A Morchella growth-promoting strain Bacillus MSB09

Abstract

The present invention belongs to the field of microbial technology and specifically discloses a Morchella growth-promoting strain Bacillus MSB09, Bacillus ( Bacillus sp. )MSB09, classification and naming: Bacillus sp., deposit number is GDMCC No. 65938, Morchella as a precious edible and medicinal mushroom, the cultivation scale is constantly expanding, but the soil continuous cropping problem has become the main obstacle to its soil covering cultivation and fruiting. This paper explores the soil microbial structure of Morchella and screens out the beneficial microorganism Bacillus ( Bacillus sp. ) MSB09 provides technical support for rebuilding soil microecology, alleviating the obstacles of continuous cropping of morels and biological control.

Description

Technical Field

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

[0002] Continuous cropping obstacles, also known as replant disorder or replant problems, refer to the difficulties in seedling emergence, poor seedling growth, increased pests and diseases, reduced yields, and varietal degradation that occur when the same or related crops are continuously planted on the same land under conventional cultivation management. This phenomenon widely affects the cultivation of a wide range of plants, including food crops, cash crops, vegetables, trees, and traditional Chinese medicines, and has become a major obstacle to the development of my country's planting industry. Its causes include soil microorganisms, soil enzymes, soil-borne diseases, and allelopathic autotoxicity, with a key factor being changes in the soil microbial structure.

[0003] Morel Morchella spp.) belong to the Ascomycota phylum, specifically the class Pezizomycetes, order Pezizales, and family Morchellaceae. This rare edible and medicinal fungus is named "morel" because of the distinctive, uneven surface on its head, resembling a sheep's tripe. Since the successful commercial cultivation of morels, soil and ecological problems resulting from continuous cropping have become obstacles to the sustainable development of morels. Various enzymes in the soil play a key role in the utilization of nutrients such as C, N, and P, participating in the cycling of these elements within the soil. Soil enzyme activity is closely related to the abundance and diversity of microorganisms in the habitat and is a key factor in soil ecosystem function.

[0004] Based on this, the present invention analyzes the evolution of culturable microbial communities in soils cultivated with continuous morel cultivation to isolate the dominant microorganisms in these soils. Furthermore, the present invention screens for growth-promoting morel strains and classifies each strain into species by combining morphological and physiological micro- and macroscopic characteristics with molecular biology techniques, providing technical support for the sustainable and stable development of the morel cultivation industry. Summary of the Invention

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

[0006] This study systematically isolates and identifies beneficial microorganisms in Morchella cultivation soil, providing practical experience for the sustainable cultivation and stable development of Morchella. Using a plate standoff test combined with molecular biology techniques, it rapidly screens for strains with a growth-promoting effect. Furthermore, molecular-level identification accurately identifies the strains, providing reliable technical support for the sustained, high-yield cultivation of Morchella.

[0007] Specifically, the present invention isolated and identified a Morchella growth-promoting strain Bacillus ( Bacillus sp.) MSB09, classification and nomenclature: Bacillus sp. , deposit number is GDMCC No:65938.

[0008] In one embodiment, the present invention provides a bacterial agent, wherein the bacterial agent contains the Bacillus ( Bacillus In some embodiments, the active ingredient of the bacterial agent may be Bacillus sp. MSB09 or / and a metabolite of Bacillus sp. MSB09. Optionally, the bacterial agent may be supplemented with a surfactant (such as Tween 20, Tween 80, etc.), a protective agent (such as glycerol, sodium alginate, etc.), a stabilizer (such as agar, gelatin, etc.), a buffer (such as phosphate buffer), etc.

[0009] In another embodiment, the present invention provides a composite microbial preparation containing the Bacillus ( Bacillus sp.) MSB09, the active ingredients of the composite microbial preparation may also contain other biological components or non-biological components; in one embodiment, the composite microbial preparation is made of two or more non-antagonistic microbial species, such as rhizobia, nitrogen-fixing bacteria, photosynthetic bacteria or other growth-promoting bacteria.

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

[0011] In other embodiments, the bacterial agent or composite microbial preparation provided by the present invention can be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0012] In one embodiment, the present invention provides the Bacillus ( Bacillus sp.) MSB09, or the bacterial agent, or the composite microbial preparation in promoting the growth of morels or preparing a morel growth promoter.

