Salt-tolerant bacillus GZ-HJ-BHF2 and application of fungicide thereof in prevention and treatment of pepper rust and plant diseases

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a salt-tolerant bacillus GZ-HJ-BHF2 strain and application of a microbial agent of the salt-tolerant bacillus GZ-HJ-BHF2 strain in prevention and treatment of pepper rust and plant diseases. The salt-tolerant bacillus is named as GZ-HJ-BHF2 and is preserved in the China Center for Type Culture Collection on July 31, 2024, and the preservation number of the salt-tolerant bacillus is CCTCC M 20241720. The salt-tolerant bacillus GZ-HJ-BHF2 provided by the invention shows strong bacteriostatic activity, can efficiently inhibit the growth of various pathogenic bacteria such as pepper rust disease and the like, and has a remarkable prevention and treatment effect on pepper diseases and insect pests. Meanwhile, the strain also can promote the growth of pepper plants, improve the stress resistance of the plants, and significantly improve the yield and quality of pepper. In addition, the invention also optimizes the optimal culture conditions of the fermentation culture solution of the bacillus halotolerant GZ-HJ-BHF2, and ensures the high efficiency and operability of the bacillus halotolerant GZ-HJ-BHF2 as a biological control agent in wide application. The preparation method disclosed by the invention is simple and easy to implement, low in production cost and suitable for industrial production, and has wide popularization and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to an endophyte isolated from rust-infected leaves of Zanthoxylum bungeanum-halotolerant Bacillus halotolerans, and its application in the prevention and treatment of plant diseases and insect pests, especially rust, anthracnose and wilt of Zanthoxylum bungeanum. Background Art

[0002] Zanthoxylum bungeanum Maxim. is a deciduous small tree of the genus Zanthoxylum in the family Rutaceae. It is one of the important economic tree species in my country and has a wide range of uses in spices, oils and medicinal materials. However, in recent years, with global warming and the lack of scientific management technology in the cultivation of Zanthoxylum bungeanum, Zanthoxylum bungeanum trees are facing increasingly serious problems of diseases and insect pests. These problems not only seriously affect the lifespan of Zanthoxylum bungeanum trees and the quality and yield of Zanthoxylum bungeanum, but also pose a threat to the healthy and sustainable development of the Zanthoxylum bungeanum industry.

[0003] The main pests and diseases faced by pepper cultivation at present include pepper rust, anthracnose and wilt caused by Fusarium oxysporum. Pepper rust mainly harms the leaves of pepper. In the early stage of the disease, small yellow spots will appear on the front of the leaves, and then the spots will gradually expand and turn black, eventually causing the leaves to wither and fall off. It spreads rapidly in warm and humid environments, especially in summer and autumn when there is a lot of frequent rainfall. The disease is more likely to occur and is difficult to control due to its rapid spread, high reinfection rate, and easy resistance to pesticides. Anthracnose mainly harms the leaves, fruits and stems of pepper. After infection, necrotic spots will form at the affected parts. In severe cases, it will cause fruit rot. It is particularly serious in high temperature and high humidity environments and often breaks out after summer rains. Wilt disease is transmitted through the soil, infecting the root system of pepper plants, causing the plant's ducts to be blocked, unable to transport water and nutrients normally, causing the pepper plants to wither and die. Because of its strong soil adaptability and durability, it poses a great threat to the continued cultivation of pepper.

[0004] At present, the main diseases common in pepper cultivation include pepper rust, anthracnose and wilt. Pepper rust is mainly caused by Puccinia spp., which mainly harms leaves. In the early stage of the disease, small yellow spots will appear on the front of the leaves, and then the spots will expand and gradually turn black, eventually causing the leaves to wither and fall off. The disease spreads rapidly in warm and humid environments, especially in rainy environments in summer and autumn, when the disease is more likely to occur, and it is difficult to control due to its high reinfection rate and drug resistance. Anthracnose is caused by Colletotrichum spp., which harms leaves, fruits and stems. After infection, necrotic spots are formed, which can cause fruit rot in severe cases, especially in hot and humid summers. Wilt is caused by Fusarium oxysporum, which spreads through the soil, infects the roots of the plant, hinders the normal transportation of water and nutrients, and eventually causes the plant to wither and die. Because the disease has extremely strong soil adaptability and persistence, it poses a major challenge to the continued cultivation of pepper.

