Morchella growth-promoting strain bacillus MSB09

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a morchella growth-promoting strain bacillus MSB09, which is classified and named as bacillus sp. And has a preservation number of GDMCC No: 65938. Morchella is used as a precious edible and medicinal fungus, the planting scale is continuously enlarged, but the soil continuous cropping problem has become a main obstacle for soil-covered cultivation and fruiting of the morchella, the soil microorganism structure of the morchella is studied, and the morchella can be used for promoting growth of the morchella. And beneficial microorganism bacillus (Bacillus sp.) MSB09 is screened out, so that technical support is provided for reconstruction of soil micro-ecology, alleviation of continuous cropping obstacles of morchella esculenta and biological prevention and control.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Morchella growth-promoting strain Bacillus MSB09. Background Art

[0002] Continuous cropping obstacle, also known as crop replant obstacle or replant problem, refers to the problems such as difficult emergence, poor seedling growth, increased pests and diseases, reduced yield, and deteriorated variety when the same or closely related crops are continuously planted on the same piece of land under conventional cultivation management. This phenomenon widely affects the cultivation of various plants such as food crops, cash crops, vegetables, forest trees, and traditional Chinese medicinal materials, and has become a major obstacle to the development of the planting industry in China. Its causes include soil microorganisms, soil enzymes, soil-borne diseases, allelopathic autotoxicity, etc. One important reason is the change in the soil microbial structure.

[0003] The genus Morchella Morchella spp. belongs to the large class of fungi Ascomycota, and specifically belongs to the class Pezizomycetes, the order Pezizales, and the family Morchellaceae. This rare edible and medicinal fungus is named "Morchella" because its head has a unique uneven surface, similar to the belly of a sheep. Since the successful commercial cultivation of Morchella, the soil ecological problems caused by soil and continuous cropping have become an obstacle to the sustainable development of Morchella. Various enzymes in the soil play a key role in the utilization of nutrients such as C, N, and P, and participate in the cycling of these elements in the soil. Soil enzyme activity is closely related to the abundance and diversity of microorganisms in the habitat and is a key factor in the function of the soil ecosystem.

[0004] Based on this, the present invention analyzes the evolution of culturable microbial groups in the soil of continuous cropping of Morchella, and isolates the dominant microorganisms in the continuous cropping soil of Morchella. Further screen Morchella growth-promoting strains, and classify the species of each strain by combining micro-macro characteristics such as morphology and physiology with molecular biology techniques, so as to provide technical support for the sustainable and stable development of the Morchella planting industry. Summary of the Invention

[0005] The present invention aims to provide a Morchella growth-promoting strain to solve the problems existing in the growth of Morchella described in the background art.

[0006] The system of the present invention systematically isolates and identifies beneficial microorganisms in the cultivation soil of Morchella, providing practical experience for the continuous cultivation and stable development of Morchella. By using the plate confrontation test combined with molecular biology techniques, strains with promoting effects can be quickly screened out, and the types of strains can be accurately determined through molecular-level identification, providing reliable technical support for the continuous high-yield cultivation of Morchella.

[0007] Specifically, the present invention isolates and identifies a strain of Bacillus ( Bacillus sp.) MSB09 that promotes the growth of Morchella. The taxonomic name is: Bacillus sp., and the preservation number is GDMCC No: 65938.

[0008] In one embodiment, the present invention provides a bacterial agent containing the above-mentioned Bacillus ( Bacillus sp.) MSB09. In some specific embodiments, the active ingredient of the bacterial agent can be Bacillus MSB09 or / and the metabolites of Bacillus MSB09. Optionally, surfactants (such as Tween 20, Tween 80, etc.), protectants (such as glycerol, sodium alginate, etc.), stabilizers (such as agar, gelatin, etc.), buffers (such as phosphate buffer) can be added to the bacterial agent.

[0009] In another embodiment, the present invention provides a compound microbial preparation containing the above-mentioned Bacillus ( Bacillus sp.) MSB09. The active ingredient of the compound microbial preparation can also contain other biological components or non-biological components. In one embodiment, the compound microbial preparation is made from two or more non-antagonistic microbial strains. Optionally, such as rhizobia, nitrogen-fixing bacteria, photosynthetic bacteria or other growth-promoting strains.

