Bacillus velezensis endophyte in industrial hemp and application thereof

Abstract

This invention provides a strain of endophytic Bacillus belysinus from industrial hemp ( Bacillus velezensis This strain was deposited on May 18, 2022, at the Agricultural Microbiology Center of the China Committee on Culture Collection (CGMCC) with accession number CGMCC 1.60033. The strain provided by this invention exhibits good inhibitory effects on white mold disease of industrial hemp, white mold disease of pepper, wilt disease of loofah, and various other plant pathogens. It also promotes growth in the seeds or seedlings of hemp crops and peppers. It can be applied to the prevention and control of various plant diseases and to promote plant growth, which is beneficial to the sustainable development of agriculture and has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial control technology, and more specifically, to Bacillus belyssus strains and their applications, including the strains and their fermentation broth, methods for preparing the fermentation broth, and the applications of the strains in controlling various plant diseases and promoting plant growth. Background Technology

[0002] Plant disease symptoms refer to the pathological features of infected plants in terms of physiology, tissue structure, and morphology under the interference of pathogens or adverse environmental conditions. These symptoms are mainly caused by the interference of pathogens or adverse environmental conditions.

[0003] The occurrence and prevalence of plant diseases seriously threaten agricultural production and development. Farmers' long-term reliance on chemical pesticides for disease control has caused serious harm to human health, the environment, and food safety. Therefore, developing natural, non-toxic, harmless, and ecologically beneficial biological agricultural pesticides is of great significance.

[0004] Biological control has gradually come into focus, representing a method of pest and disease control that poses no harm to the ecological environment or human health. Using beneficial microorganisms and intermediate products to control pests, diseases, and weeds can achieve significant results. Bacillus bacteria, in particular, exhibit rapid growth, simple nutritional requirements, strong resistance, and easy survival on plant surfaces. Furthermore, Bacillus can fix nitrogen and solubilize phosphorus, producing hormones that promote plant growth and development, thus controlling plant diseases, inhibiting pathogenic microorganisms, and inducing systemic resistance. Not only is it harmless to humans and animals and does not pollute the environment, but it is also very convenient to use, meeting green food production standards and improving the sustainable development of agriculture. Bacillus is a highly promising biocontrol microorganism. Summary of the Invention

[0005] Based on this, in view of the technical problems that the above-mentioned chemical control methods in the prior art have negative impacts on the ecological environment and human health, one of the objectives of the present invention is to provide a biocontrol agent that is friendly to the ecological environment and human health and can control plant diseases.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] An endophytic Bacillus belyssus of industrial hemp, with accession number 024A, was deposited on May 18, 2022, at the Agricultural Microbiology Center of the China Committee on Culture Collection of Microbial Cultures, with accession number CGMCC 1.60033.

[0008] The second objective of this invention is to provide a microbial agent containing the aforementioned Bacillus belyssus strain.

[0009] In some embodiments, the microbial agent comprises the Bacillus vesiculus strain and / or a fermentation culture of the Bacillus vesiculus strain.

[0010] In some embodiments, the fermentation culture includes fermentation supernatant and bacterial cells.

[0011] A third objective of this invention is to provide a method for preparing the microbial inoculant according to any of the above embodiments, the method comprising the following steps:

[0012] S1. Liquid seed culture: The Bacillus berberis strain is inoculated into a seed culture medium and cultured to obtain a seed culture solution;

[0013] S2. Fermentation culture: The seed culture solution is inoculated into a fermentation culture medium and cultured. The fermentation broth is collected to obtain a liquid microbial agent.

[0014] In some embodiments, before step S1, the method further includes the step of: picking a single colony of the Bacillus belye strain and placing it in LB liquid medium, culturing it overnight, mixing it with 30% sterile glycerol at a 1:1 ratio, and storing it in a -80°C freezer.

[0015] In some embodiments, the seed culture medium is LB medium, the culture conditions are 37°C, and the culture time is 24h; the liquid seed culture conditions are 30°C, 180r / min, pH 6, and the culture time is 16-24h; and / or, the fermentation culture conditions are 30°C, 180r / min, and the fermentation time is 48h; the fermentation culture medium is medium D, the composition of which is: 8g beef extract, 5g yeast extract, 10g glucose, and 1000mL sterile water.

[0016] The fourth objective of this invention is to provide the application of the Bacillus berberis strain or the microbial agent described in any of the above embodiments in the prevention and control of white rot disease.

[0017] The fifth objective of this invention is to provide the application of the above-mentioned Bacillus vesiculosus strain or the microbial agent described in any of the above embodiments in the prevention and control of at least one of the following: Phytophthora capsici, Fusarium wilt of watermelon, Anthracnose of pomelo, Soft rot of cabbage, Soft rot of konjac, Black rot of radish, Bacterial wilt of tobacco, and Bacterium blast of rice.

[0018] The sixth objective of this invention is to provide the application of the above-mentioned Bacillus leuciscus strain or the microbial agent described in any of the above embodiments in promoting the growth of hemp plants or peppers.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The Bacillus belye strain of this invention can prevent and control a variety of plant diseases, especially white rot, which has a significant effect on the control of white rot, laying the foundation for the research and development of biological control of white rot. This strain can also prevent diseases of a variety of plants (including industrial hemp, peppers and loofah) and promote plant growth, which is conducive to promoting sustainable agricultural development and has good application prospects.

[0021] The microbial agent containing the Bacillus berberis strain and its fermentation broth provided by this invention can be used to prevent and control a variety of plant diseases. It is also environmentally friendly and human health-friendly. Moreover, the preparation and use process is simple and convenient. No organic solvents are used in the production process, which greatly reduces environmental pollution. Attached Figure Description

[0022] Figure 1 The images show the colony morphology, Gram staining, and scanning electron microscopy (SEM) ultrastructure of the *Bacillus belyssus* strain of this invention; wherein, image A is the colony morphology, image B is the Gram staining effect, and image C is the scanning electron microscopy (SEM) ultrastructure.

[0023] Figure 2 This is a phylogenetic tree of the *Bacillus belyssus* strain of the present invention;

[0024] Figure 3 The image shows the inhibitory effect of the fermentation broth of Bacillus belye of the present invention on the industrial hemp white rot fungus; in which, the left plate is the control group and the right plate is the experimental group;

[0025] Figure 4 The above diagrams show the inhibitory effects of the fermentation broth of Bacillus belye of the present invention on other plant pathogens. In the diagram, A represents the white mold pathogen of pepper, B represents the Phytophthora infestans of pepper, C represents the wilt pathogen of loofah, and D represents the anthracnose pathogen of pomelo. In the diagrams A to D, the left plates of each diagram represent the control group and the right plates represent the experimental group.

[0026] Figure 5 The fermentation broth of Bacillus vesiculosus of the present invention has an inhibitory effect on other bacterial plant pathogens. Among them, Figure 1 shows the soft rot pathogen of Chinese cabbage, Figure 2 shows the black rot pathogen of radish, Figure 3 shows the bacterial blight pathogen of rice, and Figure 4 shows the bacterial wilt pathogen of tobacco.

[0027] Figure 6 This is a pot experiment diagram of the control of white rot disease in industrial hemp using the fermentation broth of Bacillus vesicatoria of the present invention.

[0028] Figure 7 The above figures illustrate the growth-promoting effect of the fermentation broth of Bacillus belyssus of the present invention on industrial hemp seeds. Figures A, B, C, and D show the growth-promoting effects of sterile water, sterile liquid culture medium, fermentation broth of strain 024A, and fermentation supernatant of strain 024A on industrial hemp seeds, respectively.

[0029] Figure 8 The above diagram shows the growth-promoting effect of the fermentation broth of Bacillus belysae on chili seeds. CK1, CK2, TI, and T2 are the growth-promoting effects of sterile water, sterile liquid culture medium, fermentation broth of strain 024A, and fermentation supernatant of strain 024A on chili seeds, respectively.

[0030] Figure 9 The above diagram illustrates the growth-promoting effect of the fermentation broth of Bacillus belysae on pepper seedlings. CK1, CK2, TI, and T2 represent the growth-promoting effects of sterile water, sterile liquid culture medium, fermentation broth of strain 024A, and fermentation supernatant of strain 024A on pepper seedlings, respectively. Detailed Implementation

[0031] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0033] Example 1: Isolation and Identification of Bacillus belyssus strains

[0034] (1) Separation and purification

[0035] LB solid medium: 5g yeast extract, 10g peptone, 10g sodium chloride, 20g agar, 1000mL deionized water, pH 7.2, sterilized at 121℃ for 30min.

[0036] The inventors collected industrial hemp leaves from industrial hemp cultivation areas in Yunnan Province. After surface disinfection, 5g of the sample was weighed, crushed, and ground, then dissolved in 95mL of sterile water to prepare a leaf tissue suspension. This suspension was then serially diluted 10-fold with sterile water, and 10... -2 10 -3 and 10 -4 100 μL of tissue suspension was evenly spread on LB fixed medium plates. The plates were then incubated at 30°C for 36–48 h. Different colonies were picked according to their morphology, color, and size and purified on new LB solid medium plates until pure colonies were obtained and numbered (024A) for storage.

