A strain of *Vitis vinifera* and its application

Abstract

This invention discloses a strain of *Albifimbria* sp. and its applications, belonging to the field of microbial technology. The strain, *Albifimbria* sp. (strain number JH96), is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231398. It was isolated from a soil sample of a lettuce field at the Shanghai Chunchang Vegetable and Fruit Professional Cooperative in Qingpu District, Shanghai. This *Albifimbria* sp. JH96 exhibits broad-spectrum antagonistic activity against fungal pathogens, inhibiting the growth of various plant pathogenic fungi, particularly suppressing sclerotium germination and mycelial growth of *Sclerotinia sclerotiorum*. It shows promising application prospects in the prevention and control of plant diseases such as sclerotinia rot in lettuce.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Varicospora verrucae* and its applications. Background Technology

[0002] *Sclerotinia sclerotiorum* is a plant pathogenic fungus with a wide host range, including plants in the Brassicaceae, Fabaceae, Solanaceae, and Asteraceae families. It easily causes sclerotinia rot in plants, significantly impacting crop production. *Sclerotinia sclerotiorum* primarily overwinters as sclerotia in diseased plant debris and soil, becoming the main primary source of infection for sclerotinia rot the following year. The sclerotia are highly resilient, surviving for many years in the soil environment. Under suitable conditions, overwintering sclerotia germinate, primarily producing apothecia, from which ascospores are dispersed by wind. These ascospores land on leaves, mainly infecting senescent or injured leaves, and then the lesions spread to healthy leaves, causing severe sclerotinia rot.

[0003] Lettuce, a variety of lettuce, is a common and popular vegetable, prevalent in both southern and northern regions. Sclerotinia stem rot in lettuce typically occurs at the base of the leaf and stem, leading to rot of the leaves, root crown, or even the death of the entire plant. After infection by the pathogen, numerous white mycelia are produced at the infected site, followed by the formation of numerous black sclerotia. The incidence of sclerotinia stem rot in lettuce is usually between 20% and 50%, resulting in a 20% to 40% reduction in yield. In severely affected areas, it can even lead to total crop failure, causing significant economic losses.

[0004] Due to the strong resistance of black sclerotia, sclerotinia rot has become one of the most difficult diseases to control in production. For a long time, chemical control has been the primary method for controlling plant diseases caused by *Sclerotinia sclerotiorum*. For example, Chinese patent document CN104322496A discloses a pesticide composition containing pyraclostrobin and propanemid. The combination of pyraclostrobin and propanemid has a significant synergistic effect, making it particularly suitable for controlling sclerotinia rot caused by pathogens of the *Sclerotinia sclerotiorum* genus.

[0005] However, the long-term use of chemical fungicides leads to increased drug resistance in pathogens and also causes environmental pollution. To reduce the use of chemical pesticides, biological control has been widely researched and applied as an alternative. Compared with chemical control methods, biological control methods have advantages such as longer effective duration, less environmental pollution, and better safety. The development and application of biocontrol bacteria will provide new strategies for controlling sclerotinia stem rot and promote the development of biological control technology.

[0006] Currently, research on the biological control of sclerotinia stem rot mainly focuses on microbial species such as Coniothyrium minitans, Aspergillus spp., and bacteria. However, in-depth and continuous exploration of highly efficient biocontrol strains and screening of biocontrol strains adapted to local soil environments remain the key to developing new biocontrol products and effectively controlling the disease. Summary of the Invention

[0007] This invention provides a strain of *Lysimachia verