CN1766091A - A strain of Bacillus subtilis and its application - Google Patents

Abstract

The invention discloses a (Bacillus subtilis) HL259 with CGMCC No.1451 aimed to provide product with broad spectrum sterilization activity and opposite microbe agent that can prevent and cure root rot and damping-off of vegetable, melon crop, peas and alfalfa brought by fusarium, sapromycetin and others. The preparation method needs simple operation, short fermentation period and low invest.

Description

A strain of Bacillus subtilis and its application

technical field

The invention relates to a strain of bacillus subtilis and its application, in particular to a microbial inoculum which uses the strain of bacillus subtilis as an active ingredient.

Background technique

With the implementation of the vegetable basket project and the adjustment of the agricultural planting structure, the planting area of beans, melons, vegetables, and alfalfa in my country has shown a trend of rapid development. At the same time, due to the increasing demand in the international and domestic markets, the cultivation area and base construction scale of pollution-free, green and organic agricultural production bases are increasing year by year. According to the annual dynamic multi-point monitoring and investigation, due to the continuous planting of vegetables in protected areas and irregular crop rotation, a large number of soil-borne pathogenic bacteria accumulate and multiply, resulting in vegetable seedling damping-off (Pythium aphanidermatum) and blight (Rhizoctoniasolani) , Fusarium oxysporum (Fusarium oxysporum) and root rot caused by compound infection of Pythium, Phytophthora and Fusarium and other soil-borne diseases are becoming more and more harmful, resulting in serious production reduction or product quality reduction, or even crop failure. Certain strains of Fusarium conflugus and Fusarium genus can also produce polyketide mycotoxins such as fumonisin, which directly affect the quality of agricultural products and endanger the health of humans and animals. Production mainly relies on the application of chemical pesticides and fertilizers, which restricts the development of high-yield and efficient agriculture.

Chemical control occupies an important position in the history of world agricultural development. For centuries, chemical pesticides have played an active role in the prevention and control of plant diseases, insect pests, weeds and rodents, and have made important contributions to the stability and increase of agricultural production, recovery of losses and improvement of quality. But at the same time, due to excessive application and abuse of pesticides, soil fertility decline and environmental pollution, affecting non-target organisms and leading to serious problems such as the emergence and rapid development of pest resistance. Especially in recent years, due to excessive pesticide residues in agricultural products, human and animal poisoning incidents have occurred continuously, which seriously threatens the quality of life of the people and limits the export of agricultural products in my country. The rampant occurrence of soil-borne diseases and the potential contamination of chemical fungicides have become the primary key issues in agricultural production safety.

With the development of social civilization, the issue of sustainable agricultural development has made a new leap in human understanding of resources and the environment, and puts forward requirements for high efficiency, low toxicity, and environmental friendliness for pesticides. Countries around the world are also taking practical steps to reduce the use of chemically synthesized pesticides. For example, the US EPA announced the cancellation of the registration of 91 chemical pesticides in the 1990s. Ten-year plan, the European Union has also formulated a similar plan. my country has also canceled the registration of more than a dozen chemical pesticides. In order to solve the contradiction between increasing the yield and ensuring the quality of agricultural products, achieving the purpose of comprehensively preventing and controlling major vegetable pests and diseases, and reducing the use of chemical pesticides, the development of biological control technology has attracted more and more attention from governments, scientific and technological workers and the public. Biological bacteria agent (BCA) has been widely concerned and valued for its low toxicity, good environmental compatibility, ability to build colonies in the rhizosphere of crop seedlings, long-lasting effect, and difficult for harmful organisms to develop resistance to it. Most of the advanced agrochemical companies in the world are actively involved in the research and marketization of biological agents. In particular, beneficial bacteria have made some progress in the prevention and control of soil-borne and seed-borne diseases, and a series of products have been successfully applied. Significant economic and ecological benefits. As an important measure of integrated control, biological control is playing an increasingly important role in the integrated management of plant diseases and insect pests.

PGPR (plant-growth-promoting rhizobacteria) is a class of biocontrol bacteria that has received widespread attention. It is also one of the main biocontrol factors used in the comprehensive control of soil-borne diseases in China. It promotes the growth of plants by transplanting to the root system and preferentially occupying the root system or inhibiting harmful rhizosphere bacteria and harmful fungi (DRMO) on the root. Among them, Bacillus spp. has become an ideal biocontrol microorganism due to its wide distribution, easy isolation and culture, the ability to produce heat-resistant and dry-resistant endospores, long storage period, and convenient use. After Johnson and others reported that Bacillus subtilis produces antibacterial substances, researchers from various countries have paid great attention to it as a biological control factor for more than half a century, and have carried out extensive disease control capabilities on a variety of crops. There are many successful applications in the test, which have shown obvious effects on disease prevention and production increase in production practice, and further confirmed the stability of Bacillus biocontrol agents in products, the compatibility with chemical pesticides and the control of different plants in different years. The consistency of efficacy is significantly better than that of non-bacillus and fungal biocontrol bacteria.

Scholars at home and abroad have successively reported the biological characteristics, colonization conditions, physical and chemical properties of antibacterial substances of different strains of Bacillus subtilis with biological control effects, and their colonization in the roots of different crops, and their effects on Pythium, Fusarium, etc. Research on the control effect of different soil-borne diseases caused by bacteria, potential induction of plant disease resistance and promotion of seedling growth. Previous studies have shown that because Bacillus can produce endophytic spores and has strong stress resistance, compared with other types of biocontrol factors, it is more conducive to the production of bacterial agents, formulation processing and survival in the environment, colonization and reproduce.

Contents of the invention

One object of the present invention is to provide a strain of Bacillus subtilis with broad-spectrum bactericidal activity.

The bacillus subtilis provided by the present invention is bacillus subtilis (Bacillus subtilis) HL259, has been preserved in China Committee for Microorganism Culture Collection General Microorganism Center (CGMCC for short) on September 1, 2005, and the preservation number is CGMCC No. 1451.

Bacillus subtilis HL259 CGMCC №1451 was isolated from soybean field soil in Keshan County, Qiqihar City, Heilongjiang Province.

The second object of the present invention is to provide a microbial agent with broad-spectrum bactericidal activity.

The microbial bacterial agent provided by the present invention has an active ingredient of Bacillus subtilis HL259 CGMCC No.1451.

The microbial inoculum can be obtained as follows: ferment Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 in YMA or corn steep liquor medium, and mix the obtained fermented liquid with the substrate to obtain the microbial inoculum.

The corn steep liquor medium contains 5 g of sucrose, 2 g of corn flour, 0.05 g of KH ₂ O ₄ , 0.5 g of (NH ₄ ) ₂ SO ₄ , 0.05 g of K ₂ HPO ₄ , 0.025 g of MgSO ₄ , 2 g of CaCO ₃ , and 1000 ml of distilled water .

YMA medium contains mannitol 10g, yeast powder 3g, K ₂ HPO ₄ 0.25g, KH ₂ PO ₄ 0.25g, 0.2g MgSO ₄ .7H ₂ O, NaCl 0.1g, CaCO ₃ 3g, agar 10g, distilled water 1000ml, pH 7.2.