[0010] In some embodiments, in the bacterial agent or compound microbial preparation provided by the present invention, Bacillus MSB09 and / or the metabolites of Bacillus MSB09 can exist in the form of cultured live microorganisms, the fermentation broth of live microorganisms, the filtrate of live microorganism cultures or a mixture of microorganisms and filtrates.

[0011] In other embodiments, the bacterial agent or compound microbial preparation provided by the present invention can be in various dosage forms, such as liquid agents, emulsions, suspensions, powders, granules, wettable powders or water-dispersible granules, etc.

[0012] In one embodiment, the present invention provides the application of the above-mentioned Bacillus ( Bacillus sp.) MSB09, or the above-mentioned bacterial agent, or the above-mentioned compound microbial preparation in promoting the growth of Morchella or preparing a growth promoter for Morchella.

[0013] In one embodiment, the present invention provides the application of the Bacillus ( Bacillus sp.) MSB09, or the bacterial agent, or the composite microbial agent in repairing the soil with continuous cropping obstacle of Morchella or in preparing a soil repair agent for continuous cropping obstacle of Morchella.

[0014] In a more specific embodiment, the present invention provides a growth promoter for Morchella, and the promoter contains the fermentation culture solution of the Bacillus ( Bacillus sp.) MSB09.

[0015] Furthermore, in the promoter, the culture medium for fermentation culture contains 10 g / L of glucose, 2 g / L of yeast, 8 g / L of peptone, 0.1 g / L of sodium chloride, 0.4 g / L of potassium dihydrogen phosphate, 0.2 g / L of magnesium sulfate, and 0.24 g / L of sodium hydroxide.

[0016] In one embodiment, the present invention provides a method for culturing Morchella strains. During the process of culturing strains, the above-mentioned growth promoter for Morchella is added. Those skilled in the art can understand that as an edible and medicinal fungus, the process of Morchella strain production includes multiple strain culturing processes such as mother strain propagation culture (primary strain), original strain propagation culture (secondary strain), and cultivated strain propagation culture (tertiary strain). By adding a growth promoter during the propagation culture process, the culture process can be shortened, the production time cost can be reduced, and the industrial production efficiency can be improved. In some specific embodiments, such as the growth promoter culture with the improved YPD medium provided by the present invention and the growth promoter culture with the Morchella wheat grain medium, the production rate of Morchella has been improved.

[0017] In one embodiment, the present invention provides a cultivation method for Morchella. During the process of covering the soil with Morchella, the above-mentioned growth promoter for Morchella is applied. Those skilled in the art know that during the cultivation process of Morchella, it is necessary to cover the strains with soil to complete the processes of mycelial growth, primordium differentiation, and fruiting body formation. In the specific implementation process, adding the growth promoter for Morchella provided by the present invention can improve the production efficiency of Morchella.

[0018] A method for improving the adaptability of continuous cropping cultivation of Morchella, applying the Bacillus ( Bacillus sp.) MSB09, or the bacterial agent, or the composite microbial agent to the soil with continuous cropping obstacle. It should be understood that the strain screened by the present invention is a growth-promoting strain obtained based on the differential strain analysis of continuous cropping soil and healthy soil. Based on this, adding the growth-promoting strain lacking in the continuous cropping obstacle soil to repair the continuous cropping obstacle soil can improve the adaptability of continuous cropping cultivation of Morchella.

[0019] The technical effects achieved by the present invention: The present invention uses high-throughput sequencing to analyze the soil samples of Morchella sextelata YMe151 cultivation, and finds that the microbial diversity of non-continuous cropping healthy soil is significantly higher than that of continuous cropping soil, and there are obvious differences in the community structure; based on the idea of reconstructing the soil flora, culturable microorganisms are isolated from the soil where Morchella grows well and there are no continuous cropping diseases; through the plate confrontation experiment with Morchella, multiple target strains that promote the mycelial growth of Morchella are obtained; its bacterial liquid is added to the plate medium and then Morchella is inoculated, and the growth-promoting strain MSB09 with a mycelial growth rate increase of 45% is screened; further, its bacterial liquid is added to plate culture and test tube culture to verify its effect on the growth of Morchella. When the concentration of MSB09 bacterial liquid is 50%, the mycelial growth rate of Morchella in plate culture is as high as 35.83 mm / d, and the biomass at 50% concentration is 1.911 g, determining the strong growth-promoting effect of strain MSB09. Morphological observation, physiological and biochemical determination and molecular identification of this growth-promoting bacterium show that it is Bacillus MSB09 ( Bacillus sp.). This strain can be applied at all stages of the industrial production process of Morchella, shortening the mycelial culture time of Morchella and reducing production costs.