[0037] The strain was deposited at the Agricultural Microbiology Center of the China Committee on Culture Collection of Microbial Cultures on May 18, 2022, with the accession number CGMCC 1.60033.

[0038] (2) Morphological and physiological identification

[0039] The strain was streaked and cultured, and Gram staining was performed using an optical microscope. Physiological and biochemical identification was performed in accordance with Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria.

[0040] Morphological characteristics: such as Figure 1 As shown in Figures A and B, the colonies on the culture medium have a smooth, opaque, whitish surface, and the bacteria are short rod-shaped. They show a positive Gram staining reaction. Figure 1 As shown in Figure C, the colonies appear rod-shaped under a scanning electron microscope, with a length greater than their width, and a size of 0.5–1 μm × 1.5–4 μm.

[0041] Secondary metabolite characteristics: The strain showed positive results for protease production, amylase production, cellulase production, and phosphate dissolution. It can also produce biofilms. Its physiological and biochemical indicators are shown in Table 1.

[0042] Table 1 Physiological and biochemical indicators of strain 024A

[0043]

[0044] (3) 16S rDNA sequence identification and phylogenetic tree alignment

[0045] DNA was extracted from the bacterial strain using a kit, and 16S rDNA was amplified by PCR using universal primers synthesized by Qingke Biotechnology Co., Ltd. (27F5'-AGA GTT TGA TCC TGG CTC AG-3' and 1492R 5'-TAC GGY TAC CTT GTT ACG ACT T-3'). The amplified products were sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0046] The amplification reaction system consisted of: 22 μL of Mix, 1 μL each of 10 μM primers, and 1 μL of template DNA.

[0047] PCR reaction conditions: 98℃ for 2 min, 98℃ for 10 s, 55℃ for 30 s, 72℃ for 30 s; 30 cycles; final extension at 72℃ for 10 min, and termination of the reaction at 4℃.

[0048] PCR products were generated by Qingke Biotechnology Co., Ltd. Sequencing results were analyzed online and compared with 16S rDNA sequences in the GenBank database. Gene sequences of typical strains with high sequence similarity were selected as references. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in Mega 7.0, as shown below. Figure 2 As shown, a systematic analysis was conducted.

[0049] Based on the 16S rDNA sequence analysis results, and taking into account the colony morphology and physiological and biochemical characteristics of the strain, this strain was identified as Bacillus velezensis.

[0050] Strain preservation: Pick a single colony of the strain and incubate it overnight in LB liquid medium. Then mix it with 30% sterile glycerol at a 1:1 ratio and store it in a -80°C freezer.

[0051] Example 2: Preparation of Microbial Fermentation Broth

[0052] Culture medium D: 8g beef extract, 5g yeast extract, 10g glucose, 1000mL sterile water, pH 7.2, sterilized at 121℃ for 20min.

[0053] (1) Liquid seed culture: 4 μL of the glycerol tube was inoculated into LB medium and cultured at 180 r / min, 30 ℃ for 24 h to obtain the seed culture solution;

[0054] (2) Fermentation culture: Inoculate the seed culture solution into culture medium D at an inoculation rate of 5%, at 30°C, at a rotation speed of 180 r / min, for a fermentation time of 48 h;

[0055] (3) Preparation of fermentation broth: Collect the fermentation broth from the fermentation culture medium and prepare it into a liquid microbial agent.

[0056] Example 3: Control effect of Bacillus belyssus strain 024A on plant pathogenic diseases.

[0057] (1) The effect of Bacillus belyssus strain 024A on the prevention and control of white rot in industrial hemp.

[0058] Potato glucose agar (PDA) medium: 200g potato, 20g glucose, 20g agar, 1000mL sterile water, pH 7.2, sterilized at 121℃ for 20min.

[0059] Preparation of fermentation supernatant: Bacillus berberis strain 024A was fermented according to the method in Example 2, and the fermentation broth was collected for later use.

[0060] A 6mm mycelial cake of *Sclerotium affine*, the pathogen of industrial hemp, was placed in the center of a PDA plate. Three symmetrical holes (6mm in diameter) were punched at a distance of 25mm from the mycelial cake. 60μL of fermentation broth from strain 024A was injected into each hole. A control group with 60μL of LB broth added to each hole served as the incubation medium. The plates were incubated at 25℃. The inhibition rate was calculated after the control group had fully colonized the plate. The inhibitory effect was as follows: Figure 3 As shown in Table 1.

