Bacillus velezensis T16-2 and application thereof in plant disease control

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to bacillus velezensis T16-2 and application of the bacillus velezensis T16-2 in plant disease control, the bacillus velezensis T16-2 is obtained by separating and purifying faeces of healthy white-head leafy monkeys and is classified and named as bacillus velezensis T16-2, and the bacillus velezensis T16-2 is named as bacillus velezensis T16-2. The strain is preserved in Guangdong Microbial Culture Collection Center on June 25, 2025, and the preservation number is GDMCC 66590. The T16-2 strain provided by the invention has a very high inhibition effect on pathogenic bacteria such as momordica grosvenori spot blight, phyllosticta and root rot, the inhibition rate can reach 74% or above, and the T16-2 strain is a safe potential biocontrol bacterium and has a good application prospect in crop disease control.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus Velezii T16-2 and application thereof in preventing and controlling plant diseases. Background Art

[0002] Leaf spot disease is a new, recently discovered disease that severely impacts the growth of monk fruit. In Guilin's main monk fruit-producing areas, the incidence rate is as high as 84%. Affected plants develop sunken spots on their leaves early on, followed by leaf wilt and plant death, severely impacting the fruit's growth. According to farmer feedback, while recommended chemical agents such as meconazole, tebuconazole, and prochloraz have somewhat controlled the disease, they require increasing concentrations and frequency of application annually. Some severely affected orchards have begun to develop resistance. Therefore, developing biological control measures for leaf spot disease is of great research significance.

[0003] Monk fruit root rot is a common disease that primarily attacks the roots and base of the stem, severely impacting plant growth and yield. Caused by the fungus Fusarium solani, root rot symptoms include rough, cracked epidermis at the stem base, browning of the vascular bundles, wilting of leaves, and plant death. Currently, fungicides such as chlorobromoisocyanuric acid are the primary preventive and treatment options for root rot in monk fruit. For severe cases, root drench with benomyl and quinoline copper can be used.

[0004] Luo Han Guo leaf spot is caused by Phyllosticta serrata, a fungal disease of the Cucurbitaceae family. This fungal disease is prone to persistent rainy weather, such as in April-May and July-August. The pathogen enters through wounds or natural openings such as stomata. It typically affects leaves, stems, and fruit. In severe cases, leaves may partially die, and harvested fruit may develop spots and rot. It can be controlled by spraying with a prochloraz aqueous solution or a difenoconazole aqueous dispersible solution combined with a pyraclostrobin suspension.

[0005] At present, although chemical fungicides still dominate the prevention and control of plant diseases, Bacillus biocontrol agents are gradually gaining attention due to their advantages such as high efficiency, strong specificity, non-toxicity and pollution-free, and are the new biocontrol bacteria with the most development potential. Summary of the Invention

[0006] In response to the above problems, the present invention provides a strain of Bacillus Velezii T16-2 and its application in plant disease prevention and control. The Bacillus Velezii T16-2 has a high inhibitory effect on pathogens such as Momordica grosvenori leaf spot, Leaf spot mold, root rot fungus, and Cuban specialization of banana Fusarium oxysporum, with an inhibition rate of more than 74%. The strain is a safe potential biocontrol bacterium and has good application prospects in crop disease prevention and control.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] Bacillus velezensis strain T16-2 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the accession number GDMCC 66590 and the deposit date of June 25, 2025.

[0009] The present invention also includes a bacterial solution containing the Bacillus velezensis T16-2 strain.

[0010] The present invention also includes the Bacillus velezensis T16-2 strain or a fermentation liquid obtained by fermenting the bacterial liquid.

[0011] The present invention also includes the use of the Bacillus velezensis T16-2, the bacterial liquid or the fermentation liquid in inhibiting the growth of Stagonosporopsis cucurbitacearum.

[0012] The present invention also includes the use of the Bacillus velezensis T16-2, the bacterial solution or the fermentation solution in inhibiting the growth of Phyllosticta cucurbitacearum.

[0013] The present invention also includes the use of the Bacillus velezensis T16-2, the bacterial liquid or the fermentation liquid in inhibiting the growth of Fusarium oxysporum, a root rot fungus.