[0005] At present, the prevention and control of pepper pests and diseases mainly relies on chemical agents, but the long-term use of chemical agents not only accelerates the resistance of pests and diseases to the agents, but also significantly reduces non-target organisms such as natural enemies of pests, seriously damaging the ecological balance of the planting area. For example, patent CN105794806A discloses a new liquid composed of a combination of multiple pesticides (including pyrimidine, pyrimidine, natron and oxadiazine) for spraying to prevent and control pepper diseases; and patent CN102870631A introduces a method for spraying chemical agents at different growth stages of pepper to prevent and control pepper rust. However, many chemical pesticides contain heavy metals such as arsenic and cadmium, and long-term use may cause heavy metal pollution in soil and agricultural products, greatly limiting the source quality of pepper products.

[0006] Based on the above situation, there is an urgent need to develop an efficient, safe and environmentally friendly biological control method to promote the healthy growth of Zanthoxylum bungeanum plants, increase their yield and quality, improve soil ecology and alleviate environmental pollution. Summary of the invention

[0007] The purpose of the present invention is to provide a salt-tolerant Bacillus in view of the deficiencies in the prior art. The salt-tolerant Bacillus shows significant effects in preventing and controlling pepper rust, inhibiting the growth of Colletotrichum gloeosporioides and Fusarium oxysporum, and promoting the healthy and sustainable development of the pepper industry.

[0008] Another object of the present invention is to provide a salt-tolerant Bacillus agent and a preparation method thereof.

[0009] In addition, the present invention also aims to provide a preparation method of halotolerant Bacillus and its specific application in plant disease and insect pest control.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0011] 1. Provision of halotolerans: The present invention provides a halotolerans strain, specifically halotolerans (Bacillus halotolerans), named GZ-HJ-BHF2. The strain was deposited in the China Center for Type Culture Collection on July 31, 2024, with a deposit number of CCTCC M 20241720, and the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0012] 2.16S rDNA sequence: The 16S rDNA sequence of the halotolerant Bacillus of the present invention is as follows Figure 2 As shown, the classification and characteristics of the strains were confirmed.

[0013] 3. Halophilic Bacillus agent: The present invention provides a bacterial agent comprising the halophilic Bacillus GZ-HJ-BHF2, which has a significant antibacterial effect and is suitable for the prevention and treatment of various plant diseases.

[0014] 4. Preparation and fermentation of bacterial agents:

[0015] (1) Culture medium preparation: The halotolerant Bacillus is inoculated into a liquid culture medium made from waste extracts. The culture medium is mainly made from waste extracts from plant extracts or agricultural byproducts, and provides rich nutrients to support the growth and metabolism of the strain.

[0016] (2) Fermentation process: The inoculated culture medium is placed at 26-30°C and fermented on a shaker for 110-130 hours. The preferred conditions are a culture temperature of 28°C and a culture time of 120 hours. During this process, the strain proliferates in large quantities through fermentation and produces metabolites with antibacterial activity.

[0017] (3) Collection of fermentation agent: After fermentation is completed, the fermentation broth is collected to obtain a halotolerant Bacillus GZ-HJ-BHF2 agent with a concentration of (0.8-1.2)×10^8 CFU / mL.

[0018] 5. Application scope of the microbial agent: The salt-tolerant Bacillus microbial agent of the present invention can be widely used in the prevention and treatment of diseases and insect pests of Zanthoxylum bungeanum, especially in the comprehensive prevention and treatment of rust, anthracnose and wilt of Zanthoxylum bungeanum.

