Burkholderia polluting strain and application of burkholderia polluting strain in prevention and treatment of litchi diseases

Abstract

The invention discloses a Burkholderia polluting strain LZ816 and an application of the Burkholderia polluting strain LZ816 in the aspect of preventing and treating litchi diseases, and particularly discloses a Burkholderia polluting strain LZ816 and an application of the Burkholderia polluting strain LZ816. The polluted burkholderia sp. LZ816 is preserved in Guangdong Microbial Culture Collection Center on March 4, 2025, and the preservation number is GDMCC No: 65968. The bacterium and a fermentation product of the bacterium have relatively good bacteriostatic activity on peronophythora litchii and colletotrichum frutescens, and can be used for preventing and treating common diseases of litchi, such as peronophythora litchii, anthracnose of litchi and the like. The bacterium can be used for preventing and treating common diseases of litchi, such as peronophythora litchii and colletotrichum frutescens. The burkholderia sp. LZ816 disclosed by the invention can be used for preparing a novel, efficient and broad-spectrum biocontrol microbial inoculum, and the burkholderia sp. Is high in propagation speed, low in requirement on nutrition, high in colonization capability and high in application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural biocontrol, and more specifically relates to a Burkholderia contaminans strain LZ816 and its application in preventing and controlling litchi diseases. Background Art

[0002] Lychee (Litchi chinensis Sonn.) is an evergreen tree of the genus Litchi in the Sapindaceae family, native to subtropical regions, and a cultivated fruit tree. Lychee boasts unique advantages among fruits and is deeply loved by people. Historically, it has been known as the "King of Fruits," making it one of the fruits with the richest cultural heritage in China. Lychee is also a traditional Chinese medicinal herb with diverse benefits, both medicinal and edible. The Compendium of Materia Medica records that lychee nourishes the liver and spleen, regulates qi, replenishes blood, warms the middle and relieves pain, and nourishes the heart and calms the mind. It regulates qi, dissipates stagnation, relieves pain, and can also prevent diarrhea. Pharmacological studies have shown that lychee seeds have anti-viral and anti-hepatic effects. Traditional Chinese medicine (TCM) theory holds that lychee seeds have the effects of promoting qi, dispersing stagnation, dispelling cold, and relieving pain. The lychee cultural heritage is uniquely Chinese and has a global impact, making research on its preservation and development of significance.

[0003] The most serious diseases affecting the litchi industry are litchi downy mildew and litchi anthracnose. Litchi downy mildew, also known as litchi downy mildew or litchi blight, is caused by the fungus Peronophythora litchii and is the most serious disease of litchi. It is devastating, with an incidence rate exceeding 20%, and in severe cases reaching 60%, causing large amounts of fruit rot and drop. In years with prolonged spring and plum rains, fruit rot rates can reach as high as 30-50%, severely impacting yield and quality, as well as the storage and transportation of fresh fruit. Litchi anthracnose, caused by the fungus Colletotrichum spp. (the dominant species is Colletotrichum fructicola), can affect young shoots, leaves, inflorescences, branches, and fruit. Fruit nearing or reaching maturity is particularly affected, causing shoot dieback, branch dieback, leaf and flower drop, and significant fruit drop during the ripening phase. It is also a significant cause of fruit rot during storage and transportation.

[0004] Currently, litchi disease control primarily relies on the use of chemical agents. Given the potential safety risks of chemical residues and the pressure of drug resistance, the safer and more environmentally friendly biocontrol technology using microbial biocontrol bacteria is more in line with green development.

[0005] Currently, most reported biocontrol agents for litchi downy mildew and litchi anthracnose are Bacillus species. Other studies have shown that Burkholderia gladioli and Burkholderia cenocepacia also have efficacy against these diseases. Due to the varying efficacy of different biocontrol agents and the potential for degradation, continuously enriching the biocontrol agent library is of vital industrial importance. Summary of the Invention

[0006] The present invention aims to explore new biocontrol bacteria resources for preventing and controlling litchi diseases.

[0007] The present invention aims to provide a contaminating Burkholderia LZ816 and a bacterial agent thereof.

[0008] Another object of the present invention is to provide the use of the contaminating Burkholderia LZ816 and its fermentation liquid in preventing and controlling litchi diseases.

