Bacillus altitudinis CL6 with effect of resisting continuous cropping of morchella esculenta and application of bacillus altitudinis CL6
By using Bacillus CL6 as a seed dressing, the problem of continuous cropping effect in morel cultivation was solved, resulting in a significant increase in morel yield and providing a green and efficient biological control measure.
Patent Information
- Application Number
- CN202511608238.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-05
- Publication Date
- 2026-01-13
AI Technical Summary
Morel cultivation suffers from frequent disease outbreaks and significant yield decline due to continuous cropping issues, and existing solutions lack effective biological control measures.
A strain of Bacillus CL6 from the Highlands is provided. It is then combined with morel spawn through inoculation and applied to morel cultivation to promote harmonious coexistence with morel mycelium and enhance resistance to continuous cropping.
It significantly increases morel mushroom yield by 66.43% without causing damage to the ascocarps, providing a green and efficient biological solution.
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Figure CN121320179A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Bacillus CL6 that has anti-morel replanting activity and its application. Background Technology
[0002] Continuous cropping obstacles are a core problem restricting sustainable agricultural development, mainly manifested as crop yield reduction due to the enrichment of soil pathogenic microorganisms, nutrient imbalance, and accumulation of allelochemicals. Microbial control, due to its ecological compatibility, has become the preferred strategy for overcoming the bottleneck of continuous cropping, among which Bacillus spp. (…) Bacillus Due to its combined antibacterial, growth-promoting, and soil-remediation functions, it has become a major force in biological control. For example, Bacillus subtilis (… B. subtilis ) inhibits soil-borne pathogens such as Fusarium by secreting lipopeptide antibiotics; Bacillus amyloliquefaciens ( B. amyloliquefaciens It can synthesize antimicrobial proteins to competitively repel pathogen colonization; recent studies have also revealed that *Bacillus hygroscopicus* (…) B. altitudinis These beneficial bacteria possess broad-spectrum antifungal activity and can degrade soil toxins. They alleviate the damage caused by continuous cropping by mechanisms such as directly inhibiting the growth of pathogens, rebuilding beneficial soil microbial communities, and secreting growth hormones (such as IAA) to enhance crop resistance.
[0003] Morel mushrooms ( Morchella spp . The high added value of [the plant] drives its large-scale open-field cultivation, and commercial cultivation has now spread throughout the country. However, the necessary soil-covering cultivation mode makes it highly susceptible to the effects of continuous cropping, mainly manifested in: the presence of Fusarium ([…]) in the soil of continuous cropping. Fusarium spp . Trichoderma ( Trichoderma spp . The surge in biomass of pathogens such as spores and worms induces devastating diseases like stem rot, white mold, and spider web disease, leading to frequent yield reductions or even crop failures in morel cultivation. The accumulation of morel metabolites and residues from exogenous nutrient bags alter the soil microenvironment, causing competitive inhibition of nutrients and a significant decrease in mycelial biomass in continuously cropped fields. While the main methods for addressing morel replanting include contact scavenging with quicklime, crop rotation with green crops, deep plowing, sun exposure, and flooding, clear operational standards and evaluations of their actual effects are lacking. Industry statistics indicate that morel yields in continuously cropped fields decrease by an average of over 60%, severely hindering the sustainable development of production areas. Summary of the Invention
[0004] In view of this, the purpose of this invention is to provide a strain of Bacillus cereus CL6 with anti-morel replanting effect and its application. This invention is the first to isolate a beneficial bacterium, Bacillus cereus CL6, with anti-morel replanting effect from the rhizosphere soil (soil below the primordia) of morel mushrooms. This strain coexists and grows harmoniously with morel mycelium and can significantly increase the yield of replanted bases, providing a green and efficient biological solution for the morel cultivation industry to resist replanting.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a strain of Bacillus hygroscopicus with anti-morel replanting activity (…). Bacillus altitudinis CL6, the Bacillus clavatum CL6, was deposited at the China Center for Type Culture Collection on March 18, 2025, with accession number CCTCC NO: M 2025516.
[0006] Preferably, the genome of the Bacillus CL6 from the Highlands was submitted to the National Genome Data Center of China, and the obtained genome number is: GWHGQKT00000000.1.
[0007] The present invention provides a microbial inoculant, which includes the aforementioned Bacillus cereus CL6.
[0008] This invention also provides the application of the aforementioned Bacillus CL6 and the aforementioned microbial inoculant in the replanting cultivation of morel mushrooms.