[0019] 6. Method for isolating the strain: The method for isolating the halotolerant Bacillus sp. GZ-HJ-BHF2 comprises the following steps:

[0020] (1) Strain screening and isolation culture: Select diseased Zanthoxylum bungeanum leaves, soak the leaves in 1%-3% sodium hypochlorite solution for 8-12 minutes, and then rinse with sterile water; then soak the leaves in 70%-80% ethanol solution for 0.8-1.2 minutes, and finally rinse with sterile water for disinfection. Cut the disinfected Zanthoxylum bungeanum leaves into pieces and place them on ordinary nutrient agar medium. The endophytes are isolated and purified by plate enrichment culture, gradient dilution and plate coating. Subsequently, the isolated endophytes are cultured in a constant temperature incubator at 25°C-30°C for 5-7 days.

[0021] (2) Strain purification: The salt-tolerant Bacillus isolated in the above step is activated in a common nutrient agar liquid culture medium to obtain purified bacteria.

[0022] (3) Strain identification: Through physiological and biochemical characteristics analysis and 16S rDNA sequence analysis, the screened strain was determined to be halotolerant Bacillus and named GZ-HJ-BHF2.

[0023] Preferably, in step (1), the concentration of the sodium hypochlorite solution is 1%-3%, and the concentration of the ethanol solution is 70%-80%.

[0024] More preferably, in step (1), the concentration of the sodium hypochlorite solution is 2%, and the concentration of the ethanol solution is 75%.

[0025] The salt-tolerant bacillus and the bacterial agent thereof of the present invention are applied to the prevention and treatment of diseases and insect pests of Zanthoxylum bungeanum.

[0026] The salt-tolerant Bacillus and the bacterial agent thereof of the present invention are applied to the prevention and treatment of rust, anthracnose and wilt of Zanthoxylum bungeanum.

[0027] The beneficial effects of the present invention are as follows:

[0028] The invention has significant effects in preventing and controlling pepper rust, inhibiting the growth of Colletotrichum gloeosporioides and Fusarium oxysporum, and also exhibits extensive antibacterial activity against other pathogens (such as root rot, Fusarium solani, ginseng rust, wheat gibberella, Alternaria tenuifolia and Rhizoctonia solani). The invention can not only promote the growth of pepper plants, but also achieve efficient disease prevention and control while ensuring the healthy and sustainable development of the pepper industry, and has broad application prospects and significant economic and social benefits. It is specifically embodied in the following aspects:

[0029] 1. Advantages of biological control: The salt-tolerant Bacillus agent of the present invention is prepared from the endophyte GZ-HJ-BHF2 separated and purified from Zanthoxylum bungeanum, and is a microbial agent. It overcomes the problems of increased drug resistance, environmental pollution, and drug residues caused by the long-term reliance on chemical agents for the control of diseases and insect pests in Zanthoxylum bungeanum on the market. As a biological control agent, GZ-HJ-BHF2 acts on pathogens through a variety of antibacterial mechanisms, reduces the development of drug resistance, and prolongs the duration of the control effect. Its agricultural enzyme is safe and residue-free after use, does not cause environmental pollution, and helps maintain the balance of the ecosystem and protect human health.

[0030] 2. Broad-spectrum antibacterial effect: The salt-tolerant Bacillus agent of the present invention has good inhibitory effect on the main pathogens of pepper such as Colletotrichum gloeosporioides and Fusarium oxysporum, as well as other pathogens such as root rot, Fusarium scaly, ginseng rust, Gibberella tritici, Alternaria tenuissima and Rhizoctonia solani through confrontation culture experiments, spraying experiments and effect evaluation. This makes the agent not only highly effective in preventing and controlling pepper rust, anthracnose and wilt, but also plays a role in the management of other crop diseases, showing a wide range of application potential.

[0031] 3. Multiple disease control: Through confrontation culture experiments and spraying test, the inhibitory effect of the salt-tolerant Bacillus GZ-HJ-BHF2 on multiple pathogens has been confirmed. It not only has a significant control effect on pepper rust, but also can effectively inhibit the growth of Colletotrichum gloeosporioides, Fusarium oxysporum and other pathogens, showing a broad spectrum of antibacterial activity, achieving comprehensive control of multiple diseases and improving overall control efficiency.

