Application of burkholderia polluting LZ816 in prevention and treatment of fusarium and soil-borne diseases

Abstract

The invention discloses an application of burkholderia polluting LZ816 in the aspect of preventing and treating fusarium and soil-borne diseases. The contaminated Burkholderia sp. LZ816 has relatively strong inhibitory activity on Fusarium sp. Such as Fusarium pseudograminearum, Fusarium oxysporum cucumber specialization, Fusarium laminatum, Fusarium oxysporum banana specialization, Fusarium solani, Fusarium oxysporum tomato specialization, Fusarium oxysporum white gourd specialization and the like; the bacterial strain can be used for well preventing and controlling wheat basal stem rot, cucumber fusarium wilt, soybean fusarium wilt, banana fusarium wilt, strawberry root rot, tomato fusarium wilt and white gourd fusarium wilt, and has important significance and application value for agricultural production.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural biocontrol, and more specifically relates to the application of Burkholderia contagiosum LZ816 in preventing and controlling Fusarium and soil-borne diseases. Background Art

[0002] Plant diseases are an important factor affecting crop yield and quality. The vast majority of agricultural crop diseases are fungal, bacterial and some viral diseases. Their pathogens overwinter in the soil or in the soil with the help of diseased residues. When conditions are suitable, they invade crops from the roots or stems and cause diseases. They are soil-borne diseases. Among them, Fusarium sp. is widely distributed in nature and is commonly found in soil and animal and plant organisms. It can infect more than 100 plant species including food crops, cash crops, medicinal plants and ornamental plants, causing a variety of plant diseases such as wilt, root rot, stem rot, stem base rot, flower rot and ear rot, causing crops to wilt and die, affecting yield and quality. It can also produce toxins that endanger plant and human health. It is one of the most difficult important diseases to prevent and control in production. For example, wheat stem rot, cucumber wilt, soybean wilt, banana wilt, strawberry root rot, tomato wilt, and wax gourd wilt caused by false Fusarium graminearum, cucumber-specific Fusarium oxysporum, Fusarium genus, banana-specific Fusarium oxysporum, Fusarium solani, tomato-specific Fusarium oxysporum, and wax gourd-specific Fusarium oxysporum frequently occur in production, causing serious damage and resulting in severe production reduction or even crop failure.

[0003] Fusarium sp. is a typical soil-borne disease, and currently the main prevention and control measures are still chemical measures. One is soil disinfection, and the other is the use of fungicides to inhibit Fusarium infection and reproduction, or to induce plants to improve disease resistance. Common agents include oxadixyl, fludioxonil, azoxystrobin, pyraclostrobin, kasugamycin, prochloraz, amino oligosaccharides, etc. However, the use of chemical agents can easily lead to residues, soil contamination, and harm to non-target organisms. At the same time, due to inadequate drug application technology at the grassroots level, it is easy to miss the appropriate application period, resulting in poor or even ineffective prevention effects. Therefore, the development of simple, practical, and efficient prevention and control technologies is very urgent.

[0004] Using biocontrol agents to control Fusarium is a green, environmentally friendly, simple, and effective measure. In recent years, the use of bacteria such as Bacillus subtilis and Paenibacillus polymyxa has increased significantly. As a class of beneficial microorganisms, biocontrol agents (biocontrol agents) can exert their control effects through various pathways: competing with pathogens for ecological niches and nutrients; secreting antimicrobial compounds to inhibit pathogen growth; and activating plant systemic resistance mechanisms. These microorganisms offer unique advantages in ensuring healthy crop growth and are a major development direction in current disease control. Summary of the Invention

[0005] The present invention aims to explore new broad-spectrum, highly effective, environmentally adaptable biocontrol bacteria against Fusarium and related soil-borne plant diseases, especially biocontrol bacteria against difficult-to-control soil-borne diseases such as wheat stem base rot, cucumber wilt, soybean wilt, banana wilt, strawberry root rot, tomato wilt, and wax gourd wilt caused by Fusarium sp.

[0006] The purpose of the present invention is to provide the use of the contaminated Burkholderia LZ816 or its fermentation liquid or fermentation product in inhibiting Fusarium.