[0014] The present invention also includes the use of the Bacillus velezensis T16-2, the bacterial liquid or the fermentation liquid in inhibiting the growth of Fusarium oxysporum, a root rot fungus.

[0015] The present invention also includes the use of the Bacillus velezensis T16-2, the bacterial liquid or the fermentation liquid in preventing and treating Momordica grosvenori leaf spot, Momordica grosvenori root rot and Momordica grosvenori leaf spot.

[0016] The present invention also includes the use of the Bacillus velezensis strain T16-2, the bacterial liquid or the fermentation liquid in preparing biological pesticides.

[0017] Preferably, the method for inhibiting the growth of Phyllosticta cucurbitacearum is: spraying a bacterial solution or fermentation liquid containing the Bacillus velezensis T16-2 strain on plant leaves.

[0018] By adopting the above technical solution, the beneficial effects of the present invention are:

[0019] The present invention finds through experiments that in a PDA culture medium inoculated with a single colony of T16-2, pathogens such as mogrosanthes leaf spot pathogen, phyllosporin, root rot pathogen, and the Cuban-specific pathogen of Fusarium oxysporum of banana grow slowly, indicating that Bacillus Velezii T16-2 has a high inhibitory effect on pathogens such as mogrosanthes leaf spot pathogen, phyllosporin, root rot pathogen, and the Cuban-specific pathogen of Fusarium oxysporum of banana, with an inhibition rate of more than 74%. The present invention is a safe potential biocontrol bacterium and has good application prospects in the prevention and control of crop diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a phylogenetic tree diagram of the Bacillus velez T16-2 of the present invention;

[0021] Figure 2 This is a diagram showing the effect of an experiment on the inhibition of Momordica grosvenori leaf spot by the Bacillus Velezii T16-2 of the present invention;

[0022] Figure 3 This is a diagram showing the effect of an experiment on the inhibition of Phyllosticta by Bacillus Velezii T16-2 of the present invention;

[0023] Figure 4 This is a diagram showing the effect of an experiment on the inhibition of root rot pathogens by the Bacillus Velez T16-2 of the present invention;

[0024] Figure 5 This is a diagram showing the effect of an experiment on the inhibition of the banana Fusarium oxysporum strain T16-2 by the present invention. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Example 1

[0027] Isolation, Screening and Identification of Bacillus velezensis T16-2

[0028] A cellulase-producing strain, T16-2, was isolated from the feces of healthy white-headed langurs using selective culture containing 1% sodium carboxymethylcellulose as the sole carbon source and Congo red staining. The strain, designated Bacillus velezensis T16-2, was inoculated into a culture medium containing sodium carboxymethylcellulose as the sole carbon source and cultured at 37°C and 200 rpm for 72 hours to obtain a fermentation broth. Two milliliters of the fermentation broth was centrifuged at 6000 rpm for 10 minutes, and the supernatant was used as a crude enzyme solution. The CMCA enzyme activity of strain T16-2 in the crude enzyme solution was determined by DNS analysis to be 4.81 U / mL.

[0029] The colony morphology of strain T16-2 is milky white, translucent, round colonies with a smooth surface, neat edges, a milky white circle in the middle, and a viscous texture. Gram staining of strain 16-2 under an oil immersion microscope revealed a purple-red color, indicating a Gram-positive bacterium with rod-shaped cells. Some cells aggregate in a palisade arrangement or overlap to form long chains. Spores are terminal or subcentral and do not swell. Both the catalase test and the starch test were positive.

[0030] The genomic DNA of the strain was extracted using the Ezup column-based bacterial genomic DNA extraction kit. The 16S rRNA gene was amplified using universal primers 27F and 1492R. The fragment size and purity of the PCR amplification product were checked by 1% agarose gel electrophoresis. After the fragments met the sequencing requirements, bidirectional sequencing was performed. The 16S rRNA gene sequences obtained by sequencing were used to construct a phylogenetic tree using the Neighbor-Joining method of MEGA6.0 software, as shown in the following figure: Figure 1 shown.