[0009] As a preferred embodiment, the method for preventing replanting of morel mushrooms is as follows: after mixing Bacillus cereus CL6 with morel mushroom spawn, morel mushroom cultivation is carried out.
[0010] Preferably, the amount of morel spawn used is 150-200 kg per acre.
[0011] Preferably, the concentration of the highland Bacillus CL6 is 1×10⁻⁶. 8 ~10 9 per mL.
[0012] Preferably, the dosage of Bacillus CL6 is 500~2000 mL / mu.
[0013] Preferably, the cultivation includes potted plants, tiered cultivation, and field cultivation.
[0014] The present invention also provides the application of the aforementioned Bacillus CL6 and the aforementioned microbial inoculant in increasing morel mushroom yield.
[0015] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a strain of *Bacillus glabra* CL6 with anti-replanting activity against morel mushrooms and its application. This invention is the first to isolate a beneficial bacterium—*Bacillus glabra* CL6—with anti-replanting activity against morel mushrooms from the rhizosphere soil (soil below the primordia) of *Morchella esculenta*. Bacillus altitudinis CL6, preservation number: CCTCC NO: M 2025516, this strain coexists harmoniously with morel mycelium and does not cause damage to ascocarps when inoculated with high doses via puncture. Field cultivation trials show that seed treatment with CL6 inoculum (1000 mL / mu, 1×10⁻⁶) is effective. 8 ~10 9 CFU / mL can significantly increase yield in continuously cropped plantations, reaching 1958.33 g / m³. 2 Compared to the control (1176.67 g / m³), 2 The yield increased by 66.43%. The *Bacillus clavatus* CL6 strain of this invention can be used as an adjunct beneficial microbial agent in morel cultivation to resist the damage caused by continuous cropping, ultimately improving the stability of morel cultivation. Using the strain of this invention can promote the resistance to continuous cropping in morels and increase morel yield. It provides a green and efficient biological solution for the morel cultivation industry to resist continuous cropping. Attached Figure Description
[0016] Figure 1 Morphological characteristics of the beneficial bacteria CL6 strain resistant to continuous cropping are shown in Figures A-B, which represent the morphological characteristics of CL6 strain on LB medium, with A being the front and B the back. Figure C represents the morphological characteristics of CL6 strain under scanning electron microscopy. Figures D-E represent the morphological characteristics of CL6 strain under transmission electron microscopy, with D having a scale bar of 1 μm and E having a scale bar of 500 nm. Figure 2 A phylogenetic tree of ML constructed based on 16S rRNA; Figure 3 This section presents the genomic characteristics of Bacillus CL6 from highland. From the inside out, the first circle represents the scale; the second circle represents the GC skew; the third circle represents the GC content; the fourth and seventh circles represent the COG to which each CDS belongs; and the fifth and sixth circles represent the location of the CDS, tRNA, and rRNA on the genome. Figure 4 The sequence characteristics of the CL6 plasmid of Bacillus hygroscopicus are shown below. From the inside out, the first circle represents the scale; the second circle represents the GC skew; the third circle represents the GC content; the fourth and seventh circles represent the COG to which each CDS belongs; and the fifth and sixth circles represent the location of the CDS, tRNA, and rRNA on the genome. Figure 5 Statistics on the yield of morel mushrooms cultivated using Bacillus CL6 from highland areas and resistant to continuous cropping.
[0017] Biological Preservation Instructions
[0018] Highland Bacillus CL6, Latin name Bacillus altitudinis The strain is deposited at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on March 18, 2025, with accession number CCTCC NO: M 2025516. Detailed Implementation
[0019] This invention provides a strain of Bacillus hygroscopicus with anti-morel replanting activity (…). Bacillus altitudinis CL6, the Bacillus clavatum CL6, was deposited at the China Center for Type Culture Collection on March 18, 2025, with accession number CCTCC NO: M 2025516.
[0020] In this invention, the genome of *Bacillus glacialis* CL6 was submitted to the National Genome Data Center of China, and the obtained genome number is GWHGQKT00000000.1. The complete genome of *Bacillus glacialis* CL6 is circular, with a genome size of 3,731,656 bp and a GC content of 41.40%. *Bacillus glacialis* CL6 also contains a circular plasmid with a genome size of 7,347 bp and a GC content of 37.05%. The plasmid sequence was submitted to the NCBI public database and the obtained sequence number is PX119821 (https: / / www.ncbi.nlm.nih.gov / nuccore / PX119821).
