Bacillus velez, bacterial agent and application thereof in prevention and treatment of pathogenic bacteria diseases

Abstract

The present invention discloses a Bacillus Velez, a microbial agent and an application thereof in the prevention and treatment of pathogenic bacteria and diseases, and relates to the technical field of the prevention and treatment of pathogenic bacteria and diseases. The Bacillus Velez provided by the present invention exhibits a strong inhibitory effect on the growth of pathogenic bacteria hyphae. It has a significant antagonistic effect on a variety of pathogens and a strong blocking effect on the infection cycle of pathogens. Based on the above characteristics, the Bacillus Velez provided by the present invention can be developed into a biocontrol agent and a biological pesticide, and can be applied to agricultural production and pesticide research. Therefore, the present invention provides a good strain resource for the future development of microbial agents for the biological prevention and treatment of edible fungi diseases.

Description

Technical Field

[0001] The invention relates to the technical field of pathogenic bacteria disease prevention and control, and in particular to a Velez subtilis sp., a bacterial agent and application thereof in pathogenic bacteria disease prevention and control. Background Art

[0002] Bacillus Velez is isolated from a wide range of sources, and its metabolites contain a variety of active substances that antagonize plant pathogens. Therefore, in terms of biological control of plant diseases, Bacillus Velez has shown its unique value. Many studies have shown that Bacillus Velez has antagonism against fungal pathogens and promotes plant growth, and is a biocontrol bacterium with development potential. Bacillus Velez has been isolated and confirmed to have antibacterial activity in a variety of environments, but most are still in the laboratory stage and have not yet been applied to fungal disease control on a large scale and commercially. Therefore, the biocontrol effects of these Bacillus Velez in practical applications need further verification.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The invention aims to provide a Velez bacillus to prevent and treat various pathogenic bacteria and diseases, thereby providing more biocontrol bacteria options for commercial applications.

[0005] The present invention is achieved in that:

[0006] In a first aspect of the present invention, a Bacillus velezensis is provided, and Bacillus velezensis is deposited in the China Center for Type Culture Collection, and the deposit address is: Wuhan University, Wuhan, China. The deposit date is October 31, 2022, and the deposit number is CCTCC NO: M20221701. The name of the deposited culture is Bacillus velezensis SAAS-D44 (Bacillus velezensis SAAS-D44). The identification result is survival.

[0007] Bacillus Velez is isolated from Morchella tissue. The inventors have found through confrontation experiments that the isolated Bacillus Velez has obvious antagonistic effects on a variety of pathogens, has strong inhibitory effects on the mycelial growth of a variety of pathogens, and has a strong blocking effect on the infection cycle of pathogens. Based on the above characteristics, the Bacillus Velez provided by the present invention can be developed into biocontrol agents and biological pesticides, which can be applied to agricultural production and pesticide research.

[0008] The 16S rDNA sequence of Bacillus velezensis was compared with the sequence in the GenBank database, and the results showed that the Bacillus velezensis of the present invention was on the same branch as Bacillus velezensis, and the similarity of its 16S rDNA sequence with Bacillus velezensis was 99%. Combining the colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, it was identified as Bacillus velezensis.

[0009] The colonies of Bacillus Velez are oval, white, with complete edges and surface conditions: dull, smooth, opaque, dry and raised. The inhibition rate of the endophytic bacteria of Morchella edodes against pathogens can reach 72%.

[0010] The second aspect of the present invention provides a bacterial agent, which includes the above-mentioned Bacillus Velez. The active ingredient in the bacterial agent is Bacillus Velez. The bacterial agent can be prepared to prevent and control the harm of pathogens to plants, edible fungi and medicinal fungi.

[0011] The bacterial agent includes but is not limited to the following forms: liquid preparation, solid bacterial agent, paste bacterial agent, suspension, emulsion, spray or semi-solid bacterial agent, such as liquid bacterial agent or sterilized bacterial agent.

[0012] The third aspect of the present invention provides a seed soaking agent or root soaking agent, which comprises the above-mentioned Bacillus Velezii.