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

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

[0011] Our research group has been engaged in the research and development of natural pesticides for nearly 30 years. Driven by the need for technological advancement in litchi disease control, the project team isolated and screened a series of microorganisms from numerous soil samples from diverse regions, types, and crops, discovering several biocontrol strains with excellent antibacterial activity. Among them, Burkholderia contagiosum LZ816, isolated and identified from orchard soil, demonstrated excellent antibacterial spectrum and activity against litchi disease pathogens. Testing revealed that it exhibited significant inhibition against Peronophythora litchii and Colletotrichum fructicola (the dominant species of litchi anthracnose). Furthermore, the fermented components were shown to retain their excellent inhibitory effects, demonstrating its potential as a highly effective, broad-spectrum, and environmentally friendly microbial pesticide with promising development prospects.

[0012] Therefore, the present invention provides a strain of Burkholderia contaminans LZ816, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 4, 2025, with the deposit number GDMCCNo:65968.

[0013] The present invention also provides a bacterial agent containing the contaminating Burkholderia LZ816 and / or its fermentation liquid.

[0014] The bacterial agent may further contain a culture medium and / or a carrier.

[0015] The present invention also provides the fermentation broth of the contaminated Burkholderia LZ816.

[0016] The present invention also provides a plant disease inhibitor, wherein the active ingredient comprises the contaminating Burkholderia LZ816 or a fermentation product thereof.

[0017] The fermentation product refers to a product obtained by removing the bacteria from the fermentation liquid contaminated with Burkholderia LZ816.

[0018] Alternatively, the method for removing the bacterial cells may be: extracting the fermentation broth of LZ816 with dichloromethane, taking the organic phase (with the bacterial cells removed) as the fermentation product, and further evaporating the organic phase and dissolving it in LB liquid medium to obtain a fermentation product solution.

[0019] Optionally, the fermentation broth is obtained by inoculating the contaminating Burkholderia LZ816 into a fermentation medium (LB liquid medium) and then culturing and fermenting.

[0020] Preferably, the culture temperature is 28-35°C and the culture time is 1-5 days.

[0021] More preferably, the culture temperature is 32°C and the culture time is 2 days.

[0022] The present invention also claims the following:

[0023] Application of the contaminating Burkholderia LZ816, the bacterial agent, the fermentation broth, or the inhibitor in killing or inhibiting litchi downy mildew and / or fruit anthracnose.

[0024] Application of the contaminating Burkholderia LZ816, the bacterial agent, the fermentation liquid, or the inhibitor in preventing and controlling plant diseases caused by litchi downy mildew and / or fruit anthracnose.

[0025] Application of the contaminating Burkholderia LZ816, the bacterial agent, the fermentation liquid, or the inhibitor in preparing products for preventing and controlling plant diseases caused by litchi downy mildew and / or fruit anthracnose.

[0026] Application of the contaminating Burkholderia LZ816, the bacterial agent, the fermentation liquid, or the inhibitor in preparing a product for preventing and treating litchi downy mildew and / or litchi anthracnose.

[0027] The product can be a bacterial agent, a microbial ecological preparation, or a microbial pesticide.

[0028] Based on the above research, the present invention also provides a method for preventing and controlling plant diseases caused by litchi downy mildew and / or fruit anthracnose, which comprises applying the contaminating Burkholderia LZ816 or the bacterial agent or the fermentation liquid or the inhibitor to the plants.

[0029] In terms of the applicable scope of pathogens, the plants include litchi, and the plant diseases are litchi downy mildew and / or litchi anthracnose.

[0030] The present invention has the following beneficial effects:

[0031] The present invention provides a contaminating Burkholderia LZ816, which can prevent and control litchi downy mildew and litchi anthracnose, which are difficult to prevent and control and are caused by litchi downy mildew and / or fruit anthracnose, and has important significance for the litchi industry.

[0032] The present invention provides a new resource for biological control of related diseases. Compared with some similar strains, it has better antibacterial effects, providing strong support for the application of biological control and microbial fertilizer. This strain has broad application prospects in many aspects of biological control.

[0033] In addition, Burkholderia has a fast reproduction rate, low nutritional requirements, and strong colonization ability, showing greater application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the colony morphology of strain LZ816.

[0035] Figure 2 This is the phylogenetic tree of strain LZ816 constructed based on the 16S rRNA gene sequence.

[0036] Figure 3 The figure above shows the inhibitory effect of contaminating Burkholderia LZ816 on litchi pathogens; the figure above shows the control group, and the figure below shows the treatment group with LZ816 strain added.