[0020] As a precious edible and medicinal mushroom, the planting scale of Morchella is constantly expanding, but the problem of soil continuous cropping has become the main obstacle to fruiting in its covered soil cultivation. The present invention explores the soil microbial structure of Morchella and screens out the beneficial microorganism Bacillus MSB09 ( Bacillus sp.), providing technical support for reconstructing the soil microecology, alleviating the continuous cropping obstacle of Morchella and biological control. Description of the Drawings

[0021] Figure 1 Bacterial community structure diagram of non-continuous cropping soil and continuous cropping soil of Morchella at the phylum level; Figure 2 Fungal community structure diagram of non-continuous cropping soil and continuous cropping soil of Morchella at the phylum level; Figure 3 Co-culture diagram of MSB09 and Morchella; on the left side of the plate is the target strain MSB09, and on the right side is Morchella. There is no obvious gap between the mycelia of Morchella and the colonies of the target strain, which is a typical growth-promoting effect; Figure 4 Growth-promoting effect diagram of adding MSB09 bacterial liquid on the mycelia of test tube species of Morchella; Figure 5 Analysis diagram of the growth-promoting effect of plate culture and test tube culture with added MSB09 bacterial liquid on the mycelia of Morchella; Figure 6 Colony diagram of growth-promoting strain MSB09 on the plate; Figure 7 100-fold microscopic diagram of Gram staining of growth-promoting strain MSB09; Figure 8100x micrograph of spore staining of the growth-promoting strain MSB09. DETAILED DESCRIPTION

[0022] 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 is clearly and completely described below in combination with the embodiments of the present invention. The purchased commodities in the test method, if the specific conditions are not indicated, are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer can all be conventional products purchased from the market.

[0023] The Bacillus MSB09 provided by the present invention is classified and named as follows: Bacillus sp., the deposit number is GDMCC No: 65938, the deposit unit is Guangdong Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Institute of Microbiology, Guangdong Academy of Sciences, the deposit date is February 25, 2025.

[0024] Example 1 1. Test materials: Morchella lilii YMe151, culture medium as shown in Table 1.

[0025] Table 1 Experimental culture medium Test medium Composition Solid modified YPD medium Glucose 10 g / L, yeast 2 g / L, peptone 8 g / L, sodium chloride 0.1 g / L, potassium dihydrogen phosphate 0.4 g / L, magnesium sulfate 0.2 g / L, sodium hydroxide 0.24 g / L, agar 16 g / L Liquid modified YPD medium Glucose 10 g / L, yeast 2 g / L, peptone 8 g / L, sodium chloride 0.1 g / L, potassium dihydrogen phosphate 0.4 g / L, magnesium sulfate 0.2 g / L, sodium hydroxide 0.24 g / L Morel wheat grain medium Wheat grains 49.5%, buckwheat husks 49.5%, lime 1% Beef extract peptone agar medium Peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, agar 15 g / L Rose bengal medium Peptone 5 g / L, glucose 10 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, rose bengal 0.033 g / L, chloramphenicol 0.1 g / L, agar 15 g / L 2. Methods Collection of soil samples To investigate the obstacles to continuous cropping of Morchella, we selected greenhouse planting, where Morchella grew well and there was no continuous cropping disease (dNC). The five-point sampling method was used to collect the mycelial soil after the harvest of Morchella as samples. 0.5 kg was taken from each point and brought back to the laboratory in a 4°C cold chain. The five samples were mixed and divided into two equal parts for storage.