[0013] In one embodiment, the present invention provides the Bacillus ( Bacillus sp.) MSB09, or the bacterial agent, or the composite microbial preparation in repairing soil with continuous morel cropping obstacles or in preparing a soil repair agent with continuous morel cropping obstacles.

[0014] In a more specific embodiment, the present invention provides a Morchella growth promoter, the promoter containing the Bacillus ( Bacillus sp.) MSB09 fermentation broth.

[0015] Furthermore, in the promoter, the fermentation culture medium 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 spp., wherein the Morchella growth promoter is added during the culturing process. Those skilled in the art will appreciate that Morchella spp., as an edible and medicinal fungus, comprises multiple culturing processes, including mother strain propagation culture (primary strain), original strain propagation culture (secondary strain), and cultivated strain propagation culture (tertiary strain). Adding a growth promoter during the propagation process can shorten the culturing process, reduce production time costs, and improve industrial production efficiency. In some specific embodiments, the modified YPD medium and Morchella grain medium provided by the present invention promote growth and culture, both of which increase the production rate of Morchella spp.

[0017] In one embodiment, the present invention provides a method for cultivating morels, wherein the morel growth promoter is applied during the morel cultivation process of covering the soil. Those skilled in the art will appreciate that during the cultivation process of morels, the fungus needs to be covered with soil to complete the mycelial growth, primordium differentiation, and fruiting body formation. In a specific implementation, the addition of the morel growth promoter provided by the present invention can improve the production efficiency of morels.

[0018] A method for improving the adaptability of continuous cropping cultivation of Morchella edulis, comprising applying the Bacillus sp. ( Bacillus sp.) MSB09, or the bacterial agent, or the composite microbial preparation. It should be understood that the strains screened in the present invention are growth-promoting strains obtained based on differential bacterial strain analysis between continuously cropped soil and healthy soil. Based on this, the growth-promoting strains with differential deficiencies are added to the soil with continuous cropping obstacles to repair the soil with continuous cropping obstacles, thereby improving the adaptability of continuous cropping cultivation of Morchella.

[0019] The technical effects achieved by the present invention are:

[0020] The present invention uses high-throughput sequencing to analyze soil samples of Morchella lilii YMe151 cultivation, and finds that the microbial diversity of non-continuously cropped healthy soil is significantly higher than that of continuously cropped soil, and there are obvious differences in community structure; based on the idea of soil flora reconstruction, cultivable microorganisms are isolated from soil where Morchella grows well and no continuous cropping diseases occur; multiple target strains that promote the growth of Morchella mycelium are obtained through plate confrontation experiments with Morchella; its bacterial solution is added to the plate culture medium and then inoculated with Morchella to screen and obtain the growth-promoting strain MSB09 with a mycelium growth rate of 45%; its bacterial solution is further added to plate culture and test tube culture to verify its effect on the growth of Morel; when the MSB09 bacterial solution is added at a concentration of 50%, the mycelium growth rate of Morchella cultured on the plate is as high as 35.83mm / d, and the biomass reaches 1.911g at a concentration of 50%, confirming the strong growth-promoting effect of strain MSB09. The growth-promoting bacteria were subjected to morphological observation, physiological and biochemical determination and molecular identification as follows: Bacillus MSB09 ( Bacillus sp.). The strain can be applied in all stages of the industrial production process of Morchella edodes, shortening the mycelial culture time of Morchella edodes and reducing production costs.

[0021] As a precious edible and medicinal mushroom, the cultivation scale of Morchella is constantly expanding, but the problem of continuous cropping in soil has become the main obstacle to its fruiting in soil-covered cultivation. This paper explores the soil microbial structure of Morchella and screens out the beneficial microorganism Bacillus MSB09 ( Bacillus sp.), providing technical support for rebuilding soil microecology, alleviating the obstacles of continuous cropping of morels and biological control. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Bacterial community structure at the phylum level in non-continuously cropped soil and continuous cropped soil of Morchella;

[0023] Figure 2 Fungal community structure at the phylum level in non-continuously cropped soil and continuous cropped soil of Morchella;

[0024] Figure 3 This is a picture of the co-culture of MSB09 and Morchella. The target strain MSB09 is on the left side of the plate, and Morchella on the right side. There is no obvious distance between the Morchella hyphae and the target strain colonies, which is a typical growth-promoting effect.