In the fermentation conditions, the culture temperature can be 25-30°C, the ventilation rate can be 1:0.5-1:1.6, the stirring speed can be 200-400rpm, and the cultivation time can be 24-48h; the ventilation rate is The ratio of the volume of air passed into the fermenter to the volume of the fermentation broth in the fermenter.

Wherein, the culture temperature is preferably 28-30° C., the ventilation rate is preferably 1:1-1:1.2, the stirring speed is preferably 250-300 rpm, and the culture time is preferably 24-30 h.

Vegetable oil or silicone oil can be added as defoamer during fermentation.

The mass percentage of the vegetable oil may be 1-2%; the mass percentage of the organosilicon oil may be 0.04-0.1%.

The substrate can be peat, micronized calcium carbonate, attapulgite or bentonite.

The number of viable bacteria of Bacillus subtilis HL259 CGMCC No. 1451 in the microbial agent can be 5×10 ⁹ cfu/g, and the pH value can be 6.0-7.8.

The experimental results show that Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №.1451 is effective against 22 species of plant pathogenic fungi and representative pathogenic bacteria of 5 important genera in 13 genera of four fungal subphyla except Pseudomonas Obvious growth inhibitory effect, among which Pythium melon and fruit, Phytophthora capsici, Fusarium colloides, Rhizoctonia graminearum, Fusarium alfalfa root rot, Verticillium dahliae of cotton, Fusarium wilt of cotton, Apple rot and wheat The inhibition rate of Rhizoctonia solani reached 72.76%-94.86%. Indoor and greenhouse tests show that the strain is used for soil regulation, seed treatment and aboveground spraying, and has a good control effect on root rot caused by soil-borne and seed-borne pathogens. The control experiments were carried out in Heilongjiang Province soybean stubble fields, Jilin Province and Beijing suburbs where root rot occurred more seriously and in alfalfa fields. The results showed that HL259 and its metabolites can significantly control Seed rot, damping-off, wilting, root rot and other diseases caused by Rhizoctonia, etc., and also cure some bacterial diseases, with a certain effect on increasing production, especially for the compound infection caused by multiple pathogenic bacteria after seed treatment and soil treatment Root rot has a significant effect of disease prevention and seedling preservation, and it is a biocontrol strain with broad-spectrum bactericidal activity.

Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 has no pathogenic effect on crops, and inoculation of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 can promote the stem length of tomato, pepper and zucchini Effect; Acupuncture inoculation with Bacillus subtilis HL259 CGMCC №1451 can promote cucumber stem length and fresh weight; root dipping inoculation with Bacillus subtilis HL259 CGMCC №1451 can improve melon stem length, Fresh weight and alfalfa stem length are promoted.

The experimental results show that the microbial inoculant with Bacillus subtilis HL259 CGMCC №1451 as the active ingredient can effectively control the production of vegetables, melons, beans and alfalfa caused by Fusarium, Pythium, Phytophthora and Likum Plant diseases such as root rot, damping-off and blight caused by Sclerotinia, and the preparation process of the microbial inoculant is simple, the fermentation period is short, the investment is low and the preservation is easy.

Description of drawings

Figure 1A is an optical microscope photo of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451

Figure 1B and Figure 1C are electron micrographs of Bacillus subtilis HL259 CGMCC №1451

Figure 2 shows the results of the phylogenetic tree analyzed by DNAMAN Version 4.0 based on the 16S rRNA sequence

Figure 3 is the comparison result of the 16SrRNA gene sequence origin of Bacillus subtilis HL259 CGMCC №1451 and Bacillus subtilis US116

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified.

The percentages in the following examples are all mass percentages unless otherwise specified.

Embodiment 1, the separation and identification of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451

1. Isolation of Bacillus subtilis HL259 CGMCC №1451

Collect 10g of soil samples from the soybean field in Keshan County, Qiqihar City, Heilongjiang, add them to a triangular flask filled with 100ml of sterile water and glass beads, vibrate on a shaker at 100 rpm for 20min, take 0.5ml and add them to 4.5ml of sterile water, and dilute in turn 10 ^-2 , 10 ^-3 , 10 ^-4 times, respectively take 0.1ml of the above suspension in YMA, beef extract peptone (bacterial medium), Martin's (fungus isolation medium), modified Gaoshi No. 1 (actin Bacteria medium) culture medium, each concentration was repeated 3 times, placed in a 28°C incubator for 24h, 3d and 5d, and the isolation and purification of bacteria, fungi and actinomycetes were carried out respectively. The antagonistic bacteria were screened with Pythium, Phytophthora, Fusarium and Rhizoctonia as target bacteria respectively. Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451, which has obvious antibacterial effect on each tested bacteria, was screened out.

2. Identification of Bacillus subtilis HL259 CGMCC №1451

(1) Morphological characteristics of the bacteria

Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 on the soil infusion agar medium (1kg of soil plus 1000ml of water, sterilized by high-pressure steam (121°C) for 30min. After adding talcum powder to the infusion, filter it with double-layer filter paper, and adjust the pH value To neutrality, then according to 100ml of soil infusion, 17g of agar, 900ml of water to prepare the culture medium) after 24 hours of cultivation, the colonies are round, white and opaque, and as the cultivation time increases, the colonies become thicker and dry, with irregular edges and Wrinkle-shaped protrusions that turn creamy in color, and the cell material grown on them is not easily dispersed in the liquid. Rod-shaped cells were observed under an optical microscope (Fig. 2A), most of them are motile, and the flagella are perinatal, and the cell size is 0.6-0.8 ×3～5μm, the Gram staining is uniform, and the cells can be seen to be stained with bluish-purple spores. Scanning electron microscope observation further confirmed the rod-shaped shape of the spores, with a thick and uniform slime layer outside the cell wall, producing oval-shaped spores without parasporium crystals (Figure 2B and Figure 2C).

(2) Physiological and biochemical identification of strains

Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 and Bacillus subtilis standard strain 1.504 (China Culture Collection Management Committee) were tested for physiological and biochemical properties under the same experimental conditions, and the results are shown in Table 1.

Table 1. HL259 identification items and results Test items HL259 Standard strain Test items HL259 Standard strain gram reaction + + melanin production 1% glucose - - 1% Tyrosine - - starch hydrolysis + + Spore staining + + use Citrate + + parasporal crystal - - propionate - - athleticism + + Production of NO ₂ from NO ₃ + + Catalase + + produce indole - - anaerobic growth - - break down Casein + + VP response + + Tyrosine - - yolk reaction - - Growth at pH 5.7 + + 7% NaCI growth ^a + + Growth at 50°C + + Acid production glucose + + gelatin hydrolysis + + Arabic candy + + gas production glucose - - Xylose + + Mannitol + +

Note: a indicates that HL259 can also grow in 10% NaCl. "+" indicates a positive result; "-" indicates a negative result.