[0013] The prepared seed soaking agent can be used for preventing and controlling pathogens of plant seeds, thereby reducing or preventing the harm of pathogens to plant seeds.

[0014] The above-mentioned soaking agent is used for soaking the seeds of crops. When used, the seeds are soaked in the solution of the soaking agent. After being treated with the soaking agent, the disease resistance of the crop seeds is significantly improved. The crops are, for example, selected from cash crops and food crops, and the cash crops are selected from at least one of the following cash crops: Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passifloraceae, Bromeliaceae, Araliaceae and Cactaceae; the food crops are selected from Gramineae crops; Gramineae crops are corn, wheat, rice, sorghum or barley.

[0015] The dosage form of the above-mentioned seed soaking agent includes but is not limited to liquid preparations, solid bacterial preparations, paste bacterial preparations, suspensions, emulsions, sprays or semi-solid bacterial preparations.

[0016] In addition, the seed soaking agent can be added with preservatives, antioxidants, color enhancers and other additives as needed. The above-mentioned seed soaking agent can also be added with other antibiotic agents in combination to improve the antibiotic effect and weaken or reduce the number and activity of pathogenic organisms.

[0017] The seed soaking agent is the fermentation liquid of the above-mentioned Bacillus Velez subtilis or the freeze-dried powder of Bacillus Velez subtilis. The above-mentioned fermentation liquid is the fermentation liquid cultured on the bacterial culture medium of Bacillus Velez subtilis.

[0018] The product is prepared as a root soaking agent, which can be used for preventing and controlling pathogens in the roots of plants and reducing the yellowing and withering of plant leaves caused by rot in the roots of plants.

[0019] In other embodiments, the root soaking agent may also be composed of a variety of bacterial agents to improve the prevention and control effect of pathogens and the growth promotion effect.

[0020] The fourth aspect of the present invention provides a use of Bacillus Velez in preventing and controlling plant pathogenic fungi diseases, edible fungi pathogenic fungi diseases or medicinal fungi pathogenic fungi diseases.

[0021] Pathogenic fungi diseases refer to: during the entire cultivation process of edible fungi, at various stages of their growth and development, as well as at various links of processing and storage, due to unsuitable environmental conditions or infection by other harmful microorganisms, the growth and development of mycelium or fruiting bodies are hindered, and a series of abnormal changes occur in physiology, organization, and morphology, such as slow growth and development, deformity, withering, and death, which reduces yield and quality, and may even lead to failure of the entire production.

[0022] The Bacillus Velezii isolated and identified by the present invention has a good prevention and control effect on edible fungus pathogenic bacteria diseases.

[0023] In a preferred embodiment of the present invention, the plant pathogenic bacteria diseases are selected from plant bacterial diseases, plant fungal diseases or plant soil-borne diseases.

[0024] In a preferred embodiment of the present invention, the above-mentioned edible fungi pathogenic fungi diseases or medicinal fungi pathogenic fungi diseases include edible fungi pathogenic fungi diseases or medicinal fungi pathogenic fungi diseases caused by at least one of Mucor hiemalis, Lecanicillium aphanocladii, Fusarium, Fusarium fujikuroi, Clonostachys rosea, Scytalidium lignicola, Cladobotryum semicirculare, Comatrichapulchell (C.Bab) Rost sp., Trichoderma pleuroticola, Penicillium cyclopium and Disporotrichum dimorphosporum.

[0025] In a preferred embodiment of the present invention, the above-mentioned edible fungus pathogenic fungus diseases or medicinal fungus pathogenic fungus diseases are edible fungus verrucosporium disease, edible fungus spider web disease, edible fungus oil scar disease, white rot, edible fungus nematode disease or edible fungus virus disease.

[0026] In a preferred embodiment of the present invention, the edible fungi are morels, shiitake mushrooms, flower face mushrooms, mushrooms, boletus, straw mushrooms, mushrooms, black fungus, white fungus, hericium, bamboo fungus, matsutake, trichoderma, red mushroom, ganoderma, cordyceps, truffles, white mushrooms or boletus.