[0032] 4. Simple and efficient application: The preparation and application process of the GZ-HJ-BHF2 bacterial solution of the present invention is simple, the required equipment and materials are low in cost, the preparation and spraying operation steps of the bacterial solution are simplified, the comprehensive cost of disease prevention and control is significantly reduced, and farmers can obtain significant prevention and control effects with lower investment, which is particularly suitable for large-scale promotion and application.

[0033] 5. Comprehensive benefits: Through the separation, purification and cultivation of the halotolerant Bacillus GZ-HJ-BHF2, the present invention solves the difficult problems of preventing and controlling pepper rust, anthracnose and wilt, while achieving multiple advantages of environmental protection, safety, high efficiency, low cost and reduced drug resistance. More importantly, the present invention also has a wide range of inhibitory effects on other plant pathogens, and has a wider application prospect and significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The GZ-HJ-BHF2 strain is cultured in culture medium (left) and has morphological characteristics (right);

[0035] Figure 2This is the phylogenetic tree diagram of the GZ-HJ-BHF2 strain constructed based on 16SrDNA;

[0036] Figure 3-1 This is the control effect of GZ-HJ-BHF2 bacterial agent on pepper rust, which is the water spray control group;

[0037] Figure 3-2 This is the blank group of the control effect of GZ-HJ-BHF2 bacterial agent on Zanthoxylum bungeanum rust;

[0038] Figure 3-3 This is the control effect of GZ-HJ-BHF2 bacterial agent on pepper rust. GZ-HJ-BHF2 bacterial liquid was sprayed on group 1;

[0039] Figure 3-4 This is the control effect of GZ-HJ-BHF2 bacterial agent on pepper rust. Two groups were sprayed with GZ-HJ-BHF2 bacterial solution;

[0040] Figure 4-1 to Figure 4-8 This is the antibacterial effect diagram of GZ-HJ-BHF2 bacterial agent on Colletotrichum gloeosporioides and Fusarium oxysporum in the confrontation culture experiment. Figure 4-1 The antibacterial effect of GZ-HJ-BHF2 against Colletotrichum gloeosporioides; Figure 4-2 The antibacterial effect of GZ-HJ-BHF2 against Fusarium oxysporum; Figure 4-3 The antibacterial effect of GZ-HJ-BHF2 against root rot fungi; Figure 4-4 The antibacterial effect of GZ-HJ-BHF2 against Fusarium solani; Figure 4-5 The antibacterial effect of GZ-HJ-BHF2 against ginseng rust fungus; Figure 4-6 The antibacterial effect of GZ-HJ-BHF2 against Gibberella tataricus; Figure 4-7 The antibacterial effect of GZ-HJ-BHF2 against Alternaria microphylla; Figure 4-8 The antibacterial effect of GZ-HJ-BHF2 against Rhizoctonia solani (the left side is the blank control and the right side is the confrontation group).

[0041] Figure 5 This is the Gram staining image of GZ-HJ-BHF2 bacteria. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels. The methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0043] The present invention provides a salt-tolerant Bacillus, which is a salt-tolerant Bacillus (Bacillus halotolerans, named GZ-HJ-BHF2), and the preservation unit is China Center for Type Culture Collection, the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC M 20241720, and the preservation date is July 31, 2024. The salt-tolerant Bacillus of the present invention is isolated from a pepper leaf sample.

[0044] Example 1: Isolation and identification of halotolerant Bacillus

[0045] 1.1 Strain source

[0046] Endophytes were isolated from Zanthoxylum bungeanum leaves at a time when all old leaves had fallen off and new leaves had grown after the rust disease. The pathogen burden on the leaves during this period was low and the activity of endophytes was high, which was conducive to screening out strains with disease prevention effects.

[0047] 1.2 Disinfection of pepper leaves

[0048] Soak the leaves in 1%-3% sodium hypochlorite solution for 8-12 minutes, then rinse with sterile water 1-3 times; soak the leaves in 70%-80% ethanol solution for 0.8-1.2 minutes, then rinse with sterile water 1-3 times.

