CN114891674A - Bacillus belgii for preventing and treating vegetable sclerotiniose and application thereof - Google Patents

Abstract

The invention discloses Bacillus belgii for preventing and treating vegetable sclerotiniose and application thereof, wherein the Bacillus belgii strain is classified and named as Bacillus velezensis (Bacillus velezensis), has a strain number Bv20 and has a preservation number of CCTCC M2022427. The Bacillus belgii Bv20 and the cell-free supernatant thereof have obvious inhibition effect on sclerotinia sclerotiorum, can be used for preventing and treating lettuce sclerotinia sclerotiorum caused by sclerotinia sclerotiorum, and has a control effect of 72.6% in a greenhouse pot experiment; the biocontrol agent or the biopesticide developed by utilizing the Bacillus belgii Bv20 has good application prospect.

Description

Bacillus belgii for preventing and treating vegetable sclerotiniose and application thereof

Technical Field

The application relates to the technical field of biological control of plant diseases, in particular to a Bacillus belgii strain and application thereof in controlling sclerotinia rot of lettuce.

Background

Sclerotinia sclerotiorum is a destructive soil-borne disease in lettuce production and is caused by Sclerotinia sclerotiorum (sclerotiorum) and Sclerotinia sclerotiorum (Sclerotinia minor), and the average yield loss caused by Sclerotinia sclerotiorum is 10-15 percent, and can reach 30 percent at most. Although the main dominant species in most areas of China is Sclerotinia sclerotiorum, in the Yangtze river delta area, the Sclerotinia sclerotiorum disease of lettuce caused by Sclerotinia sclerotiorum (Sclerotinia minor) is becoming more serious in recent years. Although the two pathogens are fungi of the same genus, the hosts are infected in different ways.

Sclerotinia sclerotiorum can infect more than 100 kinds of plants, including lettuce, rape and lettuce, etc. Sclerotinia sclerotiorum, which is mainly in the form of sclerotia, overwinter in soil, disease residues or mixed in seeds, exists in the form of sclerotia about 90% of the time in the life cycle. The sclerotium has stress resistance and can survive in soil for many years. However, sclerotinia sclerotiorum rarely produces sexual stages, i.e., ascospores and ascodiscs, under natural conditions. After the next year, the sclerotia germinate to produce mycelium, which infects the host as mycelium. Sclerotinia sclerotiorum can be infected at any growth stage of the plant, including seedling stage to adult stage. Under the humid and cold conditions, pathogenic bacteria can quickly spread at infected parts to form white flocculent hyphae, which can lead to plant wilting and death in severe cases, and a large amount of sclerotia are usually generated on the disease residues of plants to be used as a new infection source.

In contrast, sclerotinia has a broader host, infects over 400 plants, and produces black, asexual sclerotia, similar to sclerotinia sclerotiorum. The sclerotium can survive in soil for many years in a quiescent state in the absence of a suitable host, and can be used as an airborne pathogen or a soil-borne pathogen. The sclerotia on the surface germinate under suitable conditions to produce an ascospore disc, releasing ascospores (Clarkson et al, 2006), this germination being referred to as fruiting germination. After the ascospores are strongly ejected from the ascospores of the ascodisc, they are transmitted to the whole field or the adjacent field by wind and infect the above-ground plants. While the production of mycelium by the underground sclerotia directly infects the host, this germination is called Myceliogenic germination (Myceliogenic germination). However, a large number of studies have shown that sclerotinia sclerotiorum is mainly based on fruit body germination, and the silk body germination does not exceed 5%. Therefore, the prediction of sclerotinia, disease occurrence and prevalence caused by sclerotinia is mainly based on ascospore inoculation and environmental factors.

Obviously, the strategy of disease control is also different due to differences in the biological properties of the two pathogens and the infecting host. The method mainly aims at preventing and treating lettuce sclerotinia caused by sclerotinia sclerotiorum and mainly aims at preventing and treating the infection of protophyte hyphae, and the method mainly aims at preventing and treating the transmission and invasion of ascospores in production of lettuce sclerotinia sclerotiorum caused by sclerotinia sclerotiorum.

Since the lettuce production is limited by the land area, the sclerotinia sclerotiorum is more and more serious due to continuous cropping, and serious loss is caused to the lettuce production. Although the prevention and treatment method for popularizing disease-resistant varieties is most economical and effective, at present, no completely disease-resistant or completely immune variety exists. Therefore, the control of sclerotinia mainly depends on chemical control methods. The commonly used pesticides include carbendazim, tebuconazole, dimethachlon and the like. But due to the stress resistance of sclerotium, the prevention and treatment effect is limited, and the improvement of the drug resistance of pathogenic bacteria is easily induced. The biological control has the advantages of long lasting period, small environmental pollution, safety to crops, human and livestock and the like. The method for preventing and treating the sclerotinia rot of the lettuce by using biological prevention and treatment measures is beneficial to ensuring the safety of the vegetables and reducing the problem of ecological pollution, and has economic and ecological benefits. The development and application of biocontrol bacteria can provide wide market and prospect for preventing and treating sclerotinia rot of lettuce.

Currently, studies on the biological control of sclerotinia sclerotiorum are focused on sclerotinia sclerotiorum, and the discovered biocontrol bacteria are mainly concentrated on microorganisms such as Coniothyrium minitans (Coniothyrium minitans), Pseudomonas sp (Pseudomonas spp.) and Bacillus sp (Bacillus spp.), while the biocontrol studies on sclerotinia sclerotiorum are less. Aiming at the characteristic that sclerotinia sclerotiorum mainly germinates by mycelium and rarely produces ascospores and ascospores, the biocontrol strain which can be colonized in soil and can inhibit the germination of sclerotium sclerotiorum and the growth of mycelium is screened, and the biocontrol strain is particularly important for preventing and treating sclerotiniose caused by sclerotinia sclerotiorum.