[0027] The fifth aspect of the present invention also provides a method for preparing a biocontrol agent, which comprises the following steps:

[0028] The above-mentioned Bacillus Velez subtilis is fermented to obtain a fermentation liquid, or the fermentation liquid is sterilized to obtain a sterilized liquid.

[0029] The sixth aspect of the present invention also provides a use of Bacillus Velez in the preparation of biological pesticides or seed soaking agents for preventing and controlling plant pathogenic bacteria diseases, edible fungi pathogenic bacteria diseases or medicinal fungi pathogenic bacteria diseases.

[0030] The present invention has the following beneficial effects:

[0031] The Bacillus Velez provided by the present invention exhibits a strong inhibitory effect on the growth of pathogenic mycelium. It has a significant antagonistic effect on a variety of pathogens and a strong blocking effect on the infection cycle of pathogens. Based on the above characteristics, the Bacillus Velez provided by the present invention can be developed into a biocontrol agent and a biological pesticide, and can be applied to agricultural production and pesticide research. Therefore, the present invention provides a good strain resource for the future development of microbial agents for biological control of edible fungi diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 This is a diagram showing the antagonistic effect of the antagonistic dominant strain numbered D44 on five pathogens;

[0034] Figure 2 The results of inhibition of the growth of Morchella pathogen L.aphanocladii by endophytic bacteria A9 and D44 of Morchella;

[0035] Figure 3This is the electrophoresis result after amplifying the 16srDNA fragment of endophytic bacteria;

[0036] Figure 4 This is the electrophoresis result after amplification of the ITS region of endophytic bacteria;

[0037] Figure 5 This is the result of evolutionary tree analysis;

[0038] Figure 6 This is the result of the biocontrol effect of the endophytic bacteria D44 of Morchella on the pathogenic Morchella L.aphanocladii in a greenhouse experiment;

[0039] Figure 7 This is a graph showing the test results of the relevant antioxidant enzyme activity index data of the greenhouse experiment on Morchella oleifera by the dominant antagonistic bacteria D44 and Lecanicillium aphanocladii G1. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0041] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0042] The formula of NB medium: beef extract 5g / L, peptone 10g / L, agar 18g / L, sodium chloride (NaCl) 5g / L, add sterile water to the final volume.

[0043] The formula of PDA medium: potato 200g / L, glucose 20g / L, agar 18g / L, add sterile water to the final volume.

[0044] Example 1

[0045] This example is to separate and purify the endophytic bacteria of Morchella.

[0046] Rinse the fresh morels with sterile water for about 5 minutes. Use filter paper to absorb the surface water, wash twice with sterile water, soak in 75% alcohol for 3 minutes, wash three times with sterile water, soak in 3% sodium hypochlorite for 3 minutes, and finally wash four times with sterile water. Apply the last washing solution on NB and PDA culture medium as a control.

[0047] The grinding method was used to separate the endophytes. The sterilized Morchella epidermis was peeled off with a sterile scalpel, and the internal tissue was transferred to a mortar. Then, an appropriate amount of sterilized sand and sterile water were added for grinding. After a period of time, the supernatant was applied to NB and PDA culture medium plates, and cultured in a constant temperature box at 37°C and 28°C, with three replicates.

[0048] During the culture process, observations were made day by day, and the grown bacteria and fungi were placed in the corresponding fresh solid medium for purification. The purified endophytic bacteria, endophytic actinomycetes and endophytic fungi of Morchella were transferred to NB slant, Gao's No. 1 slant and PDA slant, numbered and stored in a 4℃ refrigerator for future use.

[0049] After the above separation and purification, 26 endophytic bacteria and 2 endophytic fungi isolated from Morchella tissue were used as test strains (i.e., primary screening strains).

[0050] Example 2

[0051] This example screens endophytes that are antagonistic to Morchella pathogenic fungi.

[0052] The method of confrontation culture between the test strain and the pathogenic bacteria is adopted to screen the dominant antagonistic strains with stronger antibacterial effect, so as to obtain the endophytic strains with the strongest antibacterial activity and broad spectrum.