[0061] Antibacterial inhibition rate calculation: Antibacterial inhibition rate (%) = (Radius of control group - Radius of treatment group) / Radius of control group * 100%

[0062] (2) Control effect of Bacillus belyssus strain 024A on other crop pathogens

[0063] Other crop pathogens, including *Phytophthora capsici*, *Fusarium oxysporum* f.sp. *niveum*, and *Colletotrichum gloeosporioides*, were selected. Fungal mycelium (6 mm) was placed in the center of a PDA plate, and three symmetrical holes (6 mm in diameter) were punched 25 mm away from the mycelium. 60 μL of bacterial fermentation broth was injected into each hole, with 60 μL of LB broth added to each hole as a control. The plates were incubated at 25℃. The inhibition rate was calculated after the control group had fully colonized the plate. The inhibition effect was as follows: Figure 4 As shown in Table 2.

[0064] Bacterial pathogens, including *Erwinia* (cabbage soft rot), *Ralstonia solanacearum* (tobacco bacterial wilt), and *Xanthomonas oryzaepv. oryzae* (rice bacterial leaf blight), were cultured overnight in LB agar. These were then mixed with melted LB solid medium to prepare plates. A well was punched in the center of each LB solid plate, and 60 μL of 024A fermentation broth was added. A control was prepared by adding 60 μL of LB medium to each well. The plates were incubated at 37°C, and the size of the clear zone was measured. The results are as follows: Figure 5 As shown in Table 3.

[0065] Table 2. Inhibition rate of strain 024A against several fungal diseases.

[0066]

[0067] Table 3. Inhibitory effects of strain 024A on several bacterial diseases (hydrolysis zone)

[0068]

[0069] Example 4: Pot experiment on the control of white mold disease in industrial hemp using fermentation broth of strain 024A

[0070] Pot experiment: When seedlings were stable, the roots were inoculated with pathogenic cakes for experimental treatment. The experimental treatments were as follows: sterile water, sterile uninoculated liquid culture medium, and biocontrol bacteria culture solution (OD200). 600 =1.0), 10 mL each of biocontrol bacteria fermentation supernatant were used for root irrigation. Seedlings were placed in a greenhouse with a photoperiod (light:dark = 18h:6h) and grown at 23℃. Disease severity was observed and recorded, and the disease index and control effect were calculated. The experimental results are as follows: Figure 6 As shown in Table 4.

[0071] Disease Grading Standards: Industrial Hemp White Silk Disease Disease Index Grading:

[0072] Grade 0: No lesions;

[0073] Level 1: Fewer than 4 branches withered;

[0074] Grade 2: Significant withering, covering 1 / 4 to 1 / 3 of the stem and root area;

[0075] Level 3: The withered area accounts for 1 / 2 to 3 / 4 of the stem and root area;

[0076] Level 4: Patches of plants wither, with signs of stem burn and root necrosis.

[0077] Disease index = ∑(number of diseased plants at each level × number of disease levels) / (total number of plants × highest disease level) × 100%.

[0078] The formula for calculating the effectiveness is:

[0079] Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0080] Table 4. Effects of strain 024A on white rot disease in industrial hemp through pot experiments.

[0081]

[0082] In summary, the results of plate confrontation and pot experiments show that the fermentation broth of strain 024A has a very good control effect on white rot disease of industrial hemp.

[0083] Example 5: Growth-promoting effect of fermentation broth of strain 024A on plants.

[0084] (1) Growth-promoting experiment on industrial hemp seeds

[0085] Select plump industrial hemp seeds, disinfect the surface, soak in sterile water for 2 hours, and then soak in equal volumes of sterile water, sterile liquid culture medium, and 024A fermentation broth (OD200). 600=0.5) and 024A fermentation broth supernatant were soaked for 4 hours, with 10 seeds per treatment and 3 replicates. The seeds were placed in a 25℃ incubator for growth. Germination rate and sprout length were calculated after 3 days of growth.

[0086] The test results are shown in Table 5 and Figure 7 As shown.

[0087] Table 5. Germination rate and seedling length of industrial hemp seeds under four treatments.

[0088]

[0089] The results of the growth promotion experiment show that the fermentation broth of strain 024A can promote the germination of industrial hemp seeds and the growth of seedlings.