[0025] Figure 4 The effect of adding MSB09 bacterial solution on the growth promotion of Morchella mycelium in test tube culture;

[0026] Figure 5 Analysis of the growth-promoting effect of adding MSB09 bacterial solution on plate culture and test tube culture of Morchella mycelium;

[0027] Figure 6 Colony image of growth-promoting strain MSB09 on the plate;

[0028] Figure 7 100x micrograph of Gram stain of growth-promoting strain MSB09;

[0029] Figure 8 100x micrograph of spore staining of the growth-promoting strain MSB09. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. For purchased commodities in the test method, if no specific conditions are specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they can all be conventional products purchased from the market.

[0031] The Bacillus MSB09 provided by the present invention is classified and named as follows: Bacillus sp., the deposit number is GDMCC No: 65938, the depositor is Guangdong Microbial 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.

[0032] Example 1

[0033] 1. Test materials: Morchella serrata YMe151, culture medium as shown in Table 1.

[0034] Table 1 Experimental culture medium

[0035] Test culture medium Element Solid modified YPD medium Glucose 10g / L, Yeast 2g / L, Peptone 8g / L, Sodium Chloride 0.1g / L, Potassium Dihydrogen Phosphate 0.4g / L, Magnesium Sulfate 0.2g / L, Sodium Hydroxide 0.24g / L, Agar 16g / L Liquid modified YPD medium Glucose 10g / L, Yeast 2g / L, Peptone 8g / L, Sodium Chloride 0.1g / L, Potassium Dihydrogen Phosphate 0.4g / L, Magnesium Sulfate 0.2g / L, Sodium Hydroxide 0.24g / L Morel wheat culture medium Wheat kernels 49.5%, buckwheat husks 49.5%, lime 1% Beef extract peptone agar medium Peptone 10g / L, beef extract powder 3g / L, sodium chloride 5g / L, agar 15g / L Bengal rose (tiger red) culture medium Peptone 5g / L, glucose 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, red Bengal 0.033g / L, chloramphenicol 0.1g / L, agar 15g / L

[0036] 2. Methods

[0037] Collection of soil samples

[0038] To investigate the impact of continuous cropping on morels, we selected greenhouse soil (dNC) with healthy morel growth and no continuous cropping disease. We collected samples from the mycelial layer of morels using a five-point sampling method. We collected 0.5 kg of soil from each location, refrigerated at 4°C, and brought it back to the laboratory. The five samples were mixed and divided equally into two portions for future use.

[0039] High-throughput Illumina sequencing analysis of soil microorganisms

[0040] Soil samples grown with Morchella continuous cultivation served as a comparison group (dCC). Soil samples from dNC and dCC were sent to Shanghai Paisonno within 24 hours. DNA was extracted from the soil using the MP FastDNA® Spin Kit for Soil (MP Biomedical, USA). PCR amplification was performed using the diluted genomic DNA as a template using barcoded specific primers. Amplified products were cloned and sequenced on the Illumina platform. Species annotation and abundance analysis were performed using assembly (FLASH software), filtering (Vsearch software), and OTU clustering to reveal the bacterial and fungal species composition of the samples.

[0041] Isolation of culturable soil microorganisms

[0042] Beef extract peptone medium was used for bacterial culture, and Bengal red (tiger red) medium was used for fungal culture. Take 1 gram of soil sample, dilute it with sterile water, and then take 10 -3 , 10 -4 , 10 -5 Evenly spread 0.1 ml of the dilution solution over the surface of the culture medium in triplicate. Invert the culture dish and place it in a 37°C incubator for bacterial culture, while fungi should be cultured at 28°C. After 24 hours of incubation, observe for the formation of single colonies; fungal culture requires 3 to 5 days.

[0043] Compare the control group CK with the experimental group T in the experimental design, further pick out the dominant single colony on the gradient plate of the T sample for pure culture, and perform isolation and purification of the dominant bacteria.

[0044] Screening of Morchella growth-promoting strains

[0045] Using the YMe151 strain of Morchella liumei as the indicator bacteria, the isolated bacterial and fungal strains were cultured on plates and co-cultured to conduct growth promotion tests, and to screen for potential beneficial bacteria in Morchella soil.