[0021] The present invention provides a microbial inoculant, which includes the aforementioned Bacillus cereus CL6.
[0022] This invention also provides the application of the aforementioned Bacillus CL6 and the aforementioned microbial inoculant in the replanting cultivation of morel mushrooms.
[0023] In this invention, the method for preventing replanting of morel mushrooms is as follows: after mixing Bacillus cereus CL6 with morel mushroom spawn, morel mushroom cultivation is carried out.
[0024] In this invention, the dosage of the morel mushroom spawn is 150-200 kg per acre, and the concentration of Bacillus cereus CL6 is 1×10⁻⁶. 8 ~10 9 The dosage of Bacillus CL6 is 500-2000 mL / mu, preferably 700-1500 mL / mu, and more preferably 1000 mL / mu. The cultivation includes pot cultivation, tiered cultivation and field cultivation.
[0025] The present invention also provides the application of the aforementioned Bacillus CL6 and the aforementioned microbial inoculant in increasing morel mushroom yield.
[0026] In this invention, the preferred method for increasing morel mushroom yield is to increase the yield of morel mushrooms in soil where morel mushrooms are continuously grown.
[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0028] Example 1: Highland Bacillus CL6 ( Bacillus altitudinis Isolation and screening of strains
[0029] At the morel mushroom cultivation base in Pengshui County, Chongqing (February 9, 2023), soil samples of approximately 30 mL volume were collected by vertically cutting downwards from a 50 mL sterile centrifuge tube with a 30 mm diameter, including the primordium and the soil at a depth of approximately 10-12 cm below the primordium. A total of three soil samples were collected, and the depth of each sample should be basically the same. After being brought back to the laboratory at a temperature below 10℃, obvious plant roots were removed in a laminar flow hood, and the samples were mixed in a sterile container before being used for isolation and culture experiments.
[0030] Take approximately 1g of fresh soil sample and mix thoroughly with 9mL of sterile water. Filter the mixture through an 8μm pore size sterile filter to remove most fungal spores, hyphal fragments, and debris, yielding 10g of the sample. -1 The sample diluent was prepared by serially diluting the sample with sterile water, each time by a factor of 10, to obtain 10 samples. -2 10 -3 10 -4 10 -5 10 -6 Diluted solution; finally, take 200 μL of solution with a concentration of 10. -4 10 -5 10 -6The sample dilutions were spread and inoculated onto LB (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15.0 g / L, deionized water 1000 mL; pH 7.4), R2A (yeast digest 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, KH2PO4 0.3 g / L, MgSO4 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L, deionized water 1000 mL; pH 7.2±0.2), and TSA (casein trypsin digest 15 g / L, soybean flour papain digest 5 g / L, NaCl 5 g / L, agar 15 g / L, deionized water 1000 mL; pH 7.4). Five different culture media were prepared on plates: 7.3±0.2, PCA (5.0 g / L tryptone, 2.5 g / L yeast extract, 1.0 g / L glucose, 15.0 g / L agar, 1000 mL deionized water; pH 7.0±0.2), and PCA + morel polysaccharide (5.0 g / L tryptone, 2.5 g / L yeast extract, 1.0 g / L glucose, 1.0 g / L morel polysaccharide, 15.0 g / L agar, 1000 mL deionized water; pH 7.0±0.2). Each gradient was repeated three times. The plates were incubated upside down at 18°C. After 3–5 days, single colonies of different colors and morphologies were picked and enriched in 1.5 mL LB liquid medium to obtain bacterial suspensions for later use.
[0031] The isolation results showed that a total of 643 bacterial strains were isolated on five different culture media, including 146 strains of LB, 102 strains of R2A, 168 strains of TSA, 96 strains of PCA, and 131 strains of PCR+polysaccharide.
[0032] A co-culture experiment of morel mushrooms and bacteria was conducted in a 50 mL aerated culture flask. 25 mL of CYM liquid medium (20 g glucose, 2 g peptone, 2 g yeast extract, 0.5 g magnesium sulfate heptahydrate, 1.0 g dipotassium hydrogen phosphate, 0.46 g potassium dihydrogen phosphate) was placed in a 50 mL culture flask. One piece of *Morchella esculenta* tissue (5 mm in diameter, China Center for Type Culture Collection, CCTCC NO: M 2023169) activated with PDA medium was inoculated, along with 50 μL of the isolated bacterial suspension. The mixture was co-cultured at 18 °C. The growth of both bacteria and morel mushrooms was observed daily for 7 consecutive days, and the interaction between the morel mushrooms and bacteria was statistically analyzed.