[0026] High-throughput Illumina sequencing analysis of soil microorganisms The Morchella continuous cropping soil was used as the comparison group (dCC). The dNC and dCC soil samples were sent to Shanghai Paisonno Company within 24 hours, and the DNA in the soil samples was extracted using the MP FastDNA® Spin Kit for Soil (MP Biomedical, USA). The diluted genomic DNA was used as a template and specific primers with barcodes were used for PCR amplification. The amplified products were cloned and sequenced on the Illumina platform. Species annotation and abundance analysis were performed through splicing (FLASH software), filtering (Vsearch software), and OTUs clustering to reveal the species composition of bacteria and fungi in the samples.

[0027] Isolation of culturable soil microorganisms For bacterial culture, nutrient broth peptone medium is used, while for fungal culture, Rose Bengal medium is selected. Take 1 gram of soil sample, perform gradient dilution with sterile water, and then take 0.1 ml of 10 -3 、10 -4 、10 -5 dilution solutions respectively, set three replicates, and evenly spread them on the surface of the medium. Invert the petri dishes and place them in a constant temperature incubator at 37°C for bacterial culture, while fungi are cultured at 28°C. After 24 hours of bacterial culture, observe the formation of single colonies; fungal culture takes 3 to 5 days.

[0028] Compare the control group CK with the experimental group T in the experimental design, and further pick the dominant single colonies on the gradient plate of the T sample for streak pure culture to isolate and purify the dominant bacteria.

[0029] Screening of growth-promoting strains of Morchella Using the Morchella sextelata strain YMe151 as the indicator bacterium, the growth-promoting experiments of the isolated bacterial and fungal strains were carried out by the plate confrontation culture method and co-culture to screen the potentially beneficial bacteria in the Morchella soil.

[0030] 2.4.1 Plate confrontation experiment Use a punch with a pore diameter of 6 mm to take a well-grown mycelium block of Morchella after activation and inoculate it at the 1 / 4 position of the line in the modified YPD plate. In the treatment group, at the other side of the Morchella mycelium block, that is, at the 3 / 4 position of the plate line, streak-inoculate the dominant bacteria that have been activated for 24 hours; while the control group does not inoculate the dominant bacteria. Set three replicates. Continue to culture at a constant temperature of 24°C. Starting from the next day, measure the diameter of the colony every day using the cross-streak method and continuously measure for ten days, and calculate the daily average growth rate (mm / d) of the mycelium. Calculate the growth rate to determine the effect of the target bacteria on the growth of Morchella mycelium.

[0031] Growth rate % = [(treatment colony growth rate - control colony growth rate) / control colony growth rate] × 100 2.4.2 Experiment of adding growth-promoting bacteria liquid and Morchella plate culture The strain MSB09 that promotes the growth of Morchella mycelium screened by the plate confrontation experiment was fermented and cultured using liquid nutrient broth peptone medium (the formula removes agar): temperature 37°C, rotation speed 150 r / min. After 24 hours of culture, the concentration of the bacterial liquid was measured by an ultraviolet spectrophotometer to reach 6.5×10 7CFU is sufficient; the bacterial liquid is filtered through sterile gauze and the filtrate is collected by a 0.22 μm sterile filter. The filtered bacterial liquid is added at five concentration levels: no filtrate (0%), 10%, 20%, 30%, and 50% to prepare new improved YPD medium plates. Each concentration is set with 3 replicates, and 0% is used as the control (CK). In the center of the prepared 9 cm solid medium, inoculate a Morchella hyphal block taken with a 6 mm puncher for culture. Starting from the 2nd day, measure the colony diameter by the cross-streaking method every day for 7 consecutive days to obtain the average daily growth rate (mm / d) of the hyphae. On the 9th day, weigh the hyphae to calculate the dry weight of the Morchella hyphal biomass to determine the effect of the dominant bacteria on the growth of Morchella hyphae.