(2) 16SrRNA sequence determination and plasmid extraction of HL259 strain

The 16SrRNA gene sequence sequencing results showed that the 16SrRNA gene sequence of the HL259 strain had the nucleotide sequence of sequence 1 in the sequence table. According to the homology comparison between the sequencing results and the 16SrRNA gene sequence of Bacillus subtilis in Genbank, the results showed that the homology of HL259 and Bacillus subtilis US116 The origin reaches 99.52% (Figure 3, in the figure, the "-" in the HL259 line indicates that it is the same as the base at the corresponding site of US116, and "g, c, a" indicates that it is different from the base at the corresponding site of US116), indicating The HL259 strain belongs to the genus Bacillus. The results of plasmid extraction and electrophoresis showed that HL259 contained a plasmid of about 4.5 kb, and could grow normally on a plate containing 50 μg/ml ampicillin (Amp).

The literature shows that Bacillus subtilis is motile, Gram-positive, spores are oval or columnar, mesozoic or partially mesozoic, positive in contact enzyme test, V-P test positive, growing in 7%-10% sodium chloride, grown from glucose, Arabidopsis Sugar, xylose and mannitol produce acid, hydrolyze starch, use citrate as carbon source, reduce nitrate to nitrite, decompose casein, yolk reaction is negative, no melanin is formed on glucose and tyrosine agar, no Utilize the characteristics of propionate and non-decomposition of tyrosine. Part of the physiological and biochemical character test results of the above HL259 are compared with the Bacillus subtilis standard bacterial strain, according to "Common Identification Methods for General Bacteria" (Bacterial Taxonomy Group, Institute of Microbiology and Bacteria, Chinese Academy of Sciences, Science Press, 1978), "Bacillus Genus" (translated by Cai Miaoying, Zhan Like, etc., Agricultural Press, 1988) and "Microbial Taxonomy" (Zhang Jizhong, Shanghai Fudan University Press, 1998) were retrieved to determine the physiological and biochemical properties of HL259 and Bacillus subtilis The corresponding traits of bacilli were consistent. At the same time, the 16SrRNA sequence and the phylogenetic tree results obtained by DNAMAN Version 4.0 analysis based on the 16SrRNA sequence (Figure 2) further confirmed the biochemical identification results, and identified HL259 as Bacillus subtilis.

Embodiment 2, the antibacterial activity of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No. 1451 (determination of antibacterial spectrum)

1. Determination of antibacterial activity

Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No.1451 strain and its metabolites were detected by mycelial growth rate assay and spore germination method, including 22 plant pathogenic fungi of four fungal subphyla and 13 genera: Rhizoctonia solani solani, Valsa mali, Fusarium graminearum, Mycosphaerella melonis, Alternaria solani, Fusarium moniliforme, Fusariun oxysporum f.sp.vasinfectum, Botrytis cinerea Botrytis cinerea, Fusarium solaniformis, Fusarium solani f.sp.pisi, Corn spot fungus Bipolaris maydis, Apple ringworm fungus Physalospora piricola, Anthracnose fungus Colletotrichumgloeosporioides, Cotton Verticillium dahliae, Rice blast fungus Piriculariaoryzae, Capsicum blight Phytophthora capsici, Pythium aphanidermatum, Fusarium subglutinans, Rhizoctonia cerealis, Fusarium avenaceam, Sporisorium holci-sorghi (Rivolta) vanky); The representative pathogenic bacteria of 5 important genera were determined by the double-layer plate culture method and the Oxford cup method: Agrobacterium k27 pathogen in the main genera of the thin-walled phylum, Ralstonia solanacearum , Pseudomonas (Pseudomonas) Xanthomonas cucumber, Xanthomonas (Xanthomonas) Xanthomonas oryzae, Xanthomonas cotton and tomato scab; Corynebacterium in the main genera of Firmicutes ( Clavibacter) antibacterial activity and antibacterial spectrum of tomato canker.

1. Detection of the effect of Bacillus subtilis HL259 CGMCC №1451 on inhibiting pathogenic fungi

Using the plate culture method, Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 was activated on a 523 slope at 28°C for 48 hours, and the bacterial suspension was prepared with 0.85% normal saline, and the final concentration of the bacterial suspension was determined to be 10 ⁹ by Michael turbidimetry. cfu/ml spare. The fungi to be tested were activated on a PDA plate at 25°C for 5 days to form colonies, and a fungus cake was prepared with a puncher (5 mm in diameter), and the fungus was inoculated in the center of the PDA plate with the mycelium facing down. Soak the high-temperature-sterilized double-layer filter paper sheets (5mm in diameter) in the suspension of Bacillus subtilis HL259 CGMCCNo1451 bacteria, and place them evenly on the PDA plate, with 3 sheets in each dish, at an even distance, so as to place and soak The PDA plate of the sterile water paper sheet was used as the control, and each treatment was repeated 4 times. After 4, 5, 6, and 7 days of dark culture at 25°C, the diameter of the fungal colony was measured, and the inhibition growth rate was calculated.

The results of the inhibitory effect of Bacillus subtilis HL259 CGMCC №1451 on the tested pathogenic fungi are shown in Table 2.

Table 2. Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 inhibits the pathogenic fungus tested Tested strain Filter paper method (colony radius cm) Inhibition rate(%) CK HL259 Rhizoctonia solani 4.00 0.76 92.57 Apple rot pathogen Valsa mali 3.80 0.85 89.39 Fusarium graminearum 2.50 1.33 58.50 Mycosphaerella melonis 3.47 1.43 68.69 Tomato early blight Alternaria solani 3.13 1.54 60.46 Fusarium moniliforme 3.23 1.33 69.60 Fusariun oxysporumf.sp.vasinfectum 2.77 1.05 75.77 Botrytis cinerea 3.97 1.45 72.62 Fusarium solani f.sp.pisi 3.62 1.09 81.09 Bipolaris maydis 2.52 1.40 55.44 Apple ringworm Physalospora piricola 2.27 1.18 61.58 Anthracnose pathogen Colletotrichumgloeosporioides 2.90 1.47 59.58 Cotton Verticillium dahliae 3.28 1.14 76.98 Rice blast fungus Piricularia oryzae 2.85 1.64 51.49 Phytophthora capsici 2.77 1.05 75.77 Pythium aphanidermatum 3.62 1.35 72.76 Fusarium subglutinans 3.25 1.18 75.27 Rhizoctonia cerealis 4.00 0.68 94.86 Fusarium avenaceam 3.62 1.09 81.09 Head smut Measured by turbidity method 70.88

Note: Maize head smut was measured by turbidity method, that is, the absorbance of different treatments was measured at 650nm wavelength, and the relative inhibition percentage was obtained by comparing with the blank treatment without chemical solution.

2. Detection of the effect of Bacillus subtilis HL259 CGMCC №1451 on inhibiting pathogenic bacteria

Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 and the target bacteria were activated for 48 hours to make a bacterial suspension, and the bacterial suspension was adjusted to 1.5×10 ⁹ cfu/ml by the Michael turbidimetric method, and 0.1ml of the target bacteria was absorbed Add the suspension to the medium plate and spread it with a spreading spatula. Put the Oxford cup (the diameter of the Oxford cup is 0.85cm) on the culture medium, add 100 μl of bacterial suspension in each Oxford cup, and use the PDA plate with 100 μl of sterile water in the Tianjin cup as a control, 3 per dish Oxford cups with uniform distance, stored at 4°C for 16h, each treatment repeated 3 times, measured the diameter of the inhibition zone after 24-48h dark culture at 28°C. The inhibitory effect of Bacillus subtilis (BACILLUSSUBTILIS) HL259 CGMCC No1451 on the tested pathogenic fungi is shown in Table 3, and the diameter of the inhibition zone of the controls used was 0 cm.