[0033] The co-culture results showed three types of interactions: 1) No bacterial growth throughout the entire culture process (i.e., no significant turbidity in the culture medium), indicating inhibition by *Morchella esculenta*. This phenotype was considered inhibited and discarded because downstream experiments were not possible; 2) The culture medium remained turbid throughout the entire culture process, but *Morchella esculenta* mycelial growth was extremely low or nonexistent. These bacteria were considered *Morchella esculenta*-inhibited and could be considered harmful bacteria, thus discarded; 3) The culture medium remained turbid throughout the entire culture process, or the medium initially became turbid and then cleared, and *Morchella esculenta* mycelia grew normally. These bacteria were considered candidate beneficial bacteria and retained for subsequent experiments. A total of 124 beneficial bacteria strains were isolated from the five culture media: 29 strains from LB, 24 strains from R2A, 37 strains from TSA, 16 strains from PCA, and 18 strains from PCR+polysaccharide. TSA and LB yielded relatively more candidate beneficial bacteria, at 29.83% and 23.38%, respectively. Based on the growth rate and interaction characteristics of the candidate beneficial bacteria co-cultured with morel mushrooms, six candidate beneficial bacteria with relatively fast growth rate and significant promotion of morel mushroom growth were selected for subsequent replanting resistance tests: ZB162, ZB328, ZB75, ZB26, ZB336 (CL6) and ZB66.
[0034] Example 2 Highland Bacillus CL6 ( Bacillus altitudinis Morphological characteristics and molecular identification of strains
[0035] The Bacillus CL6 obtained in Example 1 ( Bacillus altitudinis The bacterial strain was inoculated onto LB medium for activation culture. Gram staining was used to stain the bacterial cells, and their morphology and staining characteristics were observed under a biological microscope.
[0036] Figure 1 Results A and B showed that *Bacillus hygroscopicus* was Gram-positive, morphologically characterized as rod-shaped or short rod-shaped, with apical or subapical spores, a cell diameter of 0.5–0.7 μm, and a length of 1.2–3.07 μm, occurring singly or in short chains. Colonies were spherical structures with a diameter of 2–4 mm, a rough, dark gray, and opaque surface, and a pale yellow upper surface.
[0037] Microscopic morphology observation was performed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Freshly prepared samples were washed 2-3 times by centrifugation with PBS (Na₂HPO₄ 13.22 g / L, NaH₂PO₄ 0.6 g / L, NaCl 85.0 g / L, pH 7.4) and then fixed overnight in 2.5% glutaraldehyde. SEM samples were dehydrated in a gradient of 30%, 60%, 80%, 90%, and 100% ethanol, followed by critical point drying with carbon dioxide, then sputter-coated with gold and observed under a microscope. TEM samples, after glutaraldehyde fixation, were stained with 4% osmium tetroxide for 4 hours, dropped onto a copper grid, and observed and photographed under a TEM.
[0038] Figure 1 The C~E results showed that Bacillus hygroscopicus was rod-shaped to short rod-shaped and had obvious peritrichous flagella.
[0039] Bacterial strains were identified by homology analysis of the 16S rDNA sequence. Colony PCR was used, employing universal primers BSF5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and BSR5'-AAGGAGGTGATCCAGCCGCA-3' (SEQ ID NO.2). A 15 μL PCR amplification system contained 7.5 μL of 2×Taq MasterMix, 0.375 μL each of BSF and BSR primers, 5.75 μL of ddH2O, and 1 μL of DNA. The PCR reaction conditions were 95℃ for 3 min, 95℃ for 15 s, 58℃ for 1 min, 72℃ for 25 s, and 72℃ for 3 min, for a total of 34 cycles. After PCR amplification, the reaction products were detected by agarose gel electrophoresis and then subjected to bidirectional sequencing. The peak chromatogram file of the sequencing results was examined, low-quality bases at both ends of the sequence were removed, and the sequences were spliced to obtain complete 16S rDNA fragments. The 16S rDNA sequence of strain CL6 is 1419 bp in length. The sequence was submitted to the NCBI database and obtained the sequence number: PX022612 (https: / / www.ncbi.nlm.nih.gov / nuccore / PX022612).