[0031] The strain, Bacillus velezensis T16-2, is antibiotic-sensitive, showing sensitivity to tetracycline, rifampicin, ofloxacin, cefoperazone, vancomycin, gentamicin, and kanamycin. Table 1 shows the results of drug susceptibility testing, demonstrating that the strain is biosafe. When used as a biocontrol agent, the strain can prevent the transfer of drug-resistance genes to pathogens or environmental microorganisms, reducing the risk of the emergence of "superbugs."

[0032] Table 1 Results of drug sensitivity test of strain T16-2 to 7 antibiotics

[0033]

[0034] Example 2

[0035] Identification of the inhibitory effect of Bacillus velezensis T16-2 on Momordica grosvenori leaf spot pathogen.

[0036] The pathogenic bacteria of Momordica grosvenori, Stagonosporopsis cucurbitacearum (deposited and provided by the Guangxi Institute of Botany, Chinese Academy of Sciences, Guangxi Zhuang Autonomous Region) was inoculated into the center of a PDA plate and cultured for 24 h. A single colony of Bacillus velezensis T16-2 of Example 1 of the present invention was symmetrically inoculated into the PDA plate in a quadrilateral shape, 25 mm from the center, with three replicates and a blank control. After culture at 28° C. for 7 d, the colony diameters were measured by the cross method, and the inhibition rate was calculated.

[0037] Inhibition rate = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%

[0038] The results are shown in Table 2 and Figure 2 (Left: control, right: inhibition effect). In blank PDA culture, the pathogen grew rapidly, with colonies covering the entire plate (90 mm in diameter) after 7 days of growth. However, in PDA culture medium inoculated with a single T16-2 colony, the pathogen grew slowly, with a colony diameter of 27.67 ± 2.42 mm, and the calculated inhibition rate was 74.21 ± 1.22%.

[0039] When used in a potted biocontrol test, the method for inhibiting the growth of the Momordica grosvenori leaf spot pathogen Stagonosporopsis cucurbitacearum by the Bacillus velezensis T16-2 is as follows: spraying a bacterial solution or fermentation liquid containing the Bacillus velezensis T16-2 on the leaves of Momordica grosvenori.

[0040] Example 3

[0041] Identification of the inhibitory effect of Bacillus velezensis T16-2 on Momordica grosvenori leaf spot pathogen.

[0042] Phyllosticta cucurbitacearum (deposited and provided by the Guangxi Institute of Botany, Chinese Academy of Sciences) was inoculated into the center of a PDA plate and cultured for 24 h. A single colony of Bacillus velezensis T16-2 from Example 1 of the present invention was symmetrically inoculated into the PDA plate in a quadrilateral shape, 25 mm from the center. Three replicates and a blank control were set. After culture at 28°C for 7 days, the colony diameters were measured using the cross-hatch method, and the inhibition rate was calculated.

[0043] Inhibition rate = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%

[0044] The results are shown in Table 2 and Figure 3 (Left: control, right: inhibitory effect). In blank PDA culture, the pathogen grew rapidly, with colonies covering the entire plate (90 mm in diameter) after 7 days of growth. However, in PDA culture medium inoculated with a single T16-2 colony, the pathogen grew slowly, with a colony diameter of 12.25 ± 1.46 mm, and the calculated inhibition rate was 92.56 ± 2.55%.

[0045] When used in a potted biocontrol test, the method for inhibiting the growth of Phyllosticta cucurbitacearum by using the Bacillus velezensis T16-2 is as follows: spraying a bacterial solution or fermentation liquid containing the Bacillus velezensis T16-2 on the leaves of Momordica grosvenori.

[0046] Example 4

[0047] Identification of the inhibitory effect of Bacillus velezensis T16-2 on root rot pathogens.

[0048] Fusarium oxysporum (preserved and provided by the Guangxi Institute of Botany, Chinese Academy of Sciences) was inoculated into the center of a PDA plate and cultured for 24 h. A single colony of Bacillus velezensis T16-2 of Example 1 of the present invention was symmetrically inoculated into the PDA plate in a quadrilateral shape, 25 mm from the center. Three replicates and a blank control were set. After culture at 28°C for 7 days, the colony diameters were measured by the cross method, and the inhibition rate was calculated.