Table 3. Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 on the main diseases tested

Inhibitory effect of the original bacteria Tested strain Diameter of inhibition zone for three repetitions (cm) Average diameter of inhibition zone (cm) 1 2 3 Potato soft rot Erwinia carotovora 2.13 2.15 2.14 2.14 Xanthomonas oryzae 1.97 2.03 2.07 2.02 K27 Agrobacterium rhizogenes 1.91 1.9 1.96 1.92 Cucumber horn spot Pseudomonas syringae 0.85 0.85 0.85 0.85 Cotton horn spot Xanthomonas malvacearum 1.34 1.36 1.90 1.63 Tomato scab Xanthomonas resicatoria 1.48 1.55 1.65 1.56 Tomato canker Clavibacter michiganensissubsp.michiganensis 1.50 1.42 1.58 1.50 Solanacearum Ralstonia solanacearum 1.6 1.58 1.58 1.59

The test results showed that Bacillus subtilis HL259 CGMCC №1451 was effective on potato soft rot, rice bacterial blight, K27, tomato ulcer, eggplant sore, tomato scab, and cotton corner spot, but had no effect on cucumber corner spot.

In this embodiment, 22 kinds of phytopathogenic fungi comprising 13 genera of four fungal subphylums and representative pathogenic bacteria of 5 important genera are used as target bacteria to measure the bacteriostatic spectrum of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451; the result It shows that Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 has obvious growth inhibitory effect on other tested target bacteria except Pseudomonas, showing that Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 has a wide range of Antibacterial spectrum. Among them, the inhibitory rate of Pythium melon and fruit, Phytophthora capsici, Fusarium colloides, wheat sheath blight, Fusarium alfalfa root rot, Verticillium dahliad of cotton, Fusarium wilt of cotton, Apple rot and Rhizoctonia solani Reach 72.76% ~ 94.86%.

2. Determination of the safety of Bacillus subtilis HL259 CGMCC №1451 on crops

Firstly, indoor seed germination safety testing was carried out, that is, the germination safety of several major crops was treated with Bacillus subtilis (Bacillus subtilis) HL259 CGMCC №1451 bacterial suspension at a concentration of 10 ⁸ cfu/ml, and then the germination safety of the seeds was observed to clarify the germination safety of subtilis. The safety of Bacillus subtilis HL259 CGMCCNo1451 for seed treatment provides a theoretical basis for the application of field trials. The results showed that there was no significant difference in the germination potential and germination rate of the eight crop seeds of tomato, pepper, melon, cucumber, alfalfa, kidney bean, zucchini and eggplant after soaking in the bacterial suspension compared with the control. Bacillus subtilis (Bacillus subtilis) HL259CGMCC No1451 Seed soaking treatment also significantly promoted the germination rate of bean and the germination potential of eggplant. Further adopt root dipping inoculation method, acupuncture inoculation method and leaf cutting inoculation method to detect whether Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 is pathogenic to the above eight kinds of crops under the conditions of greenhouse or field test. The further test promotion of Bacillus subtilis HL259 CGMCC No1451 in production lays the foundation. The results show that the growth status of eight kinds of crops inoculated with Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 is compared with the control, and it can be seen that Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 has no pathogenic effect on eight kinds of crops, And leaf cutting inoculation inoculation with Bacillus subtilis HL259 CGMCC No1451 can promote the stem length of tomato, pepper and zucchini; acupuncture inoculation with Bacillus subtilis HL259 CGMCC No1451 can promote the stem length of cucumber , fresh weight; inoculation of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 by root dipping inoculation can promote melon stem length, fresh weight and alfalfa stem length.

Embodiment 3, take Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 as the preparation of the microbial bacterial agent of active ingredient

1. Determination of the most suitable nutrient medium and culture conditions

In order to determine the solid and liquid media suitable for the growth of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451, the comparison of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 in LB, YMA, YEM, PDA, King's B, sucrose-peptone medium (sucrose 2.0%, peptone 0.5%, K ₂ HPO ₄ 0.05%, MgSO ₄ 7H ₂ O 0.025%, distilled water 1000ml, agar 17.0g), YGM (glucose 2.5%, yeast extract 2.0g, K ₂ HPO ₄ 0.25g, KH ₂ PO ₄ 0.25g, MgSO ₄ 7H ₂ O 0.1g, MnSO ₄ 0.15g, NaCl 0.05g, FeSO ₄ 7H ₂ O 0.005g, agar 17.0g, distilled water 1000ml), 523, BPY on 9 media Bacillus subtilis (Bacillus subtilis) HL259CGMCC No1451 was inoculated by streaking and cultured at 28-30°C for 24-48 hours. It can grow and culture on 9 kinds of media plates, showing light yellowish brown or Pale off-white, but there are differences in the growth of colonies on different media. In the early stage of culture on YMA, the bacterial colonies are pus-like, and the colonies are round, and gradually the colonies become thicker, opaque, wrinkled, and the bacteria turn white, and the colonies expand rapidly; secondly, determine the subtilis The growth curve, OD450 value and pH value of Bacillus subtilis HL259 CGMCC No1451 in different liquid media were comprehensively analyzed to determine that the YMA medium is more suitable for liquid shaking culture, and the YMA culture medium was shaken for 12 hours. The amount has reached 10 ⁸ cfu/ml, and the period when the amount of bacteria reaches the peak is 24 hours, and the amount of bacteria is 10 ¹⁰ cfu/ml; then carry out the optimal production medium and culture conditions of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 filter. Tests have shown that the medium suitable for the production of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 is YMA and corn steep liquor medium (sucrose 5g, corn flour 2g, KH ₂ PO ₄ 0.05g, (NH ₄ ) ₂ SO ₄ 0.5 g, K ₂ HPO ₄ 0.05g, MgSO ₄ 0.025g, CaCO ₃ 2g, add water 1000ml). Furthermore, the selection test of shaking culture conditions including shaking culture temperature, initial pH, liquid volume and inoculum size was studied, and the inoculation amount of slant seeds (10 ⁸ cfu/ml) was determined to be 1-2% (volume ratio) on the shaking table. Erlenmeyer flask, under the conditions of 20-40% (volume ratio) liquid filling, 28-32°C culture temperature, 6.0 initial pH, and 180-220rpm shaker speed, shake culture for 18-24 hours, and its metabolites most significant antibacterial effect. Further from the determined shake flask conditions, the fermentation conditions from the seed tank to the fermenter were studied: the seeds (10 ¹⁰ cfu/ml) in the triangular flask were transferred to the seed tank (30-50L) with 1-2% (volume ratio) inoculum , the fermented liquid in the seed tank is inoculated into the fermenter (800-1000 L) with the inoculum amount of 3-5% (volume ratio). Cultivation conditions for seed tanks and fermenters: culture at 25-30°C for 24 to 48 hours, with an aeration rate of 1:0.5 to 1:1.6 (the aeration rate is defined as the volume of air that passes into the fermenter per minute and the volume of the fermentation liquid in the fermenter. volume ratio), the defoamer uses 1-2% vegetable oil by mass or 0.04-0.1% organic silicon oil by mass, and the stirring speed is 200-400rpm; then the bacterial liquid is absorbed by peat to make powder Bacillus subtilis (Bacillus subtilis) HL259CGMCC No1451 bacteria amount is greater than or equal to 5 × 10 ⁸ cfu/g.