However, the foreign biocontrol strains have adaptability problems in local soil, and the biocontrol effect of the biocontrol strains is often influenced. Therefore, screening of high-efficiency biocontrol strains from local soil is an important aspect and approach for the development and utilization of biocontrol agents.

Disclosure of Invention

The invention aims to solve the problem of difficult control of lettuce sclerotinia rot, and provides a Bacillus belgii strain, a biocontrol agent and application thereof in controlling lettuce sclerotinia rot. The Bacillus belgii Bv20 has a strong inhibiting effect on the growth of sclerotinia sclerotiorum hyphae, can effectively inhibit the expansion of sclerotinia sclerotiorum on lettuce leaves and stalks, and has a good application prospect in a biocontrol agent or a biopesticide developed by utilizing Bv 20.

A Bacillus velezensis strain is classified and named as Bacillus velezensis, the strain number is Bv20, and the preservation number is CCTCC M2022427.

The Bacillus beiLeisi strain is separated from a lettuce soil sample of Shanghai Chunchang vegetable and fruit professional cooperative, is classified and named as Bacillus beiLeisi (Bacillus velezensis), has the strain number of Bv20, has the preservation time of 2022 years, 4 months and 19 days, has the preservation place of China Center for Type Culture Collection (CCTCC) and has the preservation number of CCTCC M2022427.

The biological and morphological characteristics of the B.belgii strain are as follows:

after the strain Bv20 is cultured on an LB plate at constant temperature (30 ℃) for 48 hours, the bacterial colony is circular, white and opaque, indicating dryness and wrinkles, and the inoculating loop is slightly sticky when being touched.

The genetic characteristics of the B.belgii strain are as follows:

the 16s sequence of the strain Bv20 is shown as SEQ ID NO: shown at 13.

Five conserved genes of the strain Bv 20: groEL, gryA, polC, purH, rpoB. Wherein the sequence of groEL is shown in SEQ ID NO: 14 is shown in the figure; the sequence of gryA is shown as SEQ ID NO: 15 is shown in the figure; the sequence of polC is shown in SEQ ID NO: 16 is shown in the figure; the sequence of purH is shown as SEQ ID NO: 17 is shown; the sequence of rpoB is shown in SEQ ID NO: 18, respectively.

The application also provides application of the bacillus belgii strain in preparing a biocontrol product for preventing and treating vegetable sclerotinia sclerotiorum.

Optionally, the biocontrol product is a biocontrol microbial inoculum, a cell-free supernatant or a biological pesticide.

The present application also provides a biocontrol microbial inoculum whose active ingredient is the bacillus belgii of claim 1.Optionally, the concentration of bacillus belgii in the biocontrol microbial inoculum is 1 × 10 ⁹ ～1×10 ¹⁰ cfu/mL。

The biocontrol microbial inoculum is prepared from the Bacillus belgii strain separated by the method, and the preparation method comprises the following steps:

inoculating the Bacillus belgii into a culture solution and culturing until the culture solution OD ₆₀₀ 0.5-0.8, obtaining seed liquid; wherein the culture solution can be LB liquid culture medium, and the formula is as follows: 10g of tryptone, 5g of yeast extract, 10g of NaCl and deionized water are added to a constant volume of 1L, and the pH value is 7.2;

and inoculating the seed solution into an LB fermentation medium for amplification culture to obtain the biocontrol microbial inoculum.

Wherein the inoculation amount of the seed liquid is preferably 1-2%.

Optionally, the amplification culture is performed in a shaker at 30 ℃ and 180rpm for 72 hours. The concentration of the bacterial suspension obtained after the culture of the fermentation liquor is 1 multiplied by 10 ⁹ ～10 ¹⁰ cfu/mL。

Preparation of cell-free supernatant: centrifuging the biocontrol bacteria liquid fermentation liquor at 10000rpm for 10min to obtain cell-free supernatant of Bacillus belgii Bv 20. In the biocontrol product, the volume concentration of the cell-free supernatant is 1-10%.

The invention also provides application of the bacillus belgii strain or the biocontrol microbial inoculum in antagonizing sclerotinia sclerotiorum.

The invention also provides application of the bacillus belgii strain or the biocontrol microbial inoculum in preventing and treating vegetable sclerotinia sclerotiorum caused by sclerotinia sclerotiorum infection.

The Bacillus belgii strain or the biocontrol microbial inoculum achieves the aim of antagonizing sclerotinia sclerotiorum by inhibiting the growth of sclerotinia sclerotiorum hyphae.

Optionally, the vegetable is lettuce, lettuce or rape. Preferably lettuce.

The invention also provides a method for preventing and treating vegetable sclerotinia sclerotiorum caused by sclerotinia sclerotiorum infection, which comprises the following steps:

diluting the biocontrol microbial inoculum or cell-free supernatant prepared from the Bacillus belgii strain and applying the diluted biocontrol microbial inoculum or cell-free supernatant to roots or leaves of sclerotinia sclerotiorum vegetables; the sclerotinia sclerotiorum is caused by sclerotinia sclerotiorum infection.

Optionally, the vegetable is lettuce, lettuce or rape.

Optionally, the concentration of the bacillus beiLeisi strain of the bio-control microbial inoculum after being diluted is 1 × 10 ⁷ ～1×10 ⁸ cfu/mL。

Optionally, the volume percentage of the cell-free supernatant after the cell-free supernatant is diluted is 1-10%.

Optionally, the vegetables are irrigated for the first time 25-35 days after transplantation, and irrigated for the second time after 8-10 days, wherein the irrigation amount is 50-80 mL/plant each time.