[0040] Online alignment analysis was performed using NCBI. The 16S ribosomal RNAsequences (Bacteria and Archaea) database from the rRNA / ITS databases was selected for BLAST comparison analysis. The results showed that it aligned with NR_042337 in the database. B. altitudinis The 41KF2b strain showed 100% similarity, and was subsequently downloaded. BacillusPhylogenetic tree construction was performed using reference sequences from neighboring species: First, the sequences were aligned using MAFFT. The aligned files were then converted to phy format and constructed using IQtree2 software with maximum likelihood (ML) and a bootstrap value of 1000. The results are shown below. Figure 2 .
[0041] like Figure 2 The results showed that strain CL6 and all B. altitudinis Clustered together with 100% support, they form independent branches, and... B. xiamenensis as well as B. pumilus , B. australimaris Distinguishing them is possible. Based on morphological characteristics, strain CL6 can be identified as *Bacillus glacialis*. Bacillus altitudinis .
[0042] Example 3: Highland Bacillus CL6 ( Bacillus altitudinis Genome sequencing and characterization of strains
[0043] The whole genome of *Bacillus glomeratus* strain CL6 was sequenced using next-generation sequencing (NGS) and single-molecule fluorescent long segment sequencing (SML). The NGS genome library contained 400 bp inserts, and PE150 paired-end sequencing yielded 7.6 million reads (1.14 Gb). The SML genome sequencing yielded 162 kb reads (1.06 Gb). The raw sequencing data were submitted to the NCBI-SRA (https: / / www.ncbi.nlm.nih.gov / sra) public database, obtaining sequence numbers SRR34918214 and SRR34918217.
[0044] The third-generation sequencing data were assembled using Unicycler, Flye, Hifiasm, and Necat. Pilon software was used to perform base correction on the assembled results using high-quality short-sequence sequencing data from the second-generation sequencing. The assembly results showed that two circular sequences were obtained. Sequence 1 was identified as the complete genome sequence of the target bacteria, with a length of 3,731,656 bp and a GC content of 41.40%. Figure 3Sequence 2 is the complete plasmid sequence, with a length of 7,347 bp and a GC content of 37.05%. The CL6 genome data was submitted to the National Genome Data Center of China (https: / / ngdc.cncb.ac.cn / ), and the sequence number obtained was GWHGQKT00000000.1; the CL6 plasmid genome was submitted to the NCBI public database and the sequence number obtained was PX119821.
[0045] The genome sequence was compared with the Nucleotide Sequence Database (NT) to obtain species information. The results showed that the top 5 candidate species were all... B. altitudinis This is consistent with the identification results obtained by the present invention through morphological and molecular characteristics.
[0046] To assess the integrity of the assembled genome and potential contamination, the assembly results were analyzed using checkM software. The results showed that the genome integrity of *Bacillus glacialis* CL6 obtained in this invention reached 99.59%. ANI (Aspect-Oriented Nucleus Index) is an important parameter based on the whole genome sequence of a species, used to determine the genetic association between species by analyzing and comparing homologous gene sequences. It can intuitively represent the closeness of kinship between species. ANI analysis was performed using fastANI software to obtain ANI values, showing that *Bacillus glacialis* CL6 strain and GCF_000691145.1... B. altitudinis 41KF2b has the highest ANI accounting consistency, reaching 98.2642.
[0047] GeneMarkS software was used to predict protein-coding genes in the CL6 genome. This software prediction method utilizes a statistical model built using GeneMark.hmm, based on a frequency table matrix of nucleic acids used in the sequence, to predict potential coding regions, improving gene translation site identification and reducing false positive rates. Open reading frame (ORF) prediction results showed a total of 3,823 coding genes predicted, with a total ORF length of 3,271,257 bp, an average of 1.024 genes per kb sequence, and an average ORF length of 855.68 bp.
[0048] tRNA genes in the whole genome were predicted using tRNAscan-SE, and rRNA genes were predicted using Barrnap. The prediction of other non-coding RNAs was mainly obtained through comparison with the Rfam database. The results showed that a total of 8 5S, 16S, and 23S rRNA tandem units were predicted, with average lengths of 111 / 1546 and 2928 bp, respectively; a total of 82 tRNAs and 84 ncRNAs were predicted, with average lengths of 77 and 164 bp, respectively.