[0032] Growth promotion effect of growth-promoting strains For the co-culture of the growth-promoting bacteria and Morchella, the MSB09 strain is further nutrient domesticated, that is, it is fermented and cultured in a liquid modified YPD medium at a temperature of 28 °C and a rotation speed of 150 r / min. After 24 h of culture, the bacterial liquid concentration is measured by an ultraviolet spectrophotometer to reach 6.5×10 7 CFU for standby. Use a sterilized 6 mm puncher to inoculate a Morchella hyphal block into a sterilized test tube of Morchella wheat grain medium. At the same time, inoculate the growth-promoting bacterial liquid at five concentration levels: sterile liquid (0%), 10%, 20%, 30%, and 50%, and supplement the water content. Each concentration of the growth-promoting strain is set with 3 groups of replicates, and observe the growth of Morchella hyphae in the test tube under room temperature conditions. Measure the length of the hyphal layer at 3 - 4 d and calculate the growth rate (mm / d) of Morchella hyphae.

[0033] Identification of growth-promoting strains Inoculate the screened growth-promoting bacterium MSB09 onto a conventional beef extract peptone medium plate for bacteria, and use the streak plate method to isolate single colonies. Observe the size, color, surface characteristics, edge shape, etc. of the colonies at the end of the culture to preliminarily determine the strain type; perform Gram staining and spore staining respectively, and observe the individual morphology and staining conditions of the growth-promoting strain under a 100× oil immersion microscope.

[0034] Detection of physiological and biochemical indicators: Use an HBI microbial biochemical identification strip (purchased from Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park) to perform physiological and biochemical detection on the above growth-promoting bacteria.

[0035] Molecular biological identification: The strains were purified, and DNA was extracted using a bacterial genomic DNA extraction kit (purchased from Beijing TransGen Biotech Co., Ltd.). The test strains were amplified using the bacterial universal primers 16s 27F / 1492R. The synthesis of PCR primers and the sequencing of amplification products were completed by Tsingke Biological (Kunming). The sequences were bidirectionally sequenced and spliced, compared on NCBI, and the most similar sequences were selected for tree construction. Multiple sequence alignment was performed using Clustalx, and the results were imported into MEGA 6.0 software for cluster analysis and construction of a phylogenetic tree diagram to determine their species and genera.

[0036] Universal primer pair for bacterial 16S rDNA: 27F 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R 5'-GGTTACCTTGTTACGACTT-3'.

[0037] Results and analysis Analysis of soil microbial diversity in Morchella cultivation soil and continuous cropping soil As Figure 1 , Figure 2 , taking the continuous cropping soil of Morchella as the comparison group (dCC), the composition characteristics of the microbial flora in the healthy soil of non-continuous cropping Morchella soil (dNC) were analyzed. The results showed that the microbial diversity of non-continuous cropping soil was higher than that of continuous cropping soil, and the community structure was significantly different.

[0038] For bacteria, at the phylum level, the relative abundances of Chloroflexi, Acidobacteriota, and Bacteroidota in non-continuous cropping soil were significantly higher than those in continuous cropping soil, and they were the dominant species in non-continuous cropping healthy soil. The relative abundances of Actinobacteriota and Cyanobacteria were higher in continuous cropping soil. This indicates that Chloroflexi, Acidobacteriota, and Bacteroidota may be related to the normal growth of Morchella, and it is expected to screen probiotics for Morchella in healthy non-continuous cropping soil.

[0039] For fungi, the relative abundances of Ascomycota and Mortierellomycota in continuous cropping soil were higher than those in non-continuous cropping soil, while the relative abundances of Basidiomycota and Rozellomycota were significantly lower than those in non-continuous cropping soil.

[0040] Screening of beneficial microorganisms for Morchella 3.2.1 Isolation of culturable dominant microorganisms in soil The control group dCC and the experimental group dNC in the experimental design were compared. Dominant single colonies were picked from the gradient plate of the dNC sample and streaked for pure culture. After preliminary isolation and purification, multiple dominant microorganisms in the soil of Morchella were obtained.