[0049] Inhibition rate = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%

[0050] The results are shown in Table 2 and Figure 4 (Left: control, right: inhibitory effect). In blank PDA culture, the root rot pathogen grew rapidly, with colonies covering the entire plate (90 mm in diameter) after 7 days of growth. However, in PDA culture medium inoculated with a single T16-2 colony, the pathogen grew slowly, with a colony diameter of 17.50 ± 1.56 mm, and the calculated inhibition rate was 84.76 ± 2.71%.

[0051] When used in a potted biocontrol test, the method for inhibiting the growth of root rot Fusarium oxysporum by the Bacillus velezensis T16-2 is as follows: a bacterial solution or fermentation liquid containing the Bacillus velezensis T16-2 is poured along the roots of Momordica grosvenori seedlings.

[0052] Example 5

[0053] Identification of the inhibitory effect of Bacillus velez T16-2 on the Cuban pathogen of Fusarium oxysporum.

[0054] Fusarium oxysporum f.sp. Cubense (deposited and provided by the Guangxi Institute of Botany, Chinese Academy of Sciences) was inoculated into the center of a PDA plate and cultured for 24 h. A single colony of Bacillus velezensis T16-2 from Example 1 of the present invention was symmetrically inoculated into the PDA plate in a quadrilateral pattern, 25 mm from the center. Three replicates and a blank control were set up. After culture at 28°C for 7 days, the colony diameters were measured using the cross-hatch method, and the inhibition rate was calculated.

[0055] Inhibition rate = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%

[0056] The results are shown in Table 2 and Figure 5 (Left is the control, right is the inhibition effect) As shown in the blank PDA culture, the banana Fusarium oxysporum Cuban special type pathogen grew rapidly, and the colonies after 7 days of growth covered the entire plate (diameter 90 mm). However, in the PDA culture medium with a single colony of T16-2, the banana Fusarium oxysporum Cuban special type pathogen grew slowly, with a colony diameter of 10.75±0.25 mm, and the calculated inhibition rate was 94.35±2.06%.

[0057] Table 2 Inhibitory effect of Bacillus velezensis T16-2 on plant pathogens

[0058]

[0059] Based on the above experimental results, the Bacillus velezensis T16-2 strain can be made into a bacterial liquid or fermentation liquid or a corresponding biological pesticide and applied to biological control.

[0060] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. Bacillus velezensis T16-2, characterized in that The deposit number of the strain is GDMCC NO.66590.

2. A bacterial solution comprising the Bacillus velezensis T16-2 strain according to claim 1.

3. A fermentation liquid comprising the Bacillus velezensis T16-2 strain according to claim 1 or the fermentation liquid obtained by fermenting the bacterial liquid according to claim 2.

4. Use of the Bacillus velezensis T16-2 according to claim 1, the bacterial solution according to claim 2, or the fermentation liquid according to claim 3 in inhibiting the growth of Stagonosporopsis cucurbitacearum.

5. Use of the Bacillus velezensis T16-2 according to claim 1, the bacterial solution according to claim 2, or the fermentation liquid according to claim 3 in inhibiting the growth of Phyllosticta cucurbitacearum.

6. Use of the Bacillus velezensis T16-2 according to claim 1, the bacterial solution according to claim 2, or the fermentation liquid according to claim 3 in inhibiting the growth of Fusarium oxysporum, a root rot fungus.

7. Use of the Bacillus velezensis T16-2 strain according to claim 1, the bacterial solution according to claim 2, or the fermentation liquid according to claim 3 in inhibiting the growth of Fusarium oxysporum f. sp. Cubense.

8. Use of the Bacillus velezensis T16-2 according to claim 1, the bacterial solution according to claim 2, or the fermentation liquid according to claim 3 in preventing and treating Momordica grosvenori leaf spot, Momordica grosvenori root rot, and Momordica grosvenori leaf spot.

9. Use of the Bacillus velezensis T16-2 strain according to claim 1, the bacterial solution according to claim 2, or the fermentation liquid according to claim 3 in the preparation of biopesticides.

10. The use according to claim 5, characterized in that The method for inhibiting the growth of Phyllostictacucurbitacearum is as follows: spraying a bacterial solution or fermentation solution containing the Bacillus velezensis T16-2 strain on the leaves of plants.

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