[0049] 1.3 Strain isolation and culture

[0050] Cut the disinfected Zanthoxylum bungeanum leaves into small pieces under sterile conditions and place them on a nutrient agar medium containing 0.8% to 1.2% potassium dichromate to inhibit fungal growth. Use plate enrichment culture, gradient dilution and plate coating methods to separate and purify endophytes. Use the waste extract as culture medium and culture it in a constant temperature incubator at 25℃-30℃ for 5-7 days.

[0051] 1.4 Strain identification

[0052] Observe the morphological characteristics of the colonies and conduct morphological identification. Figure 1 and Figure 5 As shown in the figure, the strain is rod-shaped, has spores, and has a round colony morphology with a convex center, neat edges, white color, smooth surface and a certain degree of transparency. Based on these morphological characteristics, the strain can be preliminarily identified as Bacillus.

[0053] A single colony was selected from the culture medium (cultured at 28°C for 6 days) for 16S gene sequencing. The sequencing results were compared by Blast on the NCBI website and a phylogenetic tree was constructed (e.g. Figure 2 The molecular identification results showed that the single colony isolated from Zanthoxylum bungeanum leaves belonged to a new type of endophytic bacteria, which was named GZ-HJ-BHF2.

[0054] Finally, it was deposited in the China Center for Type Culture Collection on July 31, 2024, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC M 20241720.

[0055] The genomic fragment of GZ-HJ-BHF2 (SEQ ID NO.1):

[0056]

[0057]

[0058] Example 2: Application of halotolerant Bacillus sp. GZ-HJ-BHF2 in plant disease and insect pest control

[0059] 2.1 GZ-HJ-BHF2 for the control of pepper rust

[0060] 2.1.1 Preparation of bacterial solution

[0061] The purified GZ-HJ-BHF2 was inoculated into the liquid culture medium of the waste extract, and cultured in a shaking incubator at 25°C-30°C for 110-130 hours to prepare a bacterial liquid. The waste extract in the liquid culture medium of the waste extract was the paste waste after water extraction and alcohol precipitation in the process of producing Huangqi granules by Guizhou Hanfang Pharmaceutical Co., Ltd.

[0062] 2.1.2 Spraying of bacterial solution

[0063] The prepared GZ-HJ-BHF2 bacterial solution was sprayed on the leaves of Zanthoxylum bungeanum infected with rust for 5 consecutive days, once a day, for a total of 5 times. The changes in the lesions were observed every day for 20 consecutive days, and the data were recorded.

[0064] 2.1.3 Effect evaluation

[0065] A control group (sprayed with sterile water only) and a treatment group (sprayed with GZ-HJ-BHF2 bacterial solution) were set up, and the changes in lesions between the two groups were compared to evaluate the control effect of GZ-HJ-BHF2 on Zanthoxylum bungeanum rust.

[0066] The experimental results are shown in Figure 3. The results show that the treatment group ( Figure 3-3 and Figure 3-4 ) had significantly fewer rust spots and the disease index was significantly lower than that of the control group ( Figure 3-1 and Figure 3-2 ), indicating that GZ-HJ-BHF2 has a good control effect on Zanthoxylum bungeanum rust.

[0067] 2.2 Inhibition of GZ-HJ-BHF2 on Colletotrichum gloeosporioides and Fusarium oxysporum

[0068] 2.2.1 Confrontation training experiment

[0069] The inhibitory effect of GZ-HJ-BHF2 on Colletotrichum gloeosporioides and Fusarium oxysporum was evaluated by confrontation culture method.

[0070] Colletotrichum gloeosporioides or Fusarium oxysporum was inoculated in the center of the PDA medium.

[0071] GZ-HJ-BHF2 was inoculated around the pathogens, cultured at 28°C, and the diameter of the inhibition zone was observed and measured.

[0072] 2.2.2 Evaluation of antibacterial effect

[0073] The diameters of the inhibition zones formed by GZ-HJ-BHF2 against Colletotrichum gloeosporioides and Fusarium oxysporum in the confrontation culture experiment were recorded and compared to evaluate its antibacterial effect.