[0046] 2.4.1 Flat Plate Confrontation Experiment

[0047] Using a 6 mm borer, a well-grown Morchella spp. activated mycelial mass was inoculated at the 1 / 4 mark along the centerline of a modified YPD plate. In the treatment group, a dominant strain, activated for 24 hours, was streaked onto the other side of the Morchella spp., at the 3 / 4 mark along the centerline. The control group received no dominant strain. Three replicates were set up. Cultures were continued at a constant temperature of 24°C. Starting on the second day, colony diameters were measured daily using the cross-streak method for ten consecutive days. The average daily mycelial growth rate (mm / day) was calculated from this measurement. The growth rate was calculated to determine the effect of the target bacteria on mycelial growth.

[0048] Growth rate % = [(growth rate of treated colonies - growth rate of control colonies) / growth rate of control colonies] × 100

[0049] 2.4.2 Experiment on adding growth-promoting bacteria solution and culture of Morchella plate

[0050] The strain MSB09, which was screened out through the plate confrontation experiment and had a growth-promoting effect on the mycelial growth of Morchella, was fermented in liquid beef extract peptone medium (agar was removed from the formula): temperature 37°C, rotation speed 150r / min. After culturing for 24h, the concentration of the bacterial solution measured by UV spectrophotometer reached 6.5×10 7 The bacterial solution was filtered through sterile gauze and then filtered through a 0.22 μm sterile filter to collect the filtrate. Five concentrations of the filtrate were added to new modified YPD plates: no filtrate (0%), 10%, 20%, 30%, and 50%. Three replicates were set for each concentration, with 0% as the control (CK). A 6 mm diameter borer was used to inoculate a piece of Morchella mycelium in the center of the prepared 9 cm solid medium. Starting on the second day, colony diameters were measured daily using the cross-streak method for seven consecutive days to obtain the average daily mycelial growth rate (mm / day). Mycelia were weighed on the ninth day, and the dry weight of mycelial biomass was calculated to determine the effect of dominant bacteria on mycelial growth.

[0051] Growth-promoting effect of growth-promoting strains

[0052] In order to promote the co-cultivation of bacteria and Morchella, the MSB09 strain was further acclimated with nutrients, that is, liquid modified YPD medium was used for fermentation culture at a temperature of 28 °C and a rotation speed of 150 r / min. After culturing for 24 h, the concentration of the bacterial solution measured by UV spectrophotometer reached 6.5×10 7 Use a sterile 6mm borer to inoculate a piece of Morchella mycelium into a sterile Morchella wheat grain culture tube. Simultaneously, inoculate the growth-promoting bacteria at five concentrations: sterile solution (0%), 10%, 20%, 30%, and 50%, and adjust the water content to the desired level. Three replicates were set for each concentration of the growth-promoting bacteria. Incubate the tubes at room temperature and observe the growth of Morchella mycelium. Measure the length of the mycelium after 3-4 days, and calculate the mycelial growth rate (mm / day).

[0053] Identification of growth-promoting strains

[0054] The screened growth-promoting bacteria MSB09 was inoculated on a conventional beef extract peptone culture medium plate, and the single colonies were isolated by the plate streak method. After the culture was completed, the size, color, surface characteristics, edge shape, etc. of the colonies were observed to preliminarily determine the strain type; Gram staining and spore staining were performed respectively, and the individual morphology and staining of the growth-promoting strains were observed under a 100× oil microscope.

[0055] Physiological and biochemical index detection: HBI microbial biochemical identification strips (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.) were used to perform physiological and biochemical detection on the above-mentioned growth-promoting bacteria.

[0056] Molecular Biological Identification: The strains were purified, and DNA was extracted using a bacterial genomic DNA extraction kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). The test strains were amplified using the universal bacterial primers 16s 27F / 1492R. PCR primer synthesis and sequencing of the amplified products were performed by Qingke Biotechnology (Kunming). Sequences were bidirectionally sequenced and spliced, aligned at 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 to determine species.

[0057] Bacterial 16S rDNA universal primer pair: 27F 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R 5'-GGTTACCTTGTTACGACTT-3'.

[0058] Results and Analysis

[0059] Analysis of soil microbial diversity in Morchella oleifera cultivation soil and continuous cropping soil

[0060] like Figure 1 、 Figure 2 The microbial community composition characteristics of non-continuously cropped Morchella soil (dNC) were analyzed, using the continuously cropped Morchella soil as a comparison group (dCC). The results showed that the microbial diversity of non-continuously cropped soil was higher than that of the continuously cropped soil, and the community structure was significantly different.