[0007] Another object of the present invention is to provide the use of the contaminated Burkholderia LZ816 or its fermentation liquid or fermentation product in preventing and controlling plant diseases caused by Fusarium.

[0008] Another object of the present invention is to provide a product for preventing and controlling plant diseases caused by Fusarium.

[0009] Another object of the present invention is to provide a method for preventing and controlling plant diseases caused by Fusarium.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] Extensive research has shown that Burkholderia contaminans LZ816, previously isolated and identified from orchard soil and deposited with the Guangdong Provincial Microbial Culture Collection on March 4, 2025, under the accession number GDMCC No. 65968, exhibits excellent antibacterial activity against common litchi diseases, including litchi downy mildew and litchi anthracnose. Further studies have revealed that this bacterium also exhibits excellent antifungal activity against Fusarium species, including Fusarium pseudograminearum, Fusarium oxysporum cucumber-specific, Fusarium laminarum, Fusarium oxysporum banana-specific, Fusarium solani, Fusarium oxysporum tomato-specific, and Fusarium oxysporum wax gourd-specific, which are common in crops such as wheat, cucumber, soybean, banana, strawberry, tomato, and winter melon. It was further verified that the ingredients after fermentation of the bacteria still have excellent antibacterial effects, and it has the potential to become a highly effective, broad-spectrum, and environmentally friendly microbial pesticide, with broad development prospects.

[0012] Therefore, the present invention claims protection for:

[0013] Application of contaminated Burkholderia LZ816 or its fermentation liquid or fermentation product in killing or inhibiting Fusarium sp.

[0014] Said inhibition includes the use of inhibiting the spread of Fusarium in plant growth;

[0015] Said inhibition includes the use of inhibiting Fusarium in infected plants;

[0016] The inhibition includes the use in inhibiting the germination of spores of Fusarium.

[0017] Application of contaminated Burkholderia LZ816 or its fermentation liquid or fermentation products in preventing and controlling plant diseases caused by Fusarium.

[0018] The invention relates to the use of Burkholderia LZ816 or its fermentation liquid or fermentation product in the preparation of a product for preventing and treating plant diseases caused by Fusarium. The product can be a microbial agent, a microecological preparation or a microbial pesticide.

[0019] As one of the possible implementation plans: a biocontrol agent contains contaminated Burkholderia LZ816 and / or its fermentation broth.

[0020] As one of the possible embodiments: a plant disease inhibitor, wherein the active ingredient comprises contaminating Burkholderia LZ816 and / or its fermentation product.

[0021] As one of the possible implementation plans: a product for preventing and controlling plant diseases caused by Fusarium, wherein the active ingredient comprises contaminating Burkholderia LZ816 and / or its fermentation broth and / or its fermentation product.

[0022] The contaminated Burkholderia LZ816 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with the collection number GDMCC NO.65968.

[0023] Based on the above research, the present invention also provides a method for preventing and controlling plant diseases caused by Fusarium, which comprises applying a biocontrol agent to the plant, wherein the biocontrol agent contains contaminated Burkholderia LZ816 or its fermentation liquid or fermentation product.

[0024] The fermentation product refers to a product obtained by removing the bacteria from the fermentation liquid contaminated with Burkholderia LZ816.

[0025] Alternatively, the method for removing the bacterial cells is to extract the fermentation broth of strain LZ816 with dichloromethane, and the organic phase (with the bacterial cells removed) is the fermentation product. The organic phase can also be further evaporated and then dissolved in LB liquid medium to obtain a fermentation product solution.

[0026] Optionally, the fermentation broth is obtained by inoculating LZ816 into a fermentation medium and then culturing and fermenting.

[0027] Preferably, the culture temperature is 28-35°C and the culture time is 1-5 days.

[0028] More preferably, the culture temperature is 32°C and the culture time is 2 days.

[0029] Optionally, the fermentation medium may be LB liquid medium.

[0030] In terms of the applicable scope of pathogens, in the scheme of the present invention, the Fusarium includes one or more of the following: pseudograminearum Fusarium, cucumber-specific Fusarium oxysporum, layered Fusarium, banana-specific Fusarium, solanaceous Fusarium, tomato-specific Fusarium oxysporum, and wax gourd-specific Fusarium.