Compared with the prior art, the invention has at least one of the following beneficial effects:

(1) the screened Bacillus belgii Bv20 can obviously inhibit the growth of sclerotinia sclerotiorum hyphae in a plate confronting test, and the cell-free supernatant with the concentration of 8 percent has the inhibition effect on the growth of sclerotinia sclerotiorum of 100 percent.

(2) The control effect of the Bacillus beiLeisi strain Bv20 screened by the invention on the leaves of the in vitro lettuce reaches 76.3 percent.

(3) The control efficiency of the screened Bacillus beiLeisi strain Bv20 reaches 72.6 percent under the condition of a greenhouse potting experiment, and the Bacillus beiLeisi strain Bv20 can be used for developing biocontrol products and is applied to the biological control of vegetable sclerotinia rot.

(4) The Bacillus belgii Bv20 and the cell-free supernatant thereof have obvious inhibition effect on sclerotinia sclerotiorum and can be used for preventing and treating lettuce sclerotinia caused by the sclerotiorum; the biocontrol agent or the biopesticide developed by utilizing the Bacillus belgii Bv20 has good application prospect.

Drawings

FIG. 1 is a graph showing the inhibitory effect of 3 strains of biocontrol bacteria on sclerotinia sclerotiorum; a is a hypha growth chart of 3 biocontrol bacteria for inhibiting sclerotinia sclerotiorum by a diffusion method; b is the size of a bacteriostatic zone of 3 biocontrol bacteria;

FIG. 2 is a photograph showing the morphological observation of Bacillus belgii Bv20 on an LB plate;

FIG. 3 is a phylogenetic tree constructed by combining the gyrA gene, rpoB gene, purH gene, groEL gene and polC gene sequences of Bacillus belgii;

FIG. 4 shows the detection of lipopeptide compounds produced by Bacillus belgii strain Bv20 by MALDI-TOF mass spectrometry;

FIG. 5 is a graph showing the effect of different concentrations of cell-free supernatant produced by strain Bv20 on sclerotinia sclerotiorum hypha growth;

FIG. 6 shows the control effect of the microbial inoculum prepared by the strain Bv20 on sclerotinia sclerotiorum on in-vitro lettuce leaves. The upper panel is a control group (clear water); the lower panel is a treatment group, namely, the microbial inoculum prepared by the strain Bv20 for spraying;

FIG. 7 is a graph showing the effect of the Bv20 preparation on the control of sclerotinia on lettuce plants: a is control group (clear water); b is a treatment group, namely an irrigation strain Bv20 microbial inoculum; C. e is the symptom of disease amplification of the control group; D. f is the symptom of disease amplification of the treatment group;

FIG. 8 shows the effect of B.baylaxis strain Bv20 on the growth of hyphae of ten plant pathogenic bacteria. Fusarium graminearum (Fusarium graminearum), Fusarium oxysporum (Fusarium oxysporum), Curvularia vulgare (Curvularia lunata), Botrytis cinerea (Botrytis cinerea), Fusarium granatum (Fusarium fujikuroi), Fusarium proliferatum (Fusarium proliferatum), Alternaria alternata (Alternaria sp.), Camellia sinensis (Gloeosporium tea-sinesis), Rhizoctonia solani (Rhizoctonia solani) and Helminthosporium zeae (Bipolaris maydis) in this order from left to right.

Detailed Description

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

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The following examples relate to media and reagent compositions comprising:

potato dextrose agar medium (PDA medium): 200g of potato, 20g of glucose, 12g of agar powder and 1L of deionized water, and sterilizing at 121 ℃ for 20min, wherein the pH value is natural.

LB culture solution: 10g of tryptone, 5g of yeast extract, 10g of NaCl and deionized water are added until the volume is 1L, and the mixture is sterilized at 121 ℃ for 20min and has the pH value of 7.2.

NA agar medium: 2.5g of glucose, 10g of tryptone, 5g of sodium chloride, 3g of beef extract, 12g of agar powder and 1L of deionized water, and sterilizing at 121 ℃ for 20min and at the pH value of 7.0.

0.7mol/L sodium chloride solution: 41g of NaCl, constant volume to 1L by using deionized water, and sterilization at 121 ℃ for 20min, wherein the pH value is 7.0.

Example 1

Soil sample collection and biocontrol bacteria separation

Soil samples were collected from the lettuce fields of Shanghai Chunchang vegetable and fruit professional Cooperation, 3 months 2021. Weighing 1g of soil sample, adding the soil sample into a solution containing 10mL of sodium chloride (with the concentration of 0.7mol/L), oscillating the solution for 1min to prepare a soil suspension, and diluting the soil solution to the concentration of 10 by using 0.7mol/L sodium chloride solution ^-2 、10 ^-3 And 10 ^-4 Of the gradient of (c). To screen antagonistic bacteria, liquid cultured sclerotinia sclerotiorum (s.minor) mycelia were first broken into mycelia fragments on a homogenizer, 100 μ L of mycelia fragments were aspirated and spread on LB plates, excess water was removed in a fume hood, and then different gradient soil solutions (100 μ L per plate) were spread on LB plates. They were incubated in a 28 ℃ incubator. After every 24h, colonies with significant zones of inhibition were selected and potential biocontrol bacteria were streaked on LB plates for purification. The obtained purified strain was preserved in a refrigerator at 4 ℃ for use.

Second, evaluation of biocontrol bacterium effect in vitro

For further screening of antagonistic bacteria, the inhibitory effect was determined by diffusion method, i.e. by first breaking the liquid cultured sclerotinia sclerotiorum (s.minor) hyphae into hypha fragments on a homogenizer, sucking 100 μ L of hypha fragments and coating them on a PDA plate, removing excess water in a fume hood, then placing a filter paper containing the bacterial liquid in the center of the PDA plate, and determining the size of the zone of inhibition after 3 days.