[0053] The test pathogens used in this experiment were five pathogens isolated from diseased strains of Morchella, namely: Mucor hiemalis, Lecanicillium aphanocladii, Fusarium graminearum, Fusarium fujikuroi, and Clonostachysrosea, which were preserved in the Microbiology Laboratory of Shanghai Academy of Agricultural Sciences.

[0054] 1. Activation of strains

[0055] The 28 endophytic bacteria and 5 pathogenic bacteria isolated from Morchella in Example 1 were activated on NB and PDA plate culture media, wherein bacteria were activated on NB plate culture media, and fungi were activated on PDA plate culture media.

[0056] 2. Antagonism Experiment

[0057] The plate confrontation method was used to determine the inhibitory effect of endophytes of Morchella against pathogens such as Lecanicillium aphanocladii (YG-1), Fusarium graminearum (YG-2), Fusarium fujikuroi (YG-4), Clonostachys rosea (YG-5) and Mucor hiemalis (YG-6), and endophytes with antibacterial activity were screened out.

[0058] 2.1 Initial screening of antagonistic bacteria

[0059] The endophytes to be tested were reactivated on the NB plate, and a single colony was picked and inoculated into the NB medium. The culture was carried out at 28°C for 24 hours to obtain the activated endophytes. The pathogenic Morchella was inoculated on the PDA plate for activation. After culturing at 25°C for 7 days, a 5mm mycelium block was taken and inoculated into the center of a new PDA medium plate. After culturing for 48 hours, 6 strains of endophytes were symmetrically inoculated on the PDA plate inoculated with pathogens using an inoculation loop. Each endophyte was inoculated with one loop of strains, and each strain was 30mm away from the pathogens and cultured at a constant temperature of 28°C. The pathogen plate was used as a control and repeated 3 times. The strains with antagonistic effects were obtained by initial screening for use.

[0060] 2.2 Rescreening of antagonistic bacteria

[0061] Antagonistic bacteria were rescreened using the plate confrontation test. The strains with antagonistic effects obtained in the initial screening were reactivated on the NB plate, and single colonies were picked and inoculated into the NB medium and cultured at 28°C for 24 hours. The pathogen mycelium blocks and the antagonistic bacteria were inoculated symmetrically at equal distances. The pathogen was inoculated in the center of the PDA solid medium, and the antagonistic bacteria were inoculated at equal distances at the four corners of the center of the plate, and cultured at 28°C for 7-10 days. The pathogen plate was used as a control, and the test was repeated 3 times. The results were observed when the colonies filled the plate.

[0062] 2.3 Antagonistic effects between Morchella pathogenic fungi and endophytes

[0063] The preliminary screening results of 28 endophytes isolated from Morchella to inhibit pathogens showed that two endophytes of Morchella had strong antibacterial activity and broad-spectrum antibacterial activity, and the antagonistic dominant strains were numbered as A9 and D44. The pathogen control group and the confrontation culture experimental group were tested at the same time. After 7 days of culture, it was found that the growth of pathogenic fungi in the control group was normal, while A9 and D44 in the experimental group showed obvious inhibition of the growth of 5 pathogens, and obvious inhibition zones appeared.

[0064] The antagonistic effect of the antagonistic dominant strain numbered D44 on 5 pathogens is shown in the figure below. Figure 1 shown.

[0065] Figure 1 a1 and a2 are the front and back growth conditions of the pathogen in the control group YG-1, b1 and b2 are the front and back growth conditions of the pathogen in the control group YG-3, c1 and c2 are the front and back growth conditions of the pathogen in the control group YG-4, d1 and d2 are the front and back growth conditions of the pathogen in the control group YG-5, and e1 and e2 are the front and back growth conditions of the pathogen in the control group YG-6.

[0066] a5 a6 are the front and back confrontations between the dominant antagonistic endophyte of D44 strain and the YG-1 pathogen, b5 b6 are the front and back confrontations between the dominant antagonistic endophyte of D44 strain and the YG-3 pathogen, c5 c6 are the front and back confrontations between the dominant antagonistic endophyte of D44 strain and the YG-4 pathogen, d5 d6 are the front and back confrontations between the dominant antagonistic endophyte of D44 strain and the YG-5 pathogen, and e5 e6 are the front and back confrontations between the dominant antagonistic endophyte of D44 strain and the YG-6 pathogen.