[0037] Figure 4 The figure above shows the inhibitory effect of the fermentation product of contaminated Burkholderia LZ816 on litchi pathogens; the figure above shows the control group, and the figure below shows the treatment group with the fermentation product of strain LZ816 added. DETAILED DESCRIPTION

[0038] 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.

[0039] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0040] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0041] Potato Dextrose Agar (PDA): 200 g potatoes (peeled), 15 g anhydrous glucose, 20 g agar powder, and distilled water to 1 L.

[0042] Agar medium (LB solid medium): 5 g yeast powder, 10 g peptone, 10 g NaCl, 20 g agar powder, add pure water to 1000 mL, pH neutral, sterilize at 121 ° C for 20 min.

[0043] LB liquid medium: based on LB solid medium, without adding agar powder.

[0044] Example 1 Isolation and Identification of Contaminating Burkholderia LZ816

[0045] 1. Separation

[0046] Soil samples: Soil samples were collected from an orchard in Qingyuan City, Guangdong Province.

[0047] The separation method is as follows:

[0048] (1) From the soil sample, the PDA medium dilution plate method was used for separation. The process was as follows: Take five 10 mL centrifuge tubes and mark them with 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , use a pipette to draw 9 mL of sterile water and add it to each centrifuge tube;

[0049] (2) Weigh 10 g of sample, place it in a test tube filled with 90 mL of sterile water, add glass beads and place it on a shaker for 90 minutes, which is 10 -1 of soil dilution;

[0050] (3)10 -1 After shaking the soil dilution, let it rest for 10 minutes. Use a pipette to draw 1 mL of the suspension and add it to a centrifuge tube containing 9 mL of sterile water. Dilute it in sequence to make 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 diluent;

[0051] (4) Pour the sterilized PDA culture medium cooled to 50-60℃ into the culture dish, 15mL per dish; after the culture medium cools to a flat plate, use a pipette to take 10 -6 , 10 -5 , 10 -4 , 10 -3 Add 100 μL of dilution suspension to the plate, and then spread each dilution evenly with a sterile spreading stick, repeating 3 plates for each concentration;

[0052] (5) Place the culture dish upside down in a constant temperature incubator at 25°C for 3-5 days, then pick the hyphae of a single colony onto a new plate and purify.

[0053] (6) Select individual colonies of varying morphology from the LB plate. After three streaking purifications, select the pure colony (best-growing and contaminant-free) (recorded as strain LZ816) and inoculate it into LB liquid medium for 48 h. Add glycerol to the bacterial suspension and store it in a refrigerator at -20°C.

[0054] 2. Identification

[0055] (1) Morphological characteristics:

[0056] from Figure 1 It can be seen that the single colony of strain LZ816 is approximately round, slightly yellow, milky, and has a smooth colony surface.

[0057] (2) Molecular identification

[0058] After the bacterial culture was expanded, the bacterial culture was collected and sent to Sangon Biotech Co., Ltd. for 16S rRNA sequence analysis. The 16S rRNA gene sequence obtained by sequencing was submitted to the NCBI database. After selecting the BLAST comparison results, the gene family was clustered using orthoml software to screen out the common single-copy genes. The muscle tool was then used to align and align these sequences, and finally the phylogeny was constructed using phyML software (for related results, see Figure 2 ).

[0059] In summary, combined with the results of morphological and molecular identification, strain LZ816 was determined to be Burkholderia contaminans, and was deposited in the Guangdong Provincial Microbial Culture Collection (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou) on March 4, 2025, with the deposit number GDMCC NO.65968.

[0060] Example 2 Inhibitory Effect of Strain LZ816 on Litchi Disease Pathogens

[0061] Test strain LZ816's plate inhibitory effect on litchi pathogens, plate standoff test:

[0062] (1) Test bacteria

[0063] Test biocontrol bacteria: strain LZ816.

[0064] Test pathogens: Peronophythora litchii and Colletotrichum fructicola.

[0065] The above-mentioned pathogens all originated from the College of Plant Protection, South China Agricultural University / Guangdong Key Laboratory of Microbial Signaling and Crop Disease Control, and were isolated and identified by the disease prevention and control team of the National Lychee and Longan Industry Technology System from diseased lychee tissues in the main lychee producing areas of Maoming City, Guangdong Province in 2020.