Antagonistic effect as shown in fig. 1, 3 strains of bacteria with better antagonistic effect were isolated from the soil samples of the lettuce field, and numbered as bac20, bac32 and bac 45. The bac20 strain has the most obvious inhibition effect on sclerotinia sclerotiorum hyphae, and the average inhibition zone size is 3.4 cm. And after 20 days of culture, the antagonistic effect is still very obvious, and only a small amount of sclerotium is formed on the plate.

Example 2 morphological Observation and characterization of Strain bac20

Referring to FIG. 2, after incubation of strain bac20 on LB plates at constant temperature (30 ℃) for 48h, the colonies were round, white, opaque, indicating dryness, wrinkles, and slight stickiness to the touch of the inoculating loop.

For identification, the biocontrol bacterial 16S rDNA sequences were amplified by colony PCR using bacterial 16S primers (table 1). The PCR amplification reaction system is 25 mu L: ddH ₂ O8 mu L, Taq Mix 12.5 mu L, 16s-27f primer 1 mu L, 16s-1492r primer 1 mu L and DNA template 2.5 mu L. The PCR reaction program is 94 ℃ for 2 min; 30s at 94 ℃, 100s at 58 ℃, 60s at 72 ℃ and 25 cycles; storing at 16 ℃. The PCR amplification product was sent to Hangzhou Ongke Biotech for sequence analysis. The 16s sequence is shown in SEQ ID NO: 13. homology alignment was performed with known 16S rDNA sequences in GenBank using the Blast program. The bac20 strain was preliminarily identified as a bacillus bacterium.

In order to further clarify the classification status of the biocontrol bacteria, five conserved gene sequences of the biocontrol bacteria gryA, rpoB, purH, polC and groEL (primer sequences are shown in a table 1) are amplified by using a colony PCR method, and the five gene sequences are matched to construct a phylogenetic tree. The method comprises the following steps: the obtained sequence is firstly edited by Clustalx1.83, then the sequence of each gene is arranged by MAFFT 7.273 software, the arranged sequence is used for picking out fuzzy regions by Gblocks 0.91b, the best GTR + I + G nucleotide replacement model is obtained by jModel Test 2.1.7, and finally a phylogenetic tree is constructed by a Maximum Likelihood (ML) method in RaxmlGUI v.1.5 software.

TABLE 15 conserved Gene primer sequences

As shown in FIG. 3, a phylogenetic tree was constructed by sequencing five conserved genes of groEL (SEQ ID NO: 14), gryA (SEQ ID NO: 15), polC (SEQ ID NO: 16), purH (SEQ ID NO: 17), rpoB (SEQ ID NO: 18) of biocontrol bacteria and matching the five gene sequences, wherein bac20 is highly homologous to Bacillus velezensis B-23189 and Bacillus velezensis B-23190, identifying the strain as Bacillus velezensis (B.velezensis) and identifying the strain as Bv20, and preserving the strain in China Center for Type Culture Collection (CCTCC) at 2022, 4 and 19 days, with the preservation number of CCTCC M2022427.

EXAMPLE 3 lipopeptide Compound assay

And (3) detecting and analyzing the characteristics of the biocontrol bacterial lipopeptide by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). After the Bacillus belgii Bv20 is cultured for 24h at 30 ℃ on an NA agar medium, a single colony is picked and dissolved in a centrifuge tube containing a matrix solution. The matrix solution contained 10mg/mL cyano-4-hydroxycinnamic acid dissolved in 70% acetonitrile with 0.1% trifluoroacetic acid (TFA). The samples were homogenized and centrifuged at 5000 r/min. mu.L of the sample was added dropwise to a MALDI-TOF MTP 384 target disk (Bruker Daltonik GmbH, Leipzig, Germany) and allowed to dry before analysis. MALDI-TOF mass spectrometry recording is performed using an ultrafleXtreme with a configured smart beam laser ^TM MALDI-TOF (Bruker, Bremen, Germany) apparatus. The measurements were made in a reflection mode of operation and at an ion source acceleration voltage of 20 kV. Mass spectra were stored in the low mass range region between 0.1kD and 2 kD. According to the mass spectrum peak of the series of lipopeptides reported and measured in the literature, the lipopeptide compound produced by the Bacillus belgii Bv20 is analyzed, so that the possible antagonistic mechanism of Bv20 on sclerotinia sclerotiorum is explored.

As shown in FIG. 4 and Table 2, the results of MALDI-TOF-MS analysis revealed that Bacillus belgii Bv20 has two significant series of mass peaks with mass-to-charge ratios (m/z) ranging from 1043 to 1123 and 1390 to 1529, which are assigned to Iturin (Iturin A) and Fengycin (Fengycin), respectively. Therefore, Bv20 mainly synthesizes two lipopeptide compounds of iturin and fengycin. The iturin and the fengycin have the characteristics of wide antibacterial spectrum, low toxicity and the like, can act on cell walls and cell membranes of pathogenic bacteria, and can obviously inhibit the growth of fungi.

TABLE 2 detection of lipopeptides in Bv20 by MALDI-TOF Mass Spectrometry

Example 4 preparation of biocontrol microbial inoculum and cell-free supernatant

Preparing biocontrol bacteria seed liquid: selecting a single colony of Bacillus belgii Bv20 to inoculate in 10mL of liquid LB liquid culture medium, and performing shake culture at 30 ℃ and 180rpm for 24h until the culture solution OD ₆₀₀ 0.5-0.8, and is used as a liquid fermentation seed liquid of a biocontrol agent.

Liquid fermentation: inoculating 500 mu L of biocontrol bacteria seed liquid into 500mL of liquid LB culture medium, and performing shake culture at 30 ℃ and 180rpm for 72 hours to obtain biocontrol bacteria liquid fermentation liquid.