[0061] At the phylum level, the relative abundance of Chloroflexi, Acidobacteriota, and Bacteroidota was significantly higher in non-continuously cropped soils than in continuously cropped soils, making them the dominant species in healthy non-continuously cropped soils. Meanwhile, Actinobacteriota and Cyanobacteria were more abundant in continuously cropped soils. This suggests that Chloroflexi, Acidobacteriota, and Bacteroidota may be involved in the normal growth of Morchella oleracea, and suggests the potential for screening probiotic Morchella oleracea in healthy non-continuously cropped soils.

[0062] For fungi, the relative abundance of Ascomycota and Mortierellomycota in continuously cropped soils was higher than that in non-continuous cropped soils, while the relative abundance of Basidiomycota and Rozellomycota was significantly lower than that in non-continuous cropped soils.

[0063] Screening of Beneficial Microorganisms from Morchella

[0064] 3.2.1 Isolation of cultivable dominant soil microorganisms

[0065] The control group dCC was compared with the experimental group dNC, and the dominant single colony was streaked and cultured on the gradient plate of the dNC sample. After preliminary separation and purification, multiple strains of dominant soil microorganisms of Morchella fungus were obtained.

[0066] 3.2.2 Plate confrontation test between dominant strains and Morchella

[0067] Several dominant soil strains and Morchella were cultured on modified YPD medium for 2-7 days, and the mycelial growth of the control group and the treatment group was compared. The results of the comparison of the mycelial growth rate and growth rate of Morchella showed that different dominant strains had different effects on the mycelial growth of Morchella, with both promoting and inhibiting effects. The MSB09 strain promoted the mycelial growth of Morchella, and there was no obvious distance between the mycelial growth of Morchella and the colony of the target strain (see Figure 3 ), the MSB09 strain promoted the growth rate of Morchella by 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).

[0068] Strong growth-promoting effect on Morchella

[0069] The MSB09 bacterial solution was added to a Morchella wheat grain culture tube and inoculated with Morchella spores. The mycelial length was measured at different culture times and the mycelial growth rate (mm / d) of the Morchella spores in the tube was calculated. The results showed that ( Figure 4 After 4 days of culture, the mycelial growth rate of Morchella edulis was 14.31 mm / day, compared to the CK culture. The selected bacterial solution promoted the growth of Morchella edulis. In the test tube culture experiment with the addition of the MSB09 bacterial solution, the mycelial growth rate of the MSB09 bacterial solution increased with increasing concentration from 10%, reaching a maximum of 20.11 mm / day at a concentration of 50%. The growth-promoting effect is obvious.

[0070] Figure 5The results showed that when the filtrate of MSB09 strain was added to the plate, the growth rate of Morchella mycelium accelerated with the increase of the added concentration, and the growth rate reached a maximum of 35.83 mm / d at a concentration of 50%. The mycelial biomass of Morchella was 1.911 g at the MSB09 bacterial solution concentration of 50%, which showed a promotion effect, and the biomass grew fastest at a concentration of 30% to 50%.

[0071] Species identification of the microbial strain MSB09 that promotes the growth of Morchella oleracea

[0072] 3.4.1 Morphological characteristics of strains

[0073] The selected growth-promoting bacteria were observed in macroscopic and microscopic morphology. The bacterial strain MSB09 has the following morphology: Figure 6 As shown, MSB09 grows irregularly on the plate, with flat colonies, off-white opaque, dull surface, irregularly wrinkled edges, short rod shape, endospores, and Gram-positive bacteria.

[0074] 3.4.2 Physiological and biochemical identification of strains

[0075] Bacterial strain MSB09 was tested for physiological and biochemical properties using HBI microbial biochemical identification strips. The results (Table 2) showed that strain MSB09 was negative for VP, citrate, and propionate, but positive for D-xyl alcohol, L-arabinose, and D-mannitol. It also tested negative for gelatin liquefaction, growth in 75% NaCl, growth at pH 5.7, nitrate reduction, and starch hydrolysis, but negative for anaerobic growth.