[0031] The diseases caused by these pathogens include wheat stem rot, cucumber wilt, soybean wilt, banana wilt, strawberry root rot, tomato wilt, winter melon wilt, etc.

[0032] The present invention has the following beneficial effects:

[0033] The present invention provides an application of contaminating Burkholderia LZ816 in preventing and controlling wheat stem rot, cucumber wilt, soybean wilt, banana wilt, strawberry root rot, tomato wilt, and wax gourd wilt diseases that are difficult to prevent and control and are caused by pathogens such as pseudo-graminearum Fusarium, cucumber-specific Fusarium oxysporum, Fusarium laminarum, banana-specific Fusarium oxysporum, Fusarium solani, tomato-specific Fusarium oxysporum, and wax gourd-specific Fusarium oxysporum. The application has important significance for agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Colony morphology of strain LZ816 (A) and phylogenetic tree of strain LZ816 constructed based on 16SrRNA gene sequence (B).

[0035] Figure 2 This figure shows the inhibitory effect of strain LZ816 on several plant pathogens. DETAILED DESCRIPTION

[0036] The present invention is further described below with reference to specific embodiments and accompanying drawings, but the embodiments do not limit the present invention in any form.

[0037] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0039] The contaminating Burkholderia species LZ816 is described in patent application 202510745905X.

[0040] Example 1 Isolation and Identification of Contaminating Burkholderia LZ816

[0041] Strain LZ816 was isolated from a soil sample from an orchard in Qingyuan, Guangdong. Single colonies were isolated and purified using the Potato Dextrose Agar (PDA) dilution plate method. After multiple streaking purifications, the best-growing, contaminant-free colonies were selected for inoculation, culture, and storage in LB liquid medium.

[0042] The pure colonies were identified by morphology (morphological characteristics such as Figure 1 Figure A) and molecular identification (phylogenetic tree of strain LZ816 constructed based on 16SrRNA gene sequence as shown in Figure 2). Figure 1 The bacteria were identified as Burkholderia contaminans, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with the deposit number GDMCC NO.65968.

[0043] Example 2 Inhibitory Effect of Strain LZ816 on Plant Pathogenic Fungi

[0044] The plate standoff test was used to test the plate inhibitory effect of strain LZ816 on plant pathogenic fungi:

[0045] (1) Test bacteria

[0046] Biocontrol bacteria: strain LZ816.

[0047] Pathogens: Fusarium pseudograminearum, Fusarium oxysporum f.sp.cucumerinum JHOwen, Fusarium proliferatum, Fusarium oxysporum f.sp.cubense (EFSm.) Snyder & Hansen, Fusarium solani, Fusarium oxysporum f.sp.lycopersici (Sacc.) WC Snyder & H.N. Hansen, and Fusarium oxysporum f.sp.benincasae Gerlagh & Blok. Diseases caused include wheat stem rot, cucumber wilt, soybean wilt, banana wilt, strawberry root rot, tomato wilt, and winter melon wilt. All of the above-mentioned pathogens come from the National Key Laboratory of Green Pesticides of South China Agricultural University. They were isolated, purified, identified and preserved by Huang Jiguang from obviously diseased plants at the Zengcheng Teaching and Research Base of South China Agricultural University from May to October 2023.

[0048] (2) Test method:

[0049] The target pathogen was placed in the center of the culture dish and inoculated with the biocontrol strain LZ816 in a cross-shaped symmetrical manner about 2 cm away from the pathogen block. Each group of experiments was repeated three times, and a control group without LZ816 inoculation was set up. The culture was carried out at a constant temperature of 28°C for 5 days.

[0050] The growth inhibition percentage (inhibition rate) of strain LZ816 against several pathogens was calculated according to the following formula.

[0051] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%

[0052] Diameter of control colony: take the average of the two diagonal diameters of the control colony.

[0053] Diameter of treated colony: take the average diameter of the target pathogen growth between two diagonal biocontrol bacteria.

[0054] (3) Test results:

[0055] like Figure 2As shown in Table 1, strain LZ816 has good inhibitory activity against wheat stem rot pathogen, cucumber wilt pathogen, soybean wilt pathogen, banana wilt pathogen, strawberry root rot pathogen, tomato wilt pathogen, and wax gourd wilt pathogen, with inhibition rates all higher than 65%.