Preparation of cell-free supernatant: centrifuging the biocontrol bacteria liquid fermentation liquor at 10000rpm for 10min to obtain cell-free supernatant of Bacillus belgii Bv 20.

Example 5 evaluation of Effect of in vitro cell-free supernatant

0.50mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL and 5.00mL of cell-free supernatant of Bacillus belgii Bv20 were aspirated, respectively, and added to 50mL of PDA medium to obtain PDA plates having final concentrations of 1%, 2%, 4%, 6%, 8% and 10% cell-free supernatant, respectively. Sterile water was added as a negative control. After the PDA plate is solidified, a sclerotinia sclerotiorum strain sheet is inoculated to the center of the plate. The plates were incubated at 25 ℃ for 3 days and the effect of the cell-free supernatant on the inhibition of sclerotinia sclerotiorum hyphae was evaluated.

As shown in FIG. 5, the cell-free supernatant of Bacillus belgii Bv20 has high bacteriostatic activity, and 8% of the supernatant completely inhibits the growth of sclerotinia sclerotiorum hyphae. The inhibition rates of the supernatant with 1%, 2%, 4%, 6%, 8% and 10% on sclerotinia sclerotiorum hyphae were 28.77%, 42.67%, 52.09%, 58.36%, 100% and 100% respectively.

Example 6 measurement of Effect of biocontrol microbial inoculum on in vitro leaves on control of Sclerotinia sclerotiorum

Fresh lettuce leaves with similar growth vigor are taken, washed clean by sterile water and dried, and the leaves are placed in a culture dish with the bottom padded with wet filter paper. The Bacillus belgii Bv20 biocontrol agent (prepared in example 4) was diluted 100-fold (concentration 1X 10) by spraying ⁷ ～10 ⁸ cfu/mL), spraying 2mL of bacterial liquid on each lettuce leaf, and setting sterile water as negative control. After the lettuce leaves are dried in the air, a sclerotinia sclerotiorum piece with the diameter of 6mm is inoculated on each leaf, and a moist petiole is covered by a moist cotton ball. After culturing for 2d at 25 ℃ in a 12h illumination incubator, observing the disease condition of the lettuce leaves. Grading standard of sclerotinia disease spots: stage 0: no disease symptoms; level 1: the lesion spots are less than 1.5 cm; and 2, stage: the lesion spots are less than 3.5 cm; and 3, level: the lesion spots are less than 5.5 cm; 4, level: the lesion is larger than 5.5cm or most or all of the leaves are rotten.

Disease index (%) ═ Σ (number of diseased veins or leaves at each stage × relative stage)/(number of total veins or leaves × highest stage) × 100; control effect (%) - (average disease index of sterile water control-average disease index of treatment)/average disease index of sterile water control × 100.

As shown in fig. 6 and table 3, sclerotinia sclerotiorum in the control group was rapidly infected on lettuce leaves, and the affected parts of the leaf lesions were yellowish brown water stain. In the treatment group, Bv20 biocontrol microbial inoculum can efficiently inhibit the infection of sclerotinia sclerotiorum on the leaves of lettuce, the morbidity is obviously lower than that of a control group after 2 days of inoculation of sclerotinia sclerotiorum blocks, most of the leaves only suffer from the disease at the germ inoculation part, the disease index is only 20.5%, the control effect reaches 76.3%, and the biocontrol microbial inoculum has a good control effect on sclerotinia sclerotiorum.

TABLE 3 in vitro leaf Bv20 preventive and therapeutic effects on sclerotinia rot of lettuce

Example 7 Bv20 Effect test on prevention of lettuce sclerotinia in greenhouse

Sowing lettuce seeds in a hole tray containing nutrient soil, transplanting the lettuce seeds after 1 week of emergence to pot plants with the diameter of 140mm, and culturing a lettuce seedling in each pot plant. After culturing for 30 days, lettuce plants with similar growth vigor are selected, and 20 mature sclerotinia sclerotiorum sclerotia are respectively placed at the leaf of the base part of each lettuce stem. The Bv20 microbial inoculum of example 4 was diluted 100-fold with clear water to adjust the concentration of Bacillus belgii to 1X 10 ⁷ ～10 ⁸ cfu/mL. Pouring the diluted Bv20 biocontrol microbial inoculum to the roots of the treated lettuce, and pouring 50ml of biocontrol bacterial liquid into each lettuce; the lettuce in the control group was irrigated with 50mL of clear water, and 15 plants were repeated in each group. After the culture is continued for 8-10 days, 50mL of the biocontrol microbial inoculum and clear water are applied to the treatment group and the control group respectively. After 20 days of culture, observing the pathogenesis of the sclerotinia rot of the lettuce, and calculating the disease index and the prevention effect. Disease grading standard: level 0: the plant grows normally; level 1: 1-4 leaves around the lower part of the plant become brown and soft; and 2, stage: 5-10 leaves around the lower part become brown and soft, and meanwhile, the disease spots spread to the upper part of the main root, but the whole plant has no wilting symptom; and 3, level: the leaf of the whole plant has wilting symptom; 4, level: the plants fell down or withered and die.

Disease index (%) [ Σ (number of disease-grade plants × this disease-grade representative value)/(number of total investigated plants × number of highest grade) ] × 100; control effect (%) [ (control zone disease index-treatment zone disease index)/control disease index ] × 100.