[0049] Protein-coding genes were predicted using the eggnog-mapper software. EggNOG includes functional classifications from the original COG / KOG (COG, prokaryotic; KOG, eukaryotic). For bacteria, a protein sequence was annotated into a specific eggNOG (COG) by alignment. COG can be divided into twenty-one categories according to function (Table 1). The functional classification results showed that the most common category was "Function unknown," containing 909 genes, accounting for 23.78%, followed by "Amino acid transport and metabolism," containing 333 genes, accounting for 8.71%.
[0050] Table 1. Functional annotation of the eggNOG gene encoding Bacillus CL6 strain.
[0051] Gene prediction of the plasmid sequence showed that a total of 9 ORFs were predicted. Figure 4 The total ORF length was 4,233 bp, with an ORF density of 1.225 genes / kb sequence length. The longest plasmid ORF was 1,149 bp, the average plasmid ORF length was 470.33 bp, the intergenic region was 3,114 bp, and the total ORF length accounted for 57.62%. A pseudogene sequence was predicted between 1,782 and 2,800 bp of the sequence.
[0052] A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for RNA polymerase-specific binding and transcription initiation, and most are located upstream of the transcription start site of structural genes. Using PromPredict software to predict promoters in the genome, five promoter binding sites were predicted on plasmid sequences, ranging from 889 bp to 910 bp, 1,059 bp to 1,149 bp, 1,648 bp to 1,790 bp, 2,267 bp to 2,312 bp, and 3,171 bp to 3,240 bp. The promoter region length ranged from 22 bp to 143 bp, with an average length of 74.4 bp.
[0053] Experimental Example 1: Field Cultivation of Morel Mushrooms
[0054] A cultivation experiment was conducted according to the field cultivation plan for morel mushrooms. It mainly included six steps: spawn preparation, land selection and treatment, sowing, supplementary feeding techniques, fruiting induction and preservation, and harvesting.
[0055] The spawn production follows a three-tiered system: mother culture, primary culture, and spawn. The mother culture medium is PDA solid medium (200g potato juice, 20g glucose, 20g agar powder, 1000mL distilled water, natural pH, 18×180mm glass test tubes). The primary culture formula is: 20% wheat, 76.5% sawdust, 1.5% quicklime, 2% gypsum, with a moisture content of 60%–65%, using 1000mL primary culture bottles. The spawn formula is: 3% wheat... 5%, sawdust 61.5%, quicklime 1.5%, gypsum 2%, moisture content 60%~65%, using 17×35cm, 6-mil thick cultivation bags; exogenous nutrient bag formula: wheat 60%, sawdust 36.5%, quicklime 1.5%, gypsum 2%, moisture content 60%~65%, using 15×30cm, 4-mil thick cultivation bags; per acre, use 1 morel mother spawn, 2 bottles of original spawn, 150 bags of cultivation spawn, and 2000 bags of exogenous nutrient bags.
[0056] Preparation and application of anti-replanting bacteria: The activated bacteria obtained in Example 1 were inoculated into 500 mL LB liquid medium and cultured in a shake flask at 24°C and 180 rpm for 72 h; under aseptic conditions, the bacteria were collected by centrifugation at 6000 rpm for 5 min; the bacteria were washed 2-3 times with sterile PBS to obtain a bacterial suspension; the bacterial concentration was determined using a hemocytometer, and the bacterial suspension was adjusted to 1 × 10⁻⁶ using PBS. 8 ~10 9 The recommended usage concentration is 1000 mL per acre.
[0057] Cultivation and Management: Cultivation trials were conducted at bases where morel mushrooms had been cultivated in the previous year. Fifteen days before cultivation, 200 kg of quicklime per acre was applied and the land was tilled, and the land was prepared into boxes 1-1.2 m wide.
[0058] The bacterial suspension was mixed with 150 bags of morel spawn prepared earlier at a dosage of 1000 mL / acre. The mixture was then sown in the field, covered with 3-5 cm of soil, and subsequently covered with black mulch to enter the mycelium cultivation stage. Between 7 and 15 days after sowing, exogenous nutrient bags were applied, approximately 2000 bags per acre; this was followed by the mycelium cultivation stage.