[0067] Depend on Figure 1 It can be seen that the D44 strain of dominant antagonistic endophyte screened by the present invention has obvious antagonistic effects on the five pathogens.

[0068] Table 1 below shows the statistical results of the antagonistic effects of the 28 endophytes isolated and purified against the five pathogens. Table 1 shows that the dominant antagonistic endophyte D44 screened out by the present invention has obvious antagonistic effects against the five pathogens. √ means there is an antagonistic effect, and × means there is no antagonistic effect.

[0069] Table 1 Statistical results of antagonistic experiments of 28 different endophytic strains against 5 pathogens.

[0070]

[0071] Example 3

[0072] In this example, the D44 dominant antagonistic bacteria selected above were used to study the inhibitory effect on the mycelium growth of Lecanicilliumaphanocladii.

[0073] 1. Pretreatment of PDA solid culture dishes

[0074] Draw a cross on the reverse side of the PDA solid culture dish, draw marks on four lines 2.5 cm from the center point, and draw marks on four lines 0.5 cm from the center point.

[0075] 2. Inoculation of endophytic bacteria of Morchella and pathogenic bacteria L. aphanocladii.

[0076] At four marks 2.5 cm from the center of the PDA solid culture dish, 27 strains of Morchella endophytic bacteria of different species were inoculated, and 3 replicates were performed for each Morchella endophytic bacteria. At four marks 0.5 cm from the center of each PDA solid culture dish inoculated with Morchella endophytic bacteria, a 5mm puncher was used to take Morchella pathogenic bacteria L.aphanocladii bacterial cakes as the experimental group, which were placed on the front of a 28°C constant temperature incubator for culture. A 5mm puncher was used to take Morchella pathogenic bacteria L.aphanocladii bacterial cakes as the control group, which were placed on the front of a 28°C constant temperature incubator for culture.

[0077] When the hyphae of the Morchella pathogen L. aphanocladii in the control group covered the surface of the PDA culture dish, the growth diameters of the hyphae of the pathogen in the experimental group and the control group were measured.

[0078] Inhibition rate formula = (mycelium growth diameter of the control group - mycelium growth diameter of the experimental group) / mycelium growth diameter of the control group × 100%.

[0079] 3. Inhibitory effect of endophytic bacteria of Morchella on the mycelial growth of L. aphanocladii

[0080] The plate confrontation method was used to screen the endophytic bacteria of Morchella that had an inhibitory effect on the growth of its pathogens. Some of the results are shown in Figure 1 The inhibitory effects of different endophytic bacteria on the mycelial growth of the pathogen L. aphanocladi were different. The strain D44, Bacillus velezensis, had the highest average inhibition rate of 68.9±2.34i on the mycelial growth of the pathogen L. aphanocladi.

[0081] The results of inhibition of the growth of Morchella endophytic bacteria D44 on the Morchella pathogen L.aphanocladii were referenced Figure 2 A: Growth of the pathogen of Morchella fusca L.aphanocladii in the control group. G: Growth of the pathogen of Morchella fusca L.aphanocladii after inoculation with the endophytic bacteria D44.

[0082] Depend on Figure 2 It can be seen that the endophytic bacteria D44 of Morchella has a strong antagonistic activity against the pathogen of Morchella white rot L. aphanocladii, has a strong inhibitory effect on the hyphae growth of the pathogenic fungus, and has a strong blocking effect on the infection cycle of the pathogen.

[0083] Example 4

[0084] The morphological characteristics and molecular biological identification of the endophytic bacteria of Morchella oleracea are as follows.

[0085] The colonies of D44 are oval, white, with intact edges and a surface condition of: dull, smooth, opaque, dry and raised.

[0086] The 16srDNA fragment of endophytic bacteria was amplified to obtain a band of about 1500 bp in length ( Figure 3 ); from top to bottom: 15000, 8000, 5000, 3000, 2000, 1500, 1000 bp; a: 1-10 samples, endophytic bacteria YD01-YD03, A9, YD04-YD10; b: 11-26 samples, endophytic bacteria YD11-YD13, D44, YD15-YD23, YD25, YD26, YD28. The ITS region of endophytic fungi was amplified to obtain a band of about 500 bp in length ( Figure 4 ).