As shown in FIG. 7 and Table 4, after inoculation for 25 days, the control group of lettuce sclerotinia occurred seriously, and the stem base produced a large amount of white hyphae with many small sclerotia thereon; the infected leaves are soaked in water, plants are lodging or withered, and the disease index reaches 85%. Most lettuce plants applied with the Bv20 biocontrol microbial inoculum grow well, although some infected plant stem base parts have water-immersed disease spots, white hypha is not formed on the disease spots, only 1-10 leaves at the lower part of the plant become brown and soft, the disease occurrence is obviously lighter, the disease index is only 23.3%, and the control effect reaches 72.6%.

TABLE 4 prevention and treatment effects of Bv20 on sclerotinia rot of lettuce in greenhouse

Example 8Bv 20 inhibition Spectroscopy

In order to determine the bacteriostasis spectrum of the biocontrol bacteria, a bacterial sheet (6mm) is taken from the edge of a colony of the pathogenic bacteria after 3d growth and inoculated to the center of a PDA (personal digital assistant) plate, and the determined pathogenic bacteria comprise: fusarium graminearum (Fusarium graminearum), Fusarium oxysporum (Fusarium oxysporum), Curvularia lunata (Curvularia lunata), Botrytis cinerea (Botrytis cinerea), Fusarium canopium (Fusarium fujikuroi), Fusarium stratiotes (Fusarium proliferatum), Alternaria alternata (Alternaria), Fusarium theobromae (Gloeosporium theae-sinense Miyake), Rhizoctonia solani (Rhizoctonia solani), and Helminthosporium zeae (Bipolaris maydis). Then, a filter paper sheet containing the bacterial solution was placed on both sides 3cm from the center of the PDA plate. Plates cultured with pathogens alone were used as control. And after the hyphae grow to the edge of the flat plate after the control group, evaluating the bacteriostatic ability of the biocontrol bacteria.

As shown in fig. 8, among the 10 plant pathogenic bacteria, bacillus belgii Bv20 has a good broad-spectrum antibacterial property and a good antagonistic effect, and can have a significant antagonistic effect on 10 plant pathogenic bacteria such as botrytis cinerea, alternaria, rhizoctonia solani and various fusarium.

Aiming at the current situation that the sclerotinia rot of lettuce is difficult to control, a strain of Bacillus belgii for effectively controlling the sclerotinia rot is screened out, and the biocontrol agent can efficiently inhibit the growth of sclerotinia sclerotiorum hyphae; the biocontrol agent or the biological pesticide developed by utilizing the strain has good application prospect.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

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<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 4