2. Preparation of Bacillus subtilis HL259 CGMCC No1451 microbial agent (HL259 agent)

Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 was inoculated on the YMA medium by streaking, and cultured at ³⁰ °C for 24h to activate the strain; ) of the inoculum was inoculated into the YMA culture solution of the shaker conical flask (volume is 1000ml), the filling amount was 30% (volume ratio), the culture temperature was 28 ℃, the initial pH was 6.0, and the shaker speed was 200rpm, The shaking culture time is 24 hours; after that, inoculate the seeds (10 ¹⁰ cfu/ml) in the Erlenmeyer flask into the corn steep liquor medium in the seed tank (volume 50L) at an inoculum size of 2% (volume ratio), and cultivate at 28±1°C 24h, ventilation rate is 1: 1-1: 1.2, antifoaming agent uses silicone oil 0.04%, stirring speed is 260rpm; Fermentation liquid in the seed tank is inoculated to fermenter (volume is 1000L) with 5% (volume ratio) inoculum amount ) in the corn steep liquor culture solution, the culture condition is 28±1°C, the ventilation rate is 1:1-1:1.2, the antifoaming agent uses 0.04% silicone oil, the stirring speed is 260rpm, and cultured for 24h. Adsorb the bacterial solution with peat to make a powder-type bacterial agent, or mix the bacterial precipitate obtained by centrifuging the fermented liquid with micronized calcium carbonate at a mass ratio of 1:1 to prepare a biocontrol bacterial agent, among which the powder-type bacterial agent Subtilis subtilis The amount of Bacillus subtilis HL259 CGMCC No1451 was 5×10 ⁹ cfu/g. Further, microscopic observation, plate counting and other means are used to detect the effective number of viable bacteria, pH value and miscellaneous bacteria rate in the bacterial agent to determine the product quality of the bacterial agent. The quality analysis of bacterial agent products is combined with three methods: turbidimetry, microscopic examination and plate counting. Nephelometric method and microscopic examination are simple and fast, and are used in the detection of the bacterial content and purity of the fermentation broth in the fermentation process of the triangular flask, seed tank and fermenter. Get 500ml of fermented liquid during the turbidimetric test, use blank liquid culture medium as a contrast, measure the OD650 of fermented liquid, when OD650 is higher than 1.0, fermented liquid should be diluted, each sample is set 3 repetitions, according to the measured OD650 in the "containing Bacterial amount (cfu/ml)-OD650" standard curve to find out the corresponding bacterial content. Take 500ml of fermented liquid when microscopic examination of bacterial morphology, take pure strain as contrast, smear, stain with safranin, observe bacterial morphology under a microscope, and compare with pure bacterial strain. The plate counting method is used in the final quality inspection of finished products. The test results showed that the effective number of viable bacteria of the powder biocontrol agent was 8×10 ⁹ cfu/g, the pH value was 7.2, and the miscellaneous bacteria rate was 0%.

Embodiment 4, the effect of bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 bacterial agent control watermelon blight, damping-off and fusarium wilt

Blight, damping-off, and Fusarium wilt are the main diseases in the watermelon growth process. The present embodiment measures the emergence rate and growth index of watermelon (Xingnong No. 1) by field test of Bacillus subtilis (Bacillus subtilis) HL259 CGMCC No1451 bacterial agent and the control effects on damping-off, damping-off and mid-to-late stage Fusarium wilt at seedling stage. The specific method is as follows:

Drugs for testing: Bacillus subtilis (Bacillus subtilis) HL259CGMCC No1451 powder type bacterial agent prepared in embodiment 3 step 2, No. 1 of contrast biocontrol bacterial agent (purchased from Beijing Soil and Fertilizer Workstation, active ingredient is Bacillus cereus, effectively active The number of bacteria is 10 ⁸ cfu/g), and the control biocontrol agent No. 2 (purchased from Beijing Soil and Fertilizer Workstation, the active ingredient is Bacillus subtilis, and the effective number of viable bacteria is 10 ⁸ cfu/g).

In this experiment, there were 7 treatments and 4 repetitions, with a total of 28 plots. Arrange randomly.

Treatment 1: The concentration of HL259 bacterial agent 1:40 was used for seed dressing of watermelon seeds.

Treatment 2: The concentration of HL259 bacterial agent 1:50 was used for seed dressing of watermelon seeds.

Treatment 3: The concentration of HL259 bacterial agent 1:60 was used for seed dressing of watermelon seeds.

Treatment 4: Seed dressing at 1:50 as the control biocontrol fungicide No. 1.

Treatment 5: Seed dressing with the control biocontrol agent No. 2 at 1:50.

Treatment 6: 50% thiram WP 1:200 seed dressing.

Treatment 7: Seeds not treated with chemicals (control)

The area of each plot is 46.8M ² , and the spacing between plants × row spacing = 36cm×65cm. The soil of the test site is sandy loam. Organic matter (%): 1.2828 Alkaline hydrolysis nitrogen: 93.56mg/kg Available phosphorus: 55.95mg/kg Available potassium: 69.2mg/kg pH value: 8.09. The previous crop was watermelon.

Two days before sowing, according to the proportion of seed dressing or seed dressing, mix the chemicals and seeds required by each plot into plastic bags, and use manual shaking to evenly coat or adhere the chemicals on the surface of the seeds. Seeds are broadcast live. The emergence rate was investigated 7 days after sowing, 4 points were randomly sampled in each plot, 5 plants were fixed at each point, and a total of 80 plants were investigated for each treatment. Twenty days after sowing, 20 watermelon seedlings were randomly sampled from each plot, and the main growth indicators such as stem thickness, vine length, leaf length, leaf width, and root length were investigated and recorded. The effects of pesticides on watermelon emergence and watermelon growth indicators in each treatment plot are shown in Table 4 and Table 5, which show that the HL259 bacterial agent seed-dressing watermelon seeds at a concentration of 1:40 and 1:50 has a significant effect on the emergence rate and growth indicators of watermelon. The promoting effect was better than other treatments.

Table 4. Investigation results of emergence rate (%) of watermelon seedlings in different treatment plots

3 100.0 96.3 100.0 86.7 83.9 89.7 83.9 4 100.0 96.3 92.6 100.0 92.9 96.3 96.3 average 100.0 97.2 95.3 94.8 93.2 94.6 89.3

Table 5. Survey results of watermelon growth indicators in different treatment plots

7 1 0.70 74.40 14.83 16.49 21.50 2 0.71 72.80 14.53 16.38 20.68 3 0.68 70.50 14.30 17.05 25.45 4 0.66 66.30 14.50 16.55 24.15 average 0.69 70.90 14.45 16.60 22.95

21 days after sowing, 40 plants were randomly investigated in each plot, and the number of dead plants was recorded, and the control results of watermelon blight and damping-off were calculated compared with the control. 60 days after sowing, 20 plants were randomly sampled in each plot, and the rate of diseased plants was recorded, and the disease base was investigated according to the following grading standards, and the control effect of Fusarium wilt was calculated by the disease index.