[0041] 3.2.2 Plate confrontation test between dominant strains and Morchella Multiple soil dominant strains and Morchella were cultured on improved YPD medium for 2 - 7 days respectively. The growth of Morchella hyphae in the control group and the treatment group was compared. The comparison results of the growth rate and growth rate of Morchella hyphae showed that different dominant strains had different effects on the growth of Morchella hyphae, with both promoting and inhibiting effects. The MSB09 strain showed a promoting effect on the growth of Morchella hyphae, and there was no obvious spacing between the Morchella hyphae and the colony of the target strain (see Figure 3 ). The promoting growth rate of the MSB09 strain on Morchella reached 45% (Note: The growth rate of the CK group was 15.50 ± 0.29 mm / d, and the growth rate of the MSB09 group was 22.50 ± 1.04 mm / d).

[0042] Strong growth promotion effect on Morchella The bacterial liquid of MSB09 was added to the test tube of Morchella wheat grain medium, and Morchella mycelial blocks were inoculated for culture. The length of the mycelial layer was measured at different culture times, and the growth rate of Morchella hyphae in the test tube (mm / d) was calculated. The results showed ( Figure 4 ) that after 4 days of culture, compared with the growth rate of Morchella hyphae in CK which was 14.31 mm / d, the screened bacterial liquid promoted the growth of Morchella. Among them, in the test tube culture experiment with the addition of the bacterial liquid of the MSB09 strain, the growth rate of Morchella hyphae increased curvilinearly with the increase of the concentration starting from 10% for the MSB09 bacterial liquid, and the growth rate reached the highest of 20.11 mm / d at a concentration of 50%. The growth promotion effect was obvious.

[0043] Figure 5 It was shown that on the plate with the addition of the filtrate of the MSB09 strain, with the increase of the addition concentration, the growth rate of Morchella hyphae accelerated, and the growth rate of Morchella reached the highest of 35.83 mm / d at a concentration of 50%; the biomass of Morchella hyphae at the 50% level of the MSB09 bacterial liquid concentration was 1.911 g, showing a promoting effect, and the biomass increased fastest at a concentration of 30% - 50%.

[0044] Species identification of the microbial strain MSB09 with growth promotion effect on Morchella 3.4.1 Strain morphological characteristics The morphological structures of the screened growth-promoting bacteria were observed macroscopically and microscopically. The morphology of the bacterial strain MSB09 was as Figure 6As shown, MSB09 grows irregularly on the plate. The colonies are flat, grayish-white and opaque, with no luster on the surface, irregularly wrinkled edges, short rod-shaped, endospores, and Gram-positive bacteria.

[0045] 3.4.2 Physiological and biochemical identification of the strain The physiological and biochemical tests of the bacterial strain MSB09 were carried out using the HBI microbial biochemical identification strip. The results showed (Table 2): The V-P of the MSB09 strain was negative, citrate was negative, propionate was negative, D-xylitol was positive, L-arabinose was positive, D-mannitol was negative, gelatin liquefaction was negative, growth in 75% NaCl was positive, growth at pH 5.7 was positive, nitrate reduction was positive, starch hydrolysis was positive, and anaerobic growth was negative.

[0046] Table 2 HBI microbial biochemical identification +: The experimental result is positive; —: The experimental result is negative Combining the results of morphology, physiological and biochemical, and molecular identification, it can be seen from Figure 6 、 Figure 7 and Figure 8 that the isolated and screened MSB09 strain grows irregularly on the plate. The colonies are flat, grayish-white and opaque, with no luster on the surface, irregularly wrinkled edges, short rod-shaped, endospores, Gram-positive bacteria, and aerobic. The morphological structure is basically similar to that of the genus Bacillus in the family Bacillaceae of the order Bacillales Bacillus sp. The size of the 16s rDNA sequence fragment of the MSB09 strain is 1393 bp, and the sequence is as shown in SEQ ID No.1. Combining the morphological characteristics of the MSB09 strain and the 16s rDNA sequence alignment results, the isolated MSB09 strain of the present invention was identified as a Morchella growth-promoting bacterium, Bacillus, and the taxonomic name: Bacillus sp., with the deposit number of GDMCC No: 65938.