[0076] Table 2 HBI microbial biochemical identification

[0077]

[0078] +: The test result is positive; -: The test result is negative

[0079] Combining the results of morphology, physiology, biochemistry and molecular identification, Figure 6 、 Figure 7 and Figure 8 It can be seen that the isolated and screened MSB09 strain grows irregularly on the plate, with flat colonies, grayish white and opaque, dull surface, irregular wrinkles on the edges, short rods, endospores, Gram-positive bacteria, and aerobic. Bacillussp. The 16s rDNA sequence fragment of the MSB09 strain is 1393 bp in size, and the sequence is shown in SEQ ID No. 1. Combined with the morphological characteristics of the MSB09 strain and the 16s rDNA sequence comparison results, the MSB09 strain isolated in the present invention was identified as a Morchella growth-promoting bacterium, Bacillus, with a taxonomic name: Bacillus sp., the deposit number is GDMCC No:65938.

[0080] MSB09 strain 16s rDNA sequence (SEQ ID No. 1):

[0081] TACCTCACCGACTTCGGGTGTTGCAAACTCTCGTGGTTGACGGGCGGTG

[0082] TGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTAC

[0083] TAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGA

[0084] GAACAGATTTATGGGATTGGCTAAACCTTGCGGTCTTGCAGCCCTTTGTT

[0085] CTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATT

[0086] TGACGTCATCCCCACCTTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAG

[0087] TGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGG

[0088] ACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCT

[0089] GTCACTCTGTCCCCGAAGGGAAAGCCCTATCTCTAGGGTTGTCAGAGGAT

[0090] GTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCT

[0091] CCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGA

[0092] CCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGG

[0093] CGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAG

[0094] GGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTA

[0095] CAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCA

[0096] TTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTC

[0097] CCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGAC

[0098] TTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCT

[0099] TGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTT

[0100] CTGGTTAGGTACCGTCAAGGTGCGAGCAGTTACTCTCGCACTTGTTCTTC

[0101] CCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGT

[0102] TGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC

[0103] CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTC

[0104] AGGTCGGCTACGCATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGC

[0105] TAATGCGCCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATC

[0106] CTTGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCG

[0107] GAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCC

[0108] GCCGCTAACATCCGGGAGCAAGCTCCCTTCTGTCCGCCTCGACT

[0109] In recent years, with the rapid growth of consumer demand for morels, the scale of production has gradually increased, and continuous cropping has become a major problem. One of the main reasons for continuous cropping is the change in soil microbial structure. The present invention uses high-throughput sequencing to analyze soil samples of Morchella lilii YMe151 cultivation, and finds that the microbial diversity of non-continuously cropped healthy soil is significantly higher than that of continuously cropped soil, and there are obvious differences in community structure; based on the idea of soil flora reconstruction, cultivable microorganisms are isolated from soil where Morchella grows well and no continuous cropping diseases occur; multiple target strains that promote the growth of Morchella mycelium are obtained through plate confrontation experiments with Morchella; its bacterial solution is added to the plate culture medium and then inoculated with Morchella to screen and obtain the growth-promoting strain MSB09 with a mycelium growth rate of 45%; its bacterial solution is further added to plate culture and test tube culture to verify its effect on the growth of Morel; when the MSB09 bacterial solution is added at a concentration of 50%, the mycelium growth rate of Morchella cultured on the plate is as high as 35.83mm / d, and the biomass reaches 1.911g at a concentration of 50%, confirming the strong growth-promoting effect of strain MSB09. The growth-promoting bacteria were subjected to morphological observation, physiological and biochemical determination and molecular identification as follows: Bacillus MSB09 ( Bacillus sp.). This fungus can be used to promote the growth of mycelium at all stages of industrial production of morels, and to repair the soil with continuous cropping obstacles by adding differentially deleted growth-promoting strains to the soil, thereby improving the adaptability of continuous cropping cultivation of morels.

[0110] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

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

2. A bacterial agent, characterized in that The bacterial agent contains the Bacillus according to claim 1 ( 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 morel mycelium or preparing a morel mycelium growth promoter.

5. A Morchella mycelium growth promoter, characterized in that The promoter contains the Bacillus ( Bacillus sp. ) Fermentation broth of MSB09.

6. The accelerator according to claim 5, characterized in that The fermentation culture medium comprises 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.

7. A method for cultivating Morchella spp., characterized in that: During the culture process of the strain, the Morchella mycelium growth promoter according to claim 5 or 6 is added.

8. A method for cultivating morels, characterized in that: During the soil-covering cultivation of Morchella, the Morchella mycelium growth promoter according to claim 5 or 6 is applied.

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