concentrations, respectively inoculating different target pathogenic bacteria after the flat plates are condensed, taking a sterile water LB flat plate as a reference, culturing for 24 hours at 30 ℃, observing the growth condition of the pathogenic bacteria, and counting the inhibition effect.

The results of the experiment are shown in table 3.

TABLE 3 antagonistic Effect of fermentation filtrates against various pathogenic bacteria

Note: + represents the ability of the pathogenic bacteria to grow; -means that the pathogenic bacteria are unable to grow.

When the fermentation filtrate is diluted by 5 times, 5 pathogenic bacteria can not grow and can be completely inhibited; under the condition of dilution by 10 times, only the pathogen of the peach bacterial perforation disease can grow, and the growth of other pathogens is inhibited; when the solution is diluted by 20 times, the Chinese cabbage soft rot germs and the peach tree bacterial puncture germs can grow; when diluted by 50 times, only the muskmelon fruit blotch pathogen can not grow, and other pathogens can grow.

From the above, Pro6A5 microbial inoculum has the best inhibition effect on melon bacterial fruit blotch.

Example 5: research on disease prevention effect of Pro6A5 microbial inoculum on greenhouse melon fruit blotch

The test is carried out in a continuous cropping greenhouse for many years in 2019 in 3-6 months in the muskmelon Trizizazizhatjiudu (Lanceu county, Henan province). The greenhouse soil is sandy loam, the soil fertilizer is poor, and the greenhouse soil 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 method comprises the following steps of treating with clear water, treating with 750 times of liquid of sodium chloroisocyanurate wettable powder, diluting with 10 times of liquid microbial inoculum in example 3, diluting with 20 times of liquid microbial inoculum in example 3, diluting with 50 times of liquid microbial inoculum in example 3, and diluting with 100 times of liquid microbial inoculum in example 3, wherein the treatment is randomly arranged and repeated for 3 times for each 30 strains.

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.

The calculation formula is as follows:

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

control effect (%) = [1- (CK)₀Diseased finger × PT₁Diseased finger)/(CK₁Diseased finger × PT₀Disease 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.

The classification criteria of diseased leaves are as follows:

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 4.

Table 4: experimental result of Pro6A5 microbial inoculum for preventing and treating bacterial fruit blotch of muskmelon in greenhouse

。

Pro6A5 inoculum treatment results show that: the liquid microbial inoculum of the embodiment 3 has the control effect on bacterial fruit blotches from 10 times to 100 times by dilution, and has better control effect on the bacterial fruit blotches of muskmelons in greenhouses by 10 times and 20 times by spraying for 2 times.

Example 6: effect test of Pro6A5 microbial inoculum on preventing and treating melon fruit blotch

In 2019, 3 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 the steps of soaking healthy seeds and bacteria-carrying seeds for 6 hours by using different medicaments, and each group of experiments comprises 6 treatments: CK1 (healthy seed); CK2 (seed with bacteria) + CK3 (seed with bacteria 600 times gaminolone); diluting the bacteria-carrying seeds and the liquid microbial inoculum of the embodiment 3 by 10 times; diluting the bacteria-carrying seeds and the liquid microbial inoculum of the embodiment 3 by 20 times; diluting the bacteria-carrying seeds and the liquid microbial inoculum of the embodiment 3 by 50 times; the bacterium-carrying seeds and the liquid microbial inoculum of example 3 are diluted by 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%

Pro6A5 inoculum treatment results are shown in Table 5.

TABLE 5 prevention and control Effect of Pro6A5 microbial inoculum on bacteria-carrying seeds

。

Compared with healthy seeds, bacteria-carrying seeds and medicament treatment thereof, after the liquid microbial inoculum diluent in the embodiment 3 is treated, the diluent has the prevention and treatment effects of being diluted by 10 times, 20 times, 50 times and 100 times, the seedling rate can be improved, the prevention and treatment effects of the microbial inoculum diluted by 10 times are better, and the seedling rate is improved by 20 times and is larger in amplitude. 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 7: effect test of Pro6A5 microbial inoculum for overcoming continuous cropping obstacle of saline-alkali soil

The method is carried out in a muskmelon continuous cropping greenhouse in the rural area of grape trellis, village, and village, Lanceo county, Henan province, in 2019 for 9-11 months, wherein the greenhouse soil is sandy loam, the soil fertilizer is poor, the severe saline-alkali soil is severe, the soil EC value reaches 4836us/cm, more than 30 greenhouse muskmelons in 2019 spring are yellowed in the whole growth period, the growth is slow, and finally the muskmelons die due to decay and can not be harvested; the melon transplanted in autumn in 7 months has the growth height of only about 60cm, and begins to die due to premature senility.

Experimental treatment: 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), and seeds are adopted for direct seeding and planting; comparison: 1500kg of base fertilizer, i.e. thoroughly decomposed cow dung and 50kg of compound fertilizer (N: P: K =15:15: 15); and (3) treatment: 1500kg of base fertilizer, decomposed cow dung and 50kg of compound fertilizer, and the liquid microbial inoculum of the embodiment 3 is diluted by 20 times and irrigated to roots, and each hole is 100 ml. And (4) investigating the emergence rate after 7d, and investigating the growth condition of the muskmelon seedlings after 20 d.