[0059] Environmental condition control: During the sowing stage, maintain soil moisture at 28%–30%, soil temperature at 14–18℃, and air humidity at 75%–85%; during the mycelium cultivation stage, maintain soil moisture at 24%–28%, soil temperature at 10–16℃, and air humidity at 65%–85%; 60 days after sowing, initiate mushroom cultivation by removing the mulch and watering for 20 minutes; maintain soil moisture at 27%–32%, soil temperature at 6–10℃, and air humidity at 80%–95%; during the fruiting management stage, maintain soil moisture at 25%–28%, soil temperature at 8–16℃, and air humidity at 85%–95%.
[0060] Implementation of the Resistant Replanting Experiment: The replanting site was selected at a morel mushroom base in Daxing District, Beijing. This base had previously cultivated morel mushrooms in 2022-2023. In this experiment, sowing was carried out on November 25, 2023, and exogenous nutrient bags were placed on December 10. Fruiting induction treatment was performed on February 18, 2024. A large number of small mushrooms were observed to have emerged on March 10, and harvesting was completed on March 26. Results are shown below. Figure 5 .
[0061] Statistical results showed that, during cultivation, fields treated with inoculant seed dressing had higher mycelial and asexual spore production compared to the control group, while other processes showed no significant differences. The yield difference was more pronounced, with the untreated control group averaging 1176.67 g / m². 2 All six groups of experiments using candidate beneficial bacteria showed yield increases, with the group treated with Bacillus cereus CL6 inoculum showing an average yield of 1958.33 g / m³. 2 It showed a significant difference compared to the control. p <0.01), the yield increase rate reached 66.43% ( Figure 5 Secondly, ZB75 and ZB66 showed increases of 59.63% and 55.16% respectively compared to the control; the worst-performing promoting strain was ZB162, but its average yield still reached 1395.00 g / m³. 2 Compared with the control, the yield increased by 18.55%.
[0062] Example 2: Safety evaluation of *Bacillus clausti* CL6 strain against *Morchella esculenta* ascocarps.
[0063] The field puncture test was conducted in February 2025 at a morel mushroom cultivation base in Anning County, Kunming City, Yunnan Province. Under 24℃ incubation conditions, *Bacillus cereus* CL6 obtained in Example 1 was enriched in LB liquid medium. After 3 days of incubation, the culture was washed 2-3 times by centrifugation with sterile water, and the bacterial concentration was adjusted to 1×10⁻⁶. 9CFU / mL, using a sterile toothpick to pick up the bacterial suspension, puncture the middle of the stipe of healthy morel ascocarps approximately 4-5 cm in height. Five punctures were made per ascocarp, with at least five punctures per ascocarp for the *Bacillus glaber* CL6 strain. For the control treatment, sterile water was used instead of the bacterial suspension for the puncture experiment. One week later, the punctured ascocarps were examined. Results showed that after puncture with *Bacillus glaber* CL6, the morel ascocarps continued to grow vigorously, exhibiting healthy morphology and no redness or lesions at the puncture sites, suggesting that the CL6 biocontrol bacteria were harmless to the morel ascocarps.
[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A strain of Paenibacillus altiplano CL6 having an anti-Morchella crop effect, characterized in that, Bacillus altitudinis The Bacillus altitudinis CL6 was preserved in China Center for Type Culture Collection on March 18, 2025, and the preservation number was CCTCC NO: M 2025516. 2. Bacillus altitudinis CL6 according to claim 1, characterized in that, The genome of the Bacillus altitudinis CL6 was submitted to China National Genomics Data Center, and the obtained genome number was GWHGQKT00000000.
1.
3. A microbial inoculant, characterized in that, The microbial agent comprises the Bacillus altitudinis CL6 of claim 1.
4. The Bacillus altitudinis CL6 of claim 1 or 2, or the microbial agent of claim 3 is applied in anti-morel heavy cropping.
5. Use according to claim 4, characterized in that, The method for anti-morel heavy cropping is that the Bacillus altitudinis CL6 is mixed with the morel cultivation species, and then the morel cultivation is carried out.
6. Use according to claim 5, characterized in that, The use amount of the morel cultivation species is 150-200 kg per mu.
7. Use according to claim 5, characterized in that, The concentration of the Paenibacillus sp. CL6 is 1 x 10 8 ~10 9 individuals / mL.
8. Use according to claim 7, characterized in that, The use amount of the Bacillus altitudinis CL6 is 500-2000 mL per mu.
9. Use according to claim 5, characterized in that, The cultivation includes pot culture, layer frame cultivation and field cultivation.
10. The Bacillus altitudinis CL 6. The microbial agent of claim 3 is applied in improving the yield of morel.