[0074] The experimental results are shown in Figure 4-1 to Figure 4-8 The results showed that GZ-HJ-BHF2 had a certain antibacterial effect on Colletotrichum gloeosporium, Fusarium oxysporum, root rot, Fusarium scaly, ginseng rust, Gibberella tritici, Alternaria tenuissima, and Rhizoctonia solani. GZ-HJ-BHF2 formed obvious inhibition zones against Colletotrichum gloeosporium and Fusarium oxysporum, and the diameter of the inhibition zone was significantly larger than that of the control group, indicating that GZ-HJ-BHF2 had a good inhibitory effect on Colletotrichum gloeosporium and Fusarium oxysporum.

[0075] In summary, through the confrontation culture experiment and the spraying bacterial liquid test, it is verified that the salt-tolerant Bacillus GZ-HJ-BHF2 of the present invention has an inhibitory effect on a variety of pathogens, not only has a significant control effect on pepper rust, but can also effectively inhibit the growth of Colletotrichum gloeosporioides and Fusarium oxysporum, showing a broad-spectrum antibacterial activity, achieving comprehensive prevention and control of multiple diseases, and improving the prevention and control efficiency. Provides an environmentally friendly and efficient biological control solution for agricultural production.

Claims

1. A halotolerant Bacillus, named halotolerant Bacillus GZ-HJ-BHF2, characterized in that The salt-tolerant Bacillus strain was isolated from leaves suffering from pepper rust. The salt-tolerant Bacillus strain was deposited in the China Center for Type Culture Collection, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC M 20241720 and a deposit date of July 31, 2024.

2. A halotolerant Bacillus according to claim 1, characterized in that: The sequence of the 16S rDNA of the halotolerant Bacillus is shown in FIG2 .

3. A bacterial agent containing the halotolerant Bacillus GZ-HJ-BHF2 according to claim 1.

4. The method for preparing a bacterial agent containing halotolerant Bacillus GZ-HJ-BHF2 according to claim 3, characterized in that: The salt-tolerant bacillus is inoculated into a liquid culture medium made from waste extracts, and cultured in a shaking table at 26-30° C. for 110-130 hours to obtain the product.

5. The preparation method according to claim 4, characterized in that: The culture temperature was 28°C and the culture was carried out on a shaking incubator for 120 h.

6. The halotolerant Bacillus agent according to any one of claims 3 to 5, characterized in that In the halotolerant Bacillus agent, the concentration of halotolerant Bacillus GZ-HJ-BHF2 is (0.8-1.2)×10^8 CFU / mL.

7. A method for preparing a halotolerant Bacillus strain as claimed in any one of claims 1 to 6, comprising the following steps: (1) strain screening and isolation culture: select pepper leaves with rust, soak the leaves in sodium hypochlorite solution for 8 to 12 minutes, then rinse with sterile water, soak the leaves in ethanol solution for 0.8 to 1.2 minutes, and then rinse with sterile water for disinfection; cut the disinfected pepper leaves into pieces, place them on a nutrient agar medium containing 0.8% to 1.2% potassium dichromate, and separate and purify the endophytes by plate enrichment culture, gradient dilution and plate coating; then use the waste extract as culture medium and culture in a constant temperature incubator at 25° C. to 30° C. for 5 to 7 days; (2) strain purification: activating the halotolerant Bacillus obtained in step (1) in a nutrient agar liquid culture medium to obtain halotolerant Bacillus cells; (3) Strain identification: Through physiological and biochemical characteristics analysis and 16S rDNA sequence analysis, the screened strain was determined to be Bacillus halotolerans (named GZ-HJ-BHF2).

8. The method according to claim 7, characterized in that In the step (1), the concentration of the sodium hypochlorite solution is 1%-3%, and the concentration of the ethanol solution is 70%-80%.

9. A salt-tolerant Bacillus agent according to claim 8, characterized in that: In the step (1), the concentration of the sodium hypochlorite solution is 2%, and the concentration of the ethanol solution is 75%.

10. Use of the halotolerant Bacillus agent according to any one of claims 1 to 9 in the prevention and control of diseases and insect pests of Zanthoxylum bungeanum, wherein the diseases and insect pests of Zanthoxylum bungeanum include Zanthoxylum bungeanum rust, anthracnose and wilt.

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