[0056] Table 1 Inhibitory effect of strain LZ816 on common pathogens

[0057]

[0058]

[0059] Example 3 Fermentation products of strain LZ816 and their antibacterial activity

[0060] (1) The preserved LZ816 strain was activated, and a single colony was picked and streaked onto LB solid medium for culture at 28°C for 48 h.

[0061] (2) Pick a single colony after culture in step (1) and place it in LB liquid culture medium. Shake and culture at 32°C and 180 rpm for 48 hours to obtain LZ816 strain fermentation liquid.

[0062] The fermentation broth was extracted with dichloromethane to obtain an organic phase (with the bacteria removed). The organic phase was evaporated to obtain a dry extract (i.e., the fermentation product). The fermentation product was prepared into different concentrations using LB liquid culture medium, and the antibacterial activity of the fermentation product was tested according to the test method of Example 2.

[0063] The results showed that the inhibition rate was significant at a concentration of 10 mg / L (see Table 2), and the inhibition rate was close to 100% at a concentration of 100 mg / L.

[0064] Table 2 Inhibitory effects of fermentation components of strain LZ816 on common pathogens

[0065]

[0066] *: The test concentration in the table is 10 mg / L.

[0067] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The use of contaminated Burkholderia LZ816 in killing or inhibiting Fusarium sp., wherein the contaminated Burkholderia LZ816 was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with the deposit number GDMCC NO.65968.

2. The use of a bacterial agent containing contaminated Burkholderia LZ816 in killing or inhibiting Fusarium sp., wherein the contaminated Burkholderia LZ816 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with the deposit number GDMCC NO.65968.

3. The use of fermentation broth or fermentation products of contaminated Burkholderia LZ816 in killing or inhibiting Fusarium sp., wherein the contaminated Burkholderia LZ816 was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with the deposit number GDMCC NO.65968.

4. The use of contaminated Burkholderia LZ816 or a bacterial agent containing contaminated Burkholderia LZ816 in preventing and controlling plant diseases caused by Fusarium. The contaminated Burkholderia LZ816 was deposited in the Guangdong Provincial Microbiological Culture Collection on March 4, 2025, with the deposit number GDMCC NO.65968.

5. The use of fermentation liquid or fermentation products of contaminated Burkholderia LZ816 in preventing and controlling plant diseases caused by Fusarium. The contaminated Burkholderia LZ816 was deposited in Guangdong Provincial Microbiological Culture Collection on March 4, 2025, with the deposit number GDMCC NO.65968.

6. Use of contaminated Burkholderia LZ816 or its fermentation liquid in the preparation of products for preventing and treating plant diseases caused by Fusarium. The contaminated Burkholderia LZ816 was deposited in the Guangdong Provincial Microbiological Culture Collection on March 4, 2025, with the deposit number GDMCC NO.65968.

7. The use of fermentation products of contaminated Burkholderia LZ816 in the preparation of products for preventing and treating plant diseases caused by Fusarium. The contaminated Burkholderia LZ816 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with the deposit number GDMCC NO.65968.

8. A fungicide product for Fusarium or a product for preventing and treating plant diseases caused by Fusarium, characterized in that: The active ingredient contains contaminated Burkholderia LZ816, and / or the fermentation broth of LZ816, and / or the fermentation product of LZ816; the contaminated Burkholderia LZ816 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with the deposit number GDMCC NO.65968.

9. A method for preventing and controlling plant diseases caused by Fusarium spp., characterized in that: A biocontrol agent is applied to the plant, wherein the biocontrol agent contains contaminated Burkholderia LZ816 or its fermentation liquid or fermentation product, wherein the contaminated Burkholderia LZ816 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with a deposit number of GDMCC NO.65968.

10. The use according to any one of claims 1 to 7 or the method according to claim 9, characterized in that: The Fusarium is one or more of the group consisting of pseudo-graminearum Fusarium, cucumber-specific Fusarium oxysporum, Fusarium laminarum, banana-specific Fusarium oxysporum, tomato-specific Fusarium oxysporum, and wax gourd-specific Fusarium oxysporum.

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