caaggtaatg ctccaggcat tgct 24

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 5

gacgtgggat ggctacaact 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 6

attgtcgcct ttaacgatgg 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 7

acagagcttg gcgttgaagt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 8

gcttcttggc tgaatgaagg 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 9

ttgtcgctca yaatgcaagc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 10

ytcaagcatt tcrtctgtcg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 11

gagcttgaag tkgttgaagg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 12

tgagcgtgtw acttttgtwg 20

<210> 13

<211> 1631

<212> DNA

<213> Bacillus velezensis

<400> 13

gactcgtcag tattgactgc agacctgaag cttgatatcg aattcgcgtg tcgcccttct 60

acggctacct tgttacgact tcaccccaat catctgtccc accttcggcg gctggctcct 120

aaaaggttac ctcaccgact tcgggtgtta caaactctcg tggtgtgacg ggcggtgtgt 180

acaaggcccg ggaacgtatt caccgcggca tgctgatccg cgattactag cgattccagc 240

ttcacgcagt cgagttgcag actgcgatcc gaactgagaa cagatttgtg ggattggctt 300

aacctcgcgg tttcgctgcc ctttgttctg tccattgtag cacgtgtgta gcccaggtca 360

taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttgtcac cggcagtcac 420

cttagagtgc ccaactgaat gctggcaact aagatcaagg gttgcgctcg ttgcgggact 480

taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgtc actctgcccc 540

cgaaggggac gtcctatctc taggattgtc agaggatgtc aagacctggt aaggttcttc 600

gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt caattccttt 660

gagtttcagt cttgcgaccg tactccccag gcggagtgct taatgcgtta gctgcagcac 720

taaggggcgg aaacccccta acacttagca ctcatcgttt acggcgtgga ctaccagggt 780

atctaatcct gttcgctccc cacgctttcg ctcctcagcg tcagttacag accagagagt 840

cgccttcgcc actggtgttc ctccacatct ctacgcattt caccgctaca cgtggaattc 900

cactctcctc ttctgcactc aagttcccca gtttccaatg accctccccg gttgagccgg 960

gggctttcac atcagactta agaaaccgcc tgcgagccct ttacgcccaa taattccgga 1020

caacgcttgc cacctacgta ttaccgcggc tgctggcacg tagttagccg tggctttctg 1080

gttaggtacc gtcaaggtgc cgccctattt gaacggcact tgttcttccc taacaacaga 1140

gctttacgat ccgaaaacct tcatcactca cgcggcgttg ctccgtcaga ctttcgtcca 1200

ttgcggaaga ttccctactg ctgcctcccg taggagtctg ggccgtgtct cagtcccagt 1260

gtggccgatc accctctcag gtcggctacg catcgtcgcc ttggtgagcc gttacctcac 1320

caactagcta atgcgccgcg ggtccatctg taagtggtag cggaagccac cttttatgtc 1380

tgaatcatgc ggttcaaaca accatccggt attagccccg gtttcccgga gttatcccag 1440

tcttacaggc aggttaccca cgtgttactc acccgtccgc cgctaacatc agggagcaag 1500

ctcccatctg tccgctcgac ttgcatgtat taggcacgcc gccagcgttc gtcctgagcc 1560

aggatcaaac tctaagggcg acacgcgaat tcgatatcaa gcttcaggtc tgcagcgagc 1620

ctcagacact g 1631

<210> 14

<211> 1008

<212> DNA

<213> Bacillus velezensis

<400> 14

cgctgcatga gcgttatcgt atcccgggcg cttccggatg tgcgtgacgg tctgaagccg 60

gttcacaggc ggattttgta cgcaatgaat gatttaggca tgaccagtga caaaccatat 120

aaaaaatctg cccgtatcgt cggtgaagtt atcggtaagt accacccgca cggtgactca 180

gcggtttacg aatcaatggt cagaatggcg caggatttta actaccgcta catgcttgtt 240

gacggacacg gcaacttcgg ttcggttgac ggcgactcag cggccgcgat gcgttacaca 300

gaagcgagaa tgtcaaaaat cgcaatggaa atcctccggg acattacgaa agatacgatt 360

gattatcaag ataactatga cggcgcagaa agagaacctg tcgtcatgcc ttcgagattt 420

ccgaatctgc tcgtaaacgg agctgccggt attgcggtcg gaatggcgac aaatattcct 480

ccgcatcagc ttggggaagt cattgaaggc gtgcttgccg taagtgagaa tcctgagatt 540

acaaaccagg agctgatgga atacatcccg ggcccggatt ttccgactgc aggtcagatt 600

ttgggccgga gcggcatccg caaggcatat gaatccggac ggggatccat tacgatccgg 660

gctaaggctg aaatcgaaga gacatcatcg ggaaaagaaa gaattattgt cacagaactt 720

ccttatcagg tgaacaaagc gagattaatt gaaaaaatcg cagatcttgt ccgggacaaa 780

aaaatcgaag gaattaccga tctgcgtgac gaatccgacc gtaacggaat gagaatcgtc 840

attgagatcc gccgtgacgc caatgctcac gtcattttga ataacctgta caaacaaacg 900

gccctgcaga cgtctttcgg aatcaacctg ctggcgctcg ttgacggaca gccgaaagta 960

ctaatcctga agcaatgcct ggaacattac cttggattat tacgtatt 1008

<210> 15

<211> 998

<212> DNA

<213> Bacillus velezensis

<400> 15

ggtctctctg agtgacgcct tgtgaagatg atgtatacac atctattcac attgaagaat 60

atgaatcaga agcacgtgat acaaagcttg ggcctgaaga gatcacccgc gatattccaa 120

acgtagggga agacgcgctt cgcaatcttg atgaccgcgg aattatccgt atcggtgcgg 180

aagtcaacga cggagacctt ctcgtaggta aagtaacgcc taaaggtgta actgagctta 240

cggctgaaga acgccttctg catgcgatct ttggagaaaa agcgcgtgaa gtccgtgata 300

cttctctccg tgtgcctcac ggcggcggcg gaattatcca cgacgtaaaa gtcttcaacc 360

gtgaagacgg cgacgaactt cctccgggag tgaaccagct tgtacgcgta tatatcgttc 420

agaaacgtaa gatttctgaa ggtgataaaa tggccggacg tcacggaaac aaaggggtta 480

tctcgaagat tcttcctgaa gaagatatgc cttaccttcc tgacggcacg ccgatcgata 540

tcatgcttaa cccgctgggt gtaccatcac gtatgaatat cggtcaggta ttagaacttc 600

acatgggtat ggctgcccgc tacctcggca ttcacatcgc gtcacctgta tttgacggcg 660

cgcgtgaaga agatgtgtgg gaaacacttg aagaagcagg catgtcaaga gacgctaaaa 720

cagttcttta tgacggccgt acgggagaac cgtttgacaa ccgtgtatct gtcggaatca 780

tgtacatgat caaactggcg cacatggttg atgataaact tcatgcccgt tcgacaggtc 840

cttactcact tgttacgcag cagcctctcg gcggtaaagc ccaattcggc ggacagcgtt 900

tcggtgagat ggaggtttgg gcgcttgaag cttacggcgc agcttacacg cttcaagaaa 960

tcctgactgt gaagtccgat gacgtgtcgg acggtaaa 998

<210> 16

<211> 985

<212> DNA

<213> Bacillus velezensis

<400> 16

cagcaaactt tatatatata cagagggggg ttggggtgac attggccgga ggaacaaaaa 60

aacttcttca ggaaaacggt gtggatgtca tcggcatttc agaagtgacc ggatttcctg 120

aaattatgga cggacggtta aaaacgctcc atcctaatat tcacggcgga ctgcttgccg 180

taagagacaa tgaagagcat atggcgcaga tcaatgagca cggcattgcc cccattgacc 240

ttgtggtcgt caacctttac ccgtttaaag aaacgatttc aaaagaagac gtaacatacg 300

atgaagcgat agaaaacatt gatatcggcg gtcccggcat gctgcgcgcc gcctcgaaaa 360

accatcagga tgtgacggtc atcacagatc cggccgatta cagttccgtg ctcaatgaga 420

ttaaagaaca cggcggcgtt tctcttaaaa gaaaacgcga gcttgcggcc aaagtattcc 480

gccataccgc ggcatacgac gcattaatcg ctgattactt aacacgcgag gccgatgaga 540

aagaccctga gcaattcacc gttacatttg agaaaaaaca atcgctccgc tacggtgaaa 600

accctcacca agaggcggtt ttctaccaaa gcgcacttcc cgtctccggt tccatcgcgg 660

cggcaaaaca gcttcacggc aaagagcttt cttacaacaa tattaaggac gcagatgcgg 720

ccgttcaaat cgtccgggaa tttacagaac ccgcagctgt tgccgttaaa catatgaacc 780

cgtgcggagt cggtacggga gcttcaattg aggaagcatt caataaagcg tatgaagctg 840

ataaaacctc cattttcggc ggcatcatcg cgctgaaccg tgaagttgat caggcaacgg 900

ctgaagccct tcacggcatc tttttagaat catatcccta ttttttaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa cactg 985