[0087] The 16srDNA and ITS sequences of Morchella endophytes were compared by BLAST in NCBI. The 28 endophytes belonged to 10 genera. Among the 26 Morchella endophytes, YD01-YD03, A9, YD06-YD12, YD14-YD16 belonged to Bacillus, YD05 belonged to Lysinibacillus, YD17 belonged to Paenibacillus, YD19, YD22, YD25, YD26 belonged to Pseudomonas, YD18 belonged to Curtobacterium, YD20 belonged to Rhizobium, YD21 belonged to Phyllobacterium, and YD28 belonged to Enterobacter. Among the 2 endophytic fungi, YD24 belonged to Chaetomium and YD27 belonged to Clonostachys (Table 3). Strain number YD14 is strain D44.

[0088] Phylogenetic tree analysis results refer to Figure 5 As shown, strain D44 ( Figure 5 D44-3) is of the genus Bacillus velezensis.

[0089] Table 5 BLAST alignment of endophytes of Morchella

[0090]

[0091]

[0092] Table 5

[0093]

[0094]

[0095] Example 5

[0096] In this embodiment, physiological and biochemical indicators of the screened Bacillus Velezii were detected.

[0097] The test items include: starch hydrolysis, VP experiment, gelatin liquefaction, citrate, propionate, D-xylose, L-arabinose, D-mannitol, pH5.7 growth and nitrate reduction.

[0098] The results are as follows:

[0099] Biochemical reactions VP Experiment Citrate Propionate D-Xylose L-arabinose result － + － + + Biochemical reactions D-Mannitol Gelatin liquefaction Starch hydrolysis pH5.7 Nitrate reduction result + + + － +

[0100] - indicates negative, + indicates positive.

[0101] Example 6

[0102] In this example, the D44 dominant antagonistic bacteria selected above were subjected to an inhibitory effect study experiment on Lecanicilliumaphanocladii in a greenhouse experiment.

[0103] Example 3 The strain Bacillus velezii, numbered D44, whose average inhibition rate on the mycelial growth of the pathogen L.aphanocladii reached 68.9±2.34i was screened out by the plate confrontation method, and the biocontrol effect of the strain was further verified by greenhouse experiments. The experiment adopted a single-factor variable design, and set up four groups: (1) healthy Morchella inoculated with pathogen L.aphanocladii G1; (2) healthy Morchella inoculated with pathogen L.aphanocladii G1 and then applied with Morchella endophytic bacteria D44; (3) healthy Morchella only applied with Morchella endophytic bacteria D44; (4) healthy Morchella as the control group.

[0104] The results of the biocontrol effect of endophytic bacteria D44 on the pathogenic fungus L. aphanocladi in greenhouse experiments are referenced Figure 6 As shown. Label G1: The growth of healthy Morchella on the 7th day after inoculation with pathogenic bacteria L.aphanocladii G1. Label D44+G1: The growth of healthy Morchella on the 7th day after inoculation with pathogenic bacteria L.aphanocladii G1 and application of Morchella endophytic bacteria D44. Label D44: The growth of healthy Morchella on the 7th day after application of Morchella endophytic bacteria D44 only. Label CK: The growth of healthy Morchella as the control group on the 7th day.

[0105] Depend on Figure 6It can be seen that the endophytic bacteria D44 of Morchella had a significant biocontrol effect on the pathogen of Morchella white rot L.aphanocladii in the greenhouse experiment. After Morchella was infected with the white rot pathogen L.aphanocladii, the disease degree of the experimental group that applied the endophytic bacteria D44 of Morchella was significantly lower than that of the experimental group that was only infected with the white rot pathogen L.aphanocladii. Compared with the control group of healthy Morchella, there was no significant difference in the group that applied the endophytic bacteria D44 of Morchella alone, indicating that the endophytic bacteria D44 of Morchella had no obvious harm to Morchella. In summary, the greenhouse experiment once again verified that the endophytic bacteria D44 of Morchella had a good biocontrol effect on the pathogen of Morchella white rot L.aphanocladii.