[0047] 16s rDNA sequence of the MSB09 strain (SEQ ID No.1): TACCTCACCGACTTCGGGTGTTGCAAACTCTCGTGGTGTGACGGGCGGTG TGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTAC TAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGA GAACAGATTTATGGGATTGGCTAAACCTTGCGGTCTTGCAGCCCTTTGTT CTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATT TGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAG TGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGG ACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCT GTCACTCTGTCCCCGAAGGGAAAGCCCTATCTCTAGGGTTGTCAGAGGAT GTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCT CCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGA CCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGG CGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAG GGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTA CAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCA TTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTC CCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGAC TTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCT TGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTT CTGGTTAGGTACCGTCAAGGTGCGAGCAGTTACTCTCGCACTTGTTCTTC CCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGT TGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTC AGGTCGGCTACGCATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGC TAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATC CTTGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCG GAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCC GCCGCTAACATCCGGGAGCAAGCTCCCTTCTGTCCGCTCGACT In recent years, with the rapid growth of the consumption demand for Morchella, the production scale of Morchella has gradually increased, and continuous cropping obstacle has become a major problem. One of the important reasons for the occurrence of continuous cropping obstacle is the change of soil microbial structure. In this invention, high-throughput sequencing was used to analyze the soil samples of Morchella sextelata YMe151 cultivation, and it was found that the microbial diversity of non-continuous cropping healthy soil was significantly higher than that of continuous cropping soil, and there were obvious differences in the community structure; based on the idea of reconstructing soil flora, culturable microorganisms were isolated from the soil where Morchella grew well and there was no continuous cropping disease; through the plate confrontation experiment with Morchella, multiple target strains that promoted the mycelial growth of Morchella were obtained; the mycelial growth rate of the growth-promoting strain MSB09 with a growth rate increase of 45% was screened by adding its bacterial liquid to the plate medium and then inoculating Morchella; further, its bacterial liquid was added to plate culture and test tube culture to verify its effect on the growth of Morchella. When the concentration of MSB09 bacterial liquid was 50%, the mycelial growth rate of Morchella in plate culture was as high as 35.83 mm / d, and the biomass was 1.911 g at 50% concentration, determining the strong growth-promoting effect of strain MSB09. Morphological observation, physiological and biochemical determination and molecular identification of this growth-promoting bacterium were carried out as follows: Bacillus MSB09 ( Bacillussp.). This bacterium can be used for promoting the growth of mycelium at all stages of the industrial production of Morchella, and adding growth-promoting strains with differential deficiencies to the soil with continuous cropping obstacles of Morchella to repair the soil with continuous cropping obstacles, so as to improve the adaptability of continuous cropping cultivation of Morchella.

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Bacillus ( Bacillus sp.) MSB09, characterized in that Category naming: Bacillus sp. , deposit number is GDMCC No:65938.

2. A bacterial agent, characterized in that The bacterial agent contains the Bacillus ( Bacillus sp.)MSB09.

3. A composite microbial preparation, characterized in that: The composite microbial preparation contains the Bacillus ( Bacillus sp.)MSB09.

4. The Bacillus according to claim 1 ( Bacillus sp.) MSB09, or the bacterial agent described in claim 2, or the composite microbial preparation described in claim 3 in promoting the growth of morels or preparing a morel growth promoter.

5. The Bacillus according to claim 1 ( Bacillus sp.) MSB09, or the bacterial agent described in claim 2, or the composite microbial preparation described in claim 3 in repairing soil with continuous cropping of morels or in preparing a soil repair agent with continuous cropping of morels.

6. A Morchella growth promoter, characterized in that: The promoter contains the Bacillus ( Bacillus sp.) MSB09 fermentation broth.

7. The accelerator according to claim 6, characterized in that The fermentation culture medium is composed of 10 g / L glucose, 2 g / L yeast, 8 g / L peptone, 0.1 g / L sodium chloride, 0.4 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate and 0.24 g / L sodium hydroxide.

8. A method for cultivating Morchella spp., characterized in that: During the bacterial strain cultivation process, the Morchella growth promoter according to claim 6 or 7 is added.

9. A method for cultivating morels, characterized in that: During the soil-covering cultivation of Morchella, the Morchella growth promoter according to claim 6 or 7 is applied.

10. A method for improving the adaptability of continuous cultivation of Morchella, characterized in that: Applying the Bacillus sp. ( Bacillus sp.) MSB09, or the bacterial agent according to claim 2, or the composite microbial preparation according to claim 3.

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