<210> 17

<211> 821

<212> DNA

<213> Bacillus velezensis

<400> 17

gcgccccaca aacacacttg atatgggatt ttaaatgtgg cgtacaagcg tcttttgaaa 60

acggaaaaag cgaaaaatcc ggtcattgat acgctggaac tcgcgcgttt cctgtatcct 120

gagtttaaaa atcaccgctt aaatacgtta tgtaagaagt ttgatatcga attaacccag 180

catcaccgag cggtctttga cgctgaagca acgggctacc tgctgttgaa aatgctcaaa 240

gatgccgctg aaaaagacat tttttatcat gatcagctga atgagaatat gggacaatcc 300

aatgcttatc agagatcaag accttatcac gctacattgc ttgccgtgaa tgagaccggc 360

cttaaaaatc tgtttaagct cgtgtccatt tctcatattc aatatttcta cagagtgccg 420

cgcattccga ggtcgcagct taataaatac agagaaggtc tgttaatcgg ctctgcctgt 480

gacaggggtg aggtctttga aggcatgatg caaaaatctc ctgaagaggt tgaagatatc 540

gcatccttct atgattatct tgaagtgcag ccgccggaag tatacagaca ccttctgcag 600

cttgagctcg tccgagatga aaaagcgctg aaagaaatca tcgccaacat tacgaagctc 660

ggagaaaaat tgaataagcc ggtcgtggct acgggaaatg tccactattt aaacgatgag 720

gataaaattt accggaagat cttaatatct tcccaaggcg gcgccaaccc gttaaacaga 780

cacgaactgc ctaaagtgcc ttcaaacaga aaaaaggggg g 821

<210> 18

<211> 978

<212> DNA

<213> Bacillus velezensis

<400> 18

aaagaacgaa attgtatgat caaaaaaagg attgtaagta tcaattcgac cgcggatatg 60

cgtctcctta catggtgact gactctgata agatggaagc ggttcttgac aatccttaca 120

tcttaatcac agacaaaaaa atcacaaaca ttcaagaaat ccttcctgtg cttgagcaag 180

ttgtacagca aggcaaacca ttgcttctga tcgctgaaga tgttgaaggg gaagctcttg 240

ctacactcgt tgtcaacaaa cttcgcggca cattcaacgc tgttgccgtt aaagctcctg 300

gcttcggtga ccgccgtaaa gcaatgcttg aagacatctc tgttcttaca ggcggagaag 360

tgatcacaga agacttaggc cttgacctga aatctactga aatcggacaa ttgggacgcg 420

cttctaaagt tgtggtaacg aaagaaaaca caacaatcgt agaaggcgcc ggcgacactg 480

aaaaaattgc tgcacgcgtc aaccaaatcc gcgctcaagt ggaagaaaca acttctgaat 540

tcgacagaga aaaattacaa gagcgtcttg cgaaacttgc cggcggcgta gctgtcatca 600

aagtcggcgc tgcgactgaa actgagctga aagagcgtaa acttcgcatc gaagacgccc 660

tcaactcaac tcgcgcagct gttgaagaag gcatcgtatc cggcggtggt acagcgcttg 720

tcaacgtata caacaaagtc gctgcagtgg aagctgaagg cgatgcgcaa acaggtatca 780

acattgtgct tcgcgcgctt gaagagccga tccgtcaaat cgcgcacaat gcaggccttg 840

aaggatctgt catcgttgag cgcctgaaaa acgaaaaaat cggcgtaggc ttcaacgctg 900

caaccggcga atgggtaaac atgatcgaaa aaggatcgtg actagaaaaa aaaccgcccc 960

cccctcacaa aaaaagcg 978

Claims (10)

1. The Bacillus belgii strain is characterized by being classified and named as Bacillus velezensis, the strain number is Bv20, and the preservation number is CCTCC M2022427.

2. Use of a bacillus beiLeisi strain according to claim 1 for the preparation of a biocontrol product for the control of vegetable sclerotinia rot.

3. Use according to claim 2, characterized in that the biocontrol product is a biocontrol agent, a cell-free supernatant or a biopesticide.

4. A biocontrol microbial inoculum having as an active ingredient Bacillus belgii according to claim 1.

5. The biocontrol microbial inoculum of claim 4 wherein the concentration of Bacillus belgii in the biocontrol microbial inoculum is 1 x 10 ⁹ ～1×10 ¹⁰ cfu/mL。

6. Use of the bacillus beiLeisi strain of claim 1 or the biocontrol agent of claim 4 for antagonizing sclerotinia sclerotiorum.

7. Use of a bacillus beiLeisi strain according to claim 1 or a biocontrol agent according to claim 4 for controlling vegetable sclerotiniose caused by sclerotinia sclerotiorum infection.

8. Use according to claim 7, characterized in that the vegetable is lettuce, lettuce or rape.

9. A method for controlling vegetable sclerotiniose caused by sclerotinia sclerotiorum infection is characterized by comprising the following steps:

a biocontrol microbial agent or cell-free supernatant made of the Bacillus belgii strain of claim 1 is diluted and applied to roots or leaves of sclerotinia sclerotiorum vegetables caused by infection with sclerotinia sclerotiorum.

10. The control method according to claim 9,

the vegetable is lettuce, lettuce or rape;

the concentration of the bacillus beleisi strain of the biocontrol microbial inoculum after being diluted is 1 multiplied by 10 ⁷ ～1×10 ⁸ cfu/mL；

The volume percentage of the cell-free supernatant after the cell-free supernatant is diluted is 1-10%;

irrigating for the first time 25-35 days after the vegetables are transplanted, and irrigating for the second time 8-10 days later, wherein the irrigation amount is 50-80 mL/plant each time.

Images