[0066] (2) Test method:

[0067] The test pathogens were placed in the center of the culture dish, and the biocontrol bacteria LZ816 was inoculated in a cross-shaped symmetrical manner about 2 cm away from the pathogen block. Each group of experiments was repeated 3 times, and a control group without LZ816 inoculation was set up. The culture was kept at a constant temperature of 28°C for 5 days.

[0068] The inhibition rate of biocontrol bacteria LZ816 against pathogens was calculated as follows:

[0069] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%

[0070] Diameter of control colony: take the average of the two diagonal diameters of the control colony.

[0071] Diameter of treated colony: take the average diameter of the growth of the test pathogen between two diagonal biocontrol bacteria.

[0072] (3) Test results:

[0073] The results are shown in Table 1 and Figure 3 As shown in the figure, the inhibition rate of contaminating Burkholderia LZ816 against litchi pathogens was above 60%.

[0074] Table 1 Inhibitory effect of contaminating Burkholderia LZ816 on common diseases of litchi

[0075]

[0076] Example 3 Inhibitory Effects of Fermentation Products of Strain LZ816 on Litchi Disease Pathogens

[0077] The contaminated Burkholderia strain LZ816 was activated and streaked onto LB solid medium at 28°C for 48 hours. A single colony was then picked and transferred to LB liquid medium, shaken at 32°C and 180 rpm for 48 hours, and the fermentation broth of strain LZ816 was filtered.

[0078] The fermentation broth is then extracted with dichloromethane, and the organic phase (from which the bacteria have been removed) is evaporated to dryness to obtain the fermentation product.

[0079] The fermentation product was then prepared into different concentrations using LB liquid culture medium, and the antibacterial activity of the fermentation product against litchi pathogens was tested according to the test method of Example 2.

[0080] The test results are as follows Figure 4 Table 2 shows that at a concentration of 10 mg / L, the fermentation product of the contaminated Burkholderia strain LZ816 has an inhibition rate of more than 75% against litchi pathogens. At a concentration of 100 mg / L, the inhibition rate reaches 100%.

[0081] Table 2 Inhibitory effects of fermentation products of contaminated Burkholderia strain LZ816 on litchi diseases

[0082]

[0083] *: The measured concentration is 10 mg / L.

[0084] The invention not only provides a theoretical basis for the biological control research of litchi diseases, but also enriches the biocontrol bacteria resources and provides a strong basis for the development and application of new microbial pesticides.

[0085] 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. A strain of Burkholderia contaminans LZ816, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection on March 4, 2025, with the accession number GDMCC No: 65968.

2. A Burkholderia contamination agent, characterized in that Containing the contaminated Burkholderia LZ816 and / or its fermentation broth according to claim 1.

3. A contaminated Burkholderia fermentation broth, characterized in that: The fermentation broth of contaminated Burkholderia LZ816 according to claim 1.

4. A plant disease inhibitor, characterized in that The active ingredient comprises the contaminating Burkholderia LZ816 and / or its fermentation product according to claim 1.

5. Use of the Burkholderia contaminant LZ816 according to claim 1, the bacterial agent according to claim 2, the fermentation broth according to claim 3, or the inhibitor according to claim 4 in killing or inhibiting litchi downy mildew and / or fruit anthracnose.

6. Use of the Burkholderia contaminating LZ816 according to claim 1, the bacterial agent according to claim 2, the fermentation liquid according to claim 3, or the inhibitor according to claim 4 for preventing and controlling plant diseases caused by litchi downy mildew and / or fruit anthracnose.

7. Use of the Burkholderia contaminans LZ816 according to claim 1, the bacterial agent according to claim 2, the fermentation liquid according to claim 3, or the inhibitor according to claim 4 in the preparation of a product for preventing and controlling plant diseases caused by litchi downy mildew and / or fruit anthracnose.

8. Use of the Burkholderia sp. LZ816 of claim 1, the bacterial agent of claim 2, the fermentation broth of claim 3, or the inhibitor of claim 4 in the preparation of a product for preventing and treating litchi downy mildew and / or litchi anthracnose.

9. A method for preventing and controlling plant diseases caused by litchi downy mildew and / or fruit anthracnose, characterized in that: Applying the bacterial agent according to claim 2, the fermentation liquid according to claim 3, or the inhibitor according to claim 4 to plants.

10. The use according to claim 6 or 7 or the method according to claim 9, characterized in that The plant is litchi.

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