Grading standards are as follows:

Grade 0—Plant growth is normal.

Grade 1—the diseased plant is slightly shorter than normal melon seedlings, the vascular bundles turn yellowish brown when the stem is dissected, and the melon seedling production is inhibited.

Grade 2—The diseased plant is slightly shorter than normal melon seedlings, and the vascular bundles turn yellowish brown when the stem is dissected. The performance is normal in the morning and evening, and the melon seedlings wilt at noon, which affects the production of watermelon.

Grade 3—The diseased plants are significantly shorter than normal melon seedlings, wilting and dead, the roots of the diseased seedlings are pulled out and observed to turn brown, and the watermelon loses its commercial value.

The control results of HL259 fungal agent on watermelon damping-off and damping-off disease are shown in Table 6. HL259 fungal agent was treated with 1:40, 1:50 and 1:60 concentration of watermelon seeds, and it had better control of watermelon damping-off disease The effects were 81.66%, 77.08% and 72.49%, respectively; the control effects against damping-off were 88.89%, 88.89% and 72.22%. The control effects of HL259 bacterial agent on Fusarium wilt are shown in Table 6, and the disease control effects are 91.24%, 83.00% and 75.95%, respectively, which are better than other treatments.

Duncan's new multiple range test method was used to analyze and compare the control effects of damping-off, damping-off and fusarium wilt in order to evaluate the significance of differences in the control of the three main diseases of watermelon among the chemical treatments in this experiment. Analysis of variance showed that there was no significant difference in the control effects of three different seed dressing concentrations of HL259 inoculum on damping-off and blight; there were significant differences in the control effects on Fusarium wilt at the levels of 5% and 1%. , and there are also large differences with other treatments at the level of 5% and 1%.

During the whole test period, the watermelon grew normally, and no phytotoxicity was found. It shows that the HL259 bacterial agent is safe for watermelon growth when seed-dressing watermelon seeds at a concentration of 1:40 and 1:50. No adverse effects of the test agent on non-target organisms were found in the investigation, indicating that the agent is safe to the environment. The seed dressing ratio of 1:40-50 has a better control effect.

Table 6. HL259 bacterial agent control watermelon wilt, damping-off and blight test results deal with repeat Number of investigated plants Fusarium wilt Number of investigated plants Damping-off Blight level 0 Level 1 level 2 Level 3 Disease index Control effect (%) Number of diseased plants (strains) Incidence rate (%) Control effect (%) Number of diseased plants (strains) Incidence rate (%) Control effect (%) 1 1 20 19 1 0 0 0.56 94.40 40 2 5.00 63.32 0 0.00 100.00 2 20 19 1 0 0 0.56 94.4 40 1 2.50 81.66 0 0.00 100.00 3 20 18 2 0 0 1.11 89.49 40 1 2.50 81.66 2 5.00 55.56 4 20 19 0 1 0 1.11 86.67 40 0 0.00 100.00 0 0.00 100.00 average 20 0.83 91.24 40 - 2.50 81.66 - 1.25 88.89 2 1 20 18 2 0 0 1.11 88.9 40 1 2.50 81.66 1 2.50 77.78 2 20 18 1 1 0 1.67 84.19 40 1 2.50 81.66 1 2.50 77.78 3 20 17 3 0 0 2.22 78.98 40 1 2.50 81.66 0 0.00 100.00 4 20 18 1 1 0 1.67 79.95 40 2 5.00 63.32 0 0.00 100.00 average 20 1.67 83.00 40 - 3.13 77.08 - 1.25 88.89 3 1 20 17 2 1 0 2.22 77.80 40 1 2.50 81.66 3 7.50 33.33 2 20 16 3 1 0 2.78 73.67 40 3 7.50 63.32 1 2.50 77.78 3 20 17 2 1 0 2.2 78.98 40 2 5.00 63.32 1 2.50 77.78 4 20 17 2 0 1 2.22 73.35 40 1 2.50 81.66 0 0.00 100.00 average 20 2.34 75.95 40 - 4.38 72.49 - 3.13 72.22 4 1 20 16 2 2 0 333 66.7 40 2 5.00 63.32 2 5.00 55.56 2 20 16 2 1 1 3.89 63.16 40 2 5.00 63.32 4 10.00 11.11 3 20 15 3 2 0 5.00 52.65 40 3 7.50 44.97 1 2.50 77.78 4 20 15 3 2 0 3.89 53.30 40 1 2.50 81.66 2 5.00 55.56 average 20 4.03 58.95 40 - 5.00 63.32 - 5.63 50.00 5 1 20 15 2 2 1 5.0 50.00 40 2 5.00 63.32 0 0.00 100.00 2 20 15 3 1 1 4.44 57.95 40 4 10.00 26.63 1 2.50 77.78 3 20 11 5 4 0 7.22 31.63 40 3 7.50 44.97 4 10.00 11.11 4 20 14 3 2 1 5.56 33.25 40 0 0.00 100.00 1 2.50 77.78 average 20 5.56 43.21 40 - 5.63 58.73 - 3.75 66.67 6 1 20 14 3 1 2 6.11 38.90 40 0 0.00 100.00 2 5.00 55.56 2 20 12 3 3 2 8.33 21.12 40 3 7.50 44.97 2 5.00 55.56 3 20 12 4 3 1 7.22 31.63 40 4 10.00 44.97 1 2.50 77.78 4 20 13 4 2 1 6.11 26.65 40 3 7.50 63.32 2 5.00 55.56 average 20 6.94 29.58 40 - 6.25 63.32 - 4.38 61.12 7 1 20 11 3 3 3 10.00 - 40 5 12.50 - 4 10.00 - 2 20 11 2 4 3 10.56 - 40 6 15.00 - 3 7.50 - 3 20 10 3 5 2 10.56 - 40 4 10.00 - 6 15.00 - 4 20 12 3 3 2 8.33 - 40 7 17.50 - 5 12.50 - average 20 - - 40 - 13.63 - - 11.25 -

Embodiment 5, the effect of bacillus subtilis (Bacillus subtilis) HL259 CGMCC No.1451 inoculum control cucumber blight

Cucumber splinter blight is an important disease in cucumber production. It mainly damages the base of the stem, and the primary oil-stained lesions turn white on the surface, appear cracks, and sometimes overflow jelly. Spreads rapidly in wet conditions. In this example, the control effect of HL259 bacterial agent on Jinchun No. 2 (Tianjin Cucumber Research Institute) cucumber blight was determined by field test. The specific method is as follows:

Drugs for testing: Bacillus subtilis (Baciillus subtilis) HL259CGMCC No.1451 biocontrol bacterium agent prepared in embodiment 3 step 2, contrast biocontrol bacterium agent No. 1 (purchased from Beijing Soil and Fertilizer Workstation, active ingredient is Bacillus cereus, The effective viable count is 10 ⁸ cfu/g).