[0090] The fermentation broth of strain 024A was used to treat seeds and seedlings of bast fibers such as ramie, jute, flax, apocynum venetum, and hemp. The results showed that the fermentation broth had a significant growth-promoting effect on all bast fibers.

[0091] (2) Growth-promoting experiments on chili peppers

[0092] The experiment included four treatments: sterile water (CK1), sterile liquid culture medium (CK2), Bacillus 024A bacterial suspension (T1), and Bacillus 024A supernatant (T2). Bacillus was cultured at 30℃ and 180 r / min for 48 h, and the bacterial suspension was diluted to 1×10⁻⁶ with sterile water. 5 CFU / mL, centrifuged at the same volume to obtain the supernatant. 20 seeds were sown per pot using commercially available potting soil, which was sterilized twice before use. Cultivation conditions: temperature 22±1℃, daily watering to keep the soil moist. Chili seeds germinated 4-5 days after sowing, and the germination rate was recorded. After germination, each pot was maintained with 8 seedlings. Four treatments (10mL) were applied every two days, and watering was maintained at a relatively moist substrate for the remaining time, allowing the chilies to grow normally. The growth of the chilies was observed, and after 30 days, growth parameters such as plant height, stem diameter, fresh (dry) weight of the upper part, and root (dry) weight were measured.

[0093] The plant height is measured from the base of the chili plant to the top of the main stem, i.e., the growing point of the main stem. The diameter of the first node of the stem near the root node is measured as the stem diameter. The chili plant is cut off at the first node near the root node, and the upper half and the root part are weighed separately and recorded as fresh weight. Then, they are dried in a 65℃ oven until constant weight, and weighed again and recorded as dry weight.

[0094] The test results are shown in Tables 6-7 and 7-7. Figure 8-9 As shown.

[0095] Table 6. Effects of four treatments on seed germination rate and seedling growth of chili peppers.

[0096]

[0097] Table 7 shows the specific results of the increases in treatments T1 and T2 compared to CK1.

[0098]

[0099] The results of the growth-promoting experiment on chili seeds show that the fermentation broth of strain 024A can promote the germination of chili seeds and the growth of seedlings.

[0100] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0101] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis It was deposited on May 18, 2022, at the Agricultural Microbiology Center of the China Committee on Culture Collection of Microorganisms, with accession number CGMCC 1.60033.

2. A microbial inoculant containing the Bacillus berberis strain as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent includes the Bacillus vesiculus strain and / or the fermentation culture of the Bacillus vesiculus strain.

4. The microbial agent according to claim 3, characterized in that, The fermentation culture includes fermentation supernatant and bacterial cells.

5. The method for preparing the microbial inoculant according to any one of claims 2-4, characterized in that, Includes the following steps: S1. Liquid seed culture: The Bacillus berberis strain is inoculated into a seed culture medium and cultured to obtain a seed culture solution; S2. Fermentation culture: The seed culture solution is inoculated into the fermentation medium at an inoculation rate of 2-5%, and the fermentation broth is collected to obtain a liquid microbial agent.

6. The method for preparing the microbial inoculant according to claim 5, characterized in that, Before step S1, the method further includes the following steps: picking a single colony of the Bacillus belye strain and placing it in LB liquid medium, culturing it overnight, mixing it with 30% sterile glycerol at a 1:1 ratio, and storing it at a low temperature of -80°C.

7. The method for preparing the microbial inoculant according to claim 5, characterized in that, The seed culture medium is LB medium, with a culture temperature of 37°C and a culture time of 24 h; the liquid seed culture conditions are 30°C, a rotation speed of 180 r / min, a pH of 6, and a culture time of 16-24 h; and / or, the fermentation culture conditions are 30°C, a rotation speed of 180 r / min, and a fermentation time of 48 h; the fermentation medium is medium D, which consists of: 8 g beef extract, 5 g yeast extract, 10 g glucose, and 1000 mL sterile water.

8. The application of the Bacillus berberis strain of claim 1 or the microbial agent of any one of claims 2-4 in the control of white mold disease in industrial hemp or chili peppers.

9. The application of the Bacillus berberis strain of claim 1 or the microbial agent of any one of claims 2-4 in the prevention and control of at least one of the following diseases: Phytophthora in pepper, Fusarium wilt in loofah, anthracnose in pomelo, soft rot in cabbage, soft rot in konjac, black rot in radish, bacterial wilt in tobacco, and bacterial blight in rice.

10. The application of the Bacillus berberis strain of claim 1 or the microbial agent of any one of claims 2-4 in promoting the growth of industrial hemp or chili peppers.

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