The experimental results are as follows:

as shown in fig. 2, sowing for 7d, wherein the test group is subjected to centralized emergence, the emergence rate is 89.5%, the seedlings are green, the control group is subjected to small-part emergence, the emergence rate is 45%, and the bud centers of the seedlings are yellowed after emergence; sowing for 15d, wherein the first true leaf is drawn out from the test group, and the true leaf of the control group is not completely unfolded; as shown in fig. 3, sowing 20d, the root system of the treatment group reaches 27cm to the bottom layer of the ploughing, and the side roots are more and longer; the root system of the control group is 17cm at the maximum, the root tip is browned and rotted, the lateral root is rare, the root tip is browned and rotted, and partial plants are gradually yellowed and die.

The above results show that: the liquid microbial inoculum of the embodiment 3 can achieve the effect of strengthening roots and strong seedlings, and effectively solves the problem that continuous cropping and continuous cropping obstacles in saline-alkali soil affect the growth of melon seedlings.

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 Pro6A5, microbial inoculum and preparation method thereof, and application of bacillus subtilis Pro6A5 in cultivation of melons

<130>2020

<160>1

<170>PatentIn version 3.2

<210>1

<211>1456

<212>DNA

<213>Bacillus subtilis

<400>1

atctgtccca ccttcggcgg ctggctccat aaaggttacc tcaccgactt cgggtgttac 60

aaactctcgt ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgcggcat 120

gctgatccgc gattactagc gattccagct tcacgcagtc gagttgcaga ctgcgatccg 180

aactgagaac agatttgtgg gattggctta acctcgcggt ttcgctgccc tttgttctgt 240

ccattgtagc acgtgtgtag cccaggtcat aaggggcatg atgatttgac gtcatcccca 300

ccttcctccg gtttgtcacc ggcagtcacc ttagagtgcc caactgaatg ctggcaacta 360

agatcaaggg ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga 420

caaccatgca ccacctgtca ctctgccacc gaaggggacg tcctatctct aggattgtca 480

gaggatgtca agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac 540

cgcttgtgcg ggcccccgtc aattcctttg agtttcagtc ttgcgaccgt actccccagg 600

cggagtgctt aatgcgttag ctgcagcact aaggggcgga aaccccctaa cacttagcac 660

tcatcgttta cggcgtggac taccagggta tctaatcctg ttcgctcccc acgctttcgc 720

tcctcagcgt cagttacaga ccagagagtc gccttcgcca ctggtgttcc tccacatctc 780

tacgcatttc accgctacac gtggaattcc actctcctct tctgcactca agttccccag 840

tttccaatga ccctccccgg ttgagccggg ggctttcaca tcagacttaa gaaaccgcct 900

gcgagccctt tacgcccaat aattccggac aacgcttgcc acctacgtat taccgcggct 960

gctggcacgt agttagccgt ggctttctgg ttaggtaccg tcaaggtgcc gccctatttg 1020

aacggcactt gttcgtccct aacaacagag ctttacgatc cgaaaacctt catcactcac 1080

gcggcgttgc tccgtcagac tttcgtccat tgcggaagat tccctactgc tgcctcccgt 1140

aggagtctgg gccgtgtctc agtcccagtg tggccgatca ccctctcagg tcggctacgc 1200

atcgtcgcct tggtgagccg ttacctcacc aactagctaa tgcgccgcgg gtccatctgt 1260

aagtggtagc cgaagccacc ttttatgtct gaaccatgcg gttcaaacaa ccatccggta 1320

ttagccccgg tttcccggag ttatcccagt cttacaggca ggttacccac gtgttactca 1380

cccgtccgcc gctaatatca gggagcaagc tcccatctgt ccgctcgact tgcatgtatt 1440

aggcacgccg ccagcg 1456

Claims

1. The bacillus subtilis Pro6A5 has a preservation number of CCTCC NO: m2017196.

2. A microbial inoculant comprising the bacillus subtilis Pro6a5 and/or the metabolite of bacillus subtilis Pro6a5 of claim 1.

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

(1) inoculating the Bacillus subtilis Pro6A5 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 2-3 × 10⁹cfu/ml。

5. Use of the bacillus subtilis Pro6a5 of claim 1 or the microbial agent of claim 2 for controlling melon bacterial fruit blotch, tomato bacterial canker, Chinese cabbage soft rot, tobacco ralstonia solanacearum or peach bacterial punch.

6. The application of the bacillus subtilis Pro6A5 as claimed in claim 1 or the microbial agent as claimed in claim 2 in inhibiting or overcoming continuous cropping obstacles of melons in saline-alkali soil or/and saline-alkali adversity stress.

7. A method of administering bacillus subtilis Pro6a5 of claim 1 or a microbial inoculant of claim 2, comprising:

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.