There were 5 treatments and 4 repetitions in this experiment, with a total of 20 plots. Arranged in random blocks. The area of each greenhouse plot is 15.5m ² , and the spacing between plants x row spacing = 30cm x 90cm. The soil of the test site is sandy loam. The previous crop was cucumber.

Treatment 1: HL259 bacterial agent was diluted 1:20 and used to spray roots of cucumbers.

Treatment 2: HL259 bacterial agent was diluted 1:10 and used to spray roots of cucumbers.

Treatment 3: HL259 inoculum was diluted at a ratio of 1:5 and used to spray roots of cucumbers.

Treatment 4: The control biocontrol fungicide No. 1 was diluted and sprayed on the roots at a ratio of 1:5.

Treatment 5: clean water control

Investigate the disease base before spraying, investigate the results of the last spraying 20 days after spraying, and calculate the overall control effect. The investigation of the control effect of creeping blight refers to the method of "Guidelines for the efficacy test of control melon wilt" in the "Guidelines for Field Efficacy Tests of Pesticides" (2) of the Institute of Drug Control of the Ministry of Agriculture. In the experiment, the community general survey was used to record the rate of diseased plants, and the control effect was calculated based on the rate of diseased plants.

The 3 different dilution concentrations of HL259 bacterial agent and the control effect of control biocontrol agent No. 1 on cucumber blight are shown in Table 7. Applying to cucumber rhizomes, the control effects on cucumber blight were 53.31%, 60.01% and 86.60% respectively; the control effect of control biocontrol fungicide No. 1 was 53.24% after spraying with 1:5 dilution. It can be seen from the results that the control effect of HL259 bacterial agent according to 1:10 and 1:5 is obviously better than that of the control biocontrol agent No. 1 diluted with 1:5 ratio (treatment 4). The control effect of HL259 bacterial agent was equivalent to that of treatment 4 according to the 1:20 dilution dose.

The Duncan’s new multiple range test method was used to analyze and compare the control effects of creeping blight in order to evaluate the significance of differences in the control and control of cucumber blight with different doses of HL259 fungicide and the control biocontrol fungicide No. 1. Analysis of variance showed that there was a significant difference between treatment 3 (1:5) and other treatments at the 5% level, and there was no significant difference between treatment 3 (1:5) and treatment 2 (1:10) at the 1% level. Significant differences existed among biocontrol agents No. 1. Cucumbers grew normally throughout the test period, and no phytotoxicity was found. It shows that spraying HL259 bacterial agent at the test concentration to treat the roots of cucumber plants is safe for crop growth. No adverse effects of the test agent on non-target organisms were found in the investigation, indicating that the agent is safe to the environment. Spraying HL259 bacterial agent on the root and stem soil at the early stage of cucumber blight can effectively control the development and spread of the disease. It is an effective agent for the control of cucumber blight. It has no adverse effects on the normal growth of crops, and the control effect is significantly good. In conventional drug varieties and application doses. The HL259 bacterial agent can be sprayed on the cucumber rhizome soil according to the dilution ratio of 1:10 and 1:5.

Table 7. The results of HL259 bacterial agent spraying cucumber rhizome soil with different dilution ratios to control cucumber blight deal with repeat Total number of plants (plants) Number of diseased plants before spraying (strains) Diseased plant rate% Number of diseased plants after spraying (strains) Diseased plant rate% Disease prevention effect% ck 1 72 1 1.39 5 6.94 - 2 72 5 6.94 7 9.72 - 3 72 4 5.56 6 8.33 - 4 72 1 1.39 3 4.17 - average 72 2.75 3.82 5.25 7.29 - 1 1 108 1 0.93 3 2.78 46.68 2 108 4 3.70 6 5.56 46.40 3 108 3 2.78 5 4.63 46.68 4 108 3 2.78 4 3.70 73.48 average 99 2 2.55 4.50 4.17 53.31 2 1 108 3 2.78 5 4.63 46.68 2 108 1 0.93 3 2.78 46.68 3 108 3 2.78 4 3.70 73.48 4 108 0 0.00 1 0.93 73.20 average 108 1.75 1.62 3.50 3.01 60.01 3 1 108 2 1.85 3 2.78 73.20 2 108 1 0.93 1 0.93 100.00 3 108 0 0.00 1 0.93 73.20 4 108 0 0.00 0.00 0.00 100.00 average 108 0.75 0.70 1.25 1.16 86.60 4 1 108 2 1.85 4 3.70 46.68 2 108 2 1.85 3 2.78 73.20 3 108 4 3.70 6 5.56 46.40 4 108 1 0.93 3 2.78 46.68 average 108 2.25 2.08 4.00 3.71 53.24

Embodiment 6, the effect of HL259 bacterial agent preventing and treating capsicum phytophthora

Pepper blight is a devastating disease in pepper production. It is distributed in the south and north of my country. It occurs in greenhouses, greenhouses and open fields. Especially the pepper seedlings transplanted in greenhouses are the most severely affected, often resulting in the death of seedlings in patches. , damage to the adult plant will cause leaf fall and the whole plant to wilt and wither, and effective control measures must be provided during production. In this example, the control effect of HL259 bacterial agent on Jingyan Pepper No. 3 and Kemen sweet pepper pepper blight was determined by field test. The specific method is as follows:

Drugs for testing: the HL259 powdered bacterial agent prepared in step 2 of Example 3, the contrast biocontrol bacterial agent No. 1 (purchased from Beijing Soil and Fertilizer Workstation, the active ingredient is Bacillus cereus, and the effective number of viable bacteria is 10 ⁸ cfu/g ).

There were 5 treatments and 4 repetitions in this experiment, with a total of 20 plots. Arranged in random blocks. The area of each greenhouse plot is 15.5m ² , and the spacing between plants x row spacing = 30cm x 90cm. The soil of the test site is sandy loam. The previous crop was pepper.

Treatment 1: HL259 inoculum was diluted at a ratio of 1:50 for pepper leaf spraying and root irrigation treatment;

Treatment 2: HL259 inoculum was diluted at a ratio of 1:20 for pepper leaf spraying and root irrigation;

Treatment 3: HL259 inoculum was diluted in a ratio of 1:10 for pepper leaf spraying and root irrigation treatment;

Treatment 4: The control biocontrol fungicide No. 1 was diluted at a ratio of 1:50 for pepper leaf spraying and root irrigation treatment;

Treatment 5: The control biocontrol fungicide No. 1 was diluted at a ratio of 1:20 for pepper leaf spraying and root irrigation treatment;

Treatment 6: The control biocontrol fungicide No. 1 was diluted at a ratio of 1:10 for pepper leaf spraying and root irrigation treatment.

Investigate the disease base before spraying; investigate the results of the last spraying 15 days after spraying, and calculate the overall control effect. The investigation of the control effect of the epidemic disease refers to the method of the "Guidelines for the efficacy test of the prevention and treatment of melon epidemic diseases" in the "Pesticide Field Efficacy Test Guidelines" (2) of the Institute of Drug Control of the Ministry of Agriculture. The five-point sampling method was adopted in the test, the disease index was recorded, and the control effect was calculated. Among them, the disease index grading method is: 0, healthy and asymptomatic; 1, only the leaves and fruits have diseased spots on the aboveground; 3, brown rot spots on the stems and branches above the ground; 5, brown rot spots on the base of the stem; 7 Grade 9, brown rot spots on aboveground stems, branches and stem bases; Grade 9, whole plant withered.