[0106] Example 7

[0107] In this embodiment, the inhibitory effect of the D44 dominant antagonistic bacteria selected above on the Lecanicilliumaphanocladii greenhouse experiment was studied, and then the relevant antioxidant enzyme activity index data were tested. Four defense enzymes, superoxide dismutase (SOD), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and catalase (CAT) were used as plant disease resistance reaction indicators. Because SOD is a very important free radical scavenging enzyme, it can dismutate O2- into H2O2 and O2, reducing the toxic effect of O2- on plants; CAT is mainly responsible for removing peroxides such as H2O2; PPO can oxidize phenols and polyphenols to form quinones through molecular oxygen to protect the integrity of plant cells; phenylalanine ammonia-lyase (PAL) is a key enzyme in the phenol synthesis pathway and is the starting enzyme for the synthesis of many important secondary metabolites.

[0108] The experiment adopted a single-factor variable design and set up three groups: (1) healthy Morchella inoculated with the pathogen L. aphanocladi G1; (2) healthy Morchella treated with the endogenous bacteria D44; and (3) healthy Morchella as the control group.

[0109] GraphPad Prism 8 software was used to perform one-way ANOVA on the antioxidant enzyme activity index data of different groups of Morchella, and all data were tested at a significant level of p < 0.05 and GraphPad Prism 8 software was used to organize and draw the graph.

[0110] In this example, the results of the test of the relevant antioxidant enzyme activity index data of the greenhouse experiment of Morchella oleracea with the D44 dominant antagonistic bacteria and Lecanicilliumaphanocladii G1 were respectively used as reference. Figure 7 As shown. The results showed that the activities of defense enzymes in the two treatment groups were higher than those in the control group, indicating that whether inoculated with endophytic bacteria D44 or Lecanicillium aphanocladii G1, it can stimulate the increase of defense enzyme activities in Morchella, thereby achieving the effect of resisting disease infection. In terms of the increase in enzyme activity, the activities of the three defense enzymes after spraying with endophytic bacteria A9 in Morchella were higher than those inoculated with Lecanicillium aphanocladii G1, indicating that the induced resistance of endophytic bacteria D44 in Morchella is more significant than that of pathogens. The biocontrol effect of endophytic bacteria D44 in Morchella was further tested, and it has a good application prospect as a biocontrol pesticide.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus Velez, characterized in that The Bacillus Velez Bacillus velezensis ) is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20221701.

2. A bacterial agent, characterized in that It comprises the Bacillus Velezii described in claim 1.

3. A seed soaking agent or root soaking agent, characterized in that: It comprises the Bacillus Velezii described in claim 1.

4. A use of the Bacillus Velezii as claimed in claim 1 in preventing and controlling diseases, characterized in that: The disease is selected from at least one of the following: Lecanicillium aphanocladii ) caused by Morchella white rot disease, frozen soil Mucor ( Mucor hiemalis ) caused by Morchella diseases, Fusarium graminearum ( Fusarium graminearum ) caused by Morchella diseases, Fujikuro Fusarium ( Fusarium fujikuroi ) and Gliocladium roseum ( Clonostachys rosea ) caused by morel diseases.

5. A method for preparing a biocontrol agent, characterized in that: It includes the following steps: The Bacillus Velez subtilis described in claim 1 is fermented to obtain a fermentation liquid, or the fermentation liquid is sterilized to obtain a sterilized liquid.

6. A use of the Bacillus Velezii as claimed in claim 1 in the preparation of a biological pesticide or seed soaking agent for preventing and controlling diseases, characterized in that: The disease is selected from at least one of the following: Lecanicillium aphanocladii ) caused by Morchella white rot disease, frozen soil Mucor ( Mucor hiemalis ) caused by Morchella diseases, Fusarium graminearum ( Fusarium graminearum ) caused by Morchella diseases, Fujikuro Fusarium ( Fusarium fujikuroi ) and Gliocladium roseum ( Clonostachys rosea ) caused by morel diseases.

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