The control effects of three different dilution concentrations of HL259 bacterial agent and different dilution concentrations of the control biocontrol agent No. 1 on pepper blight of Jingyan Pepper No. 3 are shown in Table 8. 1:10 proportional dilution dose of root irrigation application, the control effect on pepper blight was 45.74%, 73.46% and 83.46% respectively; the control biocontrol agent No. 1 was diluted at 1:50, 1:20 and 1:10 The control effects after root irrigation were 37.60%, 56.96% and 58.70%, indicating that the control effect of HL259 bacterial agent was significantly better than that of the control biocontrol agent No. 1 according to 1:20 and 1:10 respectively. The HL259 bacterial agent sprayed the leaves according to the dilution ratio of 1:50, 1:20 and 1:10 respectively, and the control effects on pepper blight were 56.33%, 66.53% and 74.48% respectively; , 1:20 and 1:10 ratio dilutions after spraying the leaves were 42.64%, 52.25% and 59.97%, indicating that the control effect of HL259 bacterial agent was significantly better than that of the control when sprayed at 1:20 and 1:10 respectively. Antibacterial agent No. 1 treatment.

The control effects of three different dilution concentrations of the HL259 bacterial agent and different dilution concentrations of the control biocontrol agent No. 1 on the pepper blight of Solanum sweet pepper are shown in Table 9. : 10 proportional dilution dose irrigation root application, the control effect on pepper blight was 77.32%, 81.58% and 86.35%; The control effect after root was 49.19%, 54.17%, 57.47%. It shows that the control effect of HL259 bacterial agent according to the ratio of 1:50, 1:20 and 1:10 is obviously better than that of the control biocontrol agent No. 1. The HL259 bacterial agent sprayed the leaves according to the dilution ratio of 1:50, 1:20 and 1:10 respectively, and the control effects on pepper blight were 53.85%, 59.52% and 70.87% respectively; , 1:20 and 1:10 proportional dilutions after spraying leaves were 39.54%, 51.44% and 47.52%. It shows that the control effect of HL259 bacterial agent according to 1:50, 1:20 and 1:10 is obviously better than that of the control biocontrol agent No. 1.

During the whole test period, the growth of pepper was normal, and no phytotoxicity was found. It shows that spraying HL259 bacterial agent at the test concentration to treat pepper plant leaves or irrigation roots is safe for crop growth. No adverse effects of the test agent on non-target organisms were found in the investigation, indicating that the agent is safe to the environment. The HL259 bacterial agent can effectively control the development and spread of the disease by root irrigation and leaf spraying in the early stage of pepper blight. It is an effective agent for the prevention and treatment of pepper blight. Control bacterial agent No. 1. A better way to use it is to spray peppers with HL259 bacterial agent according to the dilution ratio of 1:20 and 1:10, and irrigate the roots to treat the rhizome soil.

Table 8. Control effect of HL259 bacterial agent on Jingyan Pepper No. 3 deal with spray application Root spraying Disease index (%) Control effect (%) Disease index (%) Control effect (%) CK 35.50 HL259 bacterial agent biocontrol bacteria 1 (cfu/mL) 1:501:201:101:501:201:10 14.5011.889.0620.3616.9514.21 56.3366.5374.4842.6452.2559.97 19.269.425.8722.1515.2814.66 45.7473.4683.4637.6056.9658.70

Table 9. Control effect of HL259 bacterial agent on sweet pepper deal with spray application Root spraying Disease index (%) Control effect (%) Disease index (%) Control effect (%) CK 43.60 HL259 bacterial agent 1:501:20 20.1217.65 53.8559.52 9.898.03 77.3281.58 Biocontrol bacteria 1 (cfu/mL) 1:101:501:201:10 12.7026.3621.1722.88 70.8739.5451.4447.52 5.9522.1519.9818.54 86.3549.1954.1757.47

Sequence Listing

<160>1

<210>1

<211>1448

<212>DNA

<213> Bacillus subtilis

<400>1

tgcctaatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg ttagcggcgg 60

acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccggg 120

gctaataccg gatggttgtt tgaaccgcat ggttcagaca taaaaggtgg cttcggctac 180

cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg 240

acgatgcgta gccgacctga gagggtgatc ggccaacactg ggactgagac acggcccaga 300

ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 360

gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagtg 420

ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc taactacgtg 480

ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg 540

ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 600

ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 660

gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 720

gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca cgccgtaaac 780

gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaaa cgcattaagc 840

actcccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc 900

acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga 960

catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga caggtggtgc 1020

atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080

ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg 1140

aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200

acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca caaatctgtt 1260

ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg 1320

gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380

agagtttgta acacccgaag tcggtgaggt aaccttttag gagccagccg ccgaaggtgg 1440

gacagatg 1448

Claims (10)

1, subtilis (Bacillus subtilis) HL259 CGMCC No.1451.

2, a kind of microbiobacterial agent, its activeconstituents are subtilis (Bacillus subtilis) HL259 CGMCC No.1451.

3, a kind of method for preparing the described microbiobacterial agent of claim 2, be subtilis (Bacillus subtilis) the HL259 CGMCC No.1451 that in YMA or corn steep liquor substratum, ferments, the fermented liquid that obtains mixed obtaining microbiobacterial agent with matrix.

4, method according to claim 3 is characterized in that: contain sucrose 5g in the described corn steep liquor substratum, Semen Maydis powder 2g, KH ₂PO ₄0.05g, (NH ₄) ₂SO ₄0.5g, K ₂HPO ₄0.05g, MgSO ₄0.025g, CaCO ₃2g adds water 1000ml.

5, according to claim 3 or 4 described methods, it is characterized in that: in the described fermentation condition, culture temperature is 25-30 ℃, and air flow is 1: 0.5-1: 1.6, and stirring velocity is 200-400rpm, incubation time is 24-48h; Described air flow is that per minute feeds the volume of air of fermentor tank and the volume ratio of fermentation cylinder for fermentation liquid.

6, method according to claim 5 is characterized in that: described culture temperature is 28-30 ℃, and air flow is 1: 1-1: 1.2, and stirring velocity is 250-300rpm, incubation time is 24-30h.

7, method according to claim 5 is characterized in that: in the described method, add vegetables oil or organic silicone oil during fermentation as foam killer.

8, method according to claim 7 is characterized in that: the quality percentage composition of described vegetables oil is 1-2%; The quality percentage composition of described organic silicone oil is 0.04-01%.

9, method according to claim 3 is characterized in that: described matrix is the peat composed of rotten mosses, micro mist lime carbonate or attapulgite or wilkinite.

10, method according to claim 3 is characterized in that: the viable count of subtilis in the described microbiobacterial agent (Bacillus subtilis) HL259 CGMCC No.1451 is 5 * 10 ⁹Cfu/g, the pH value is 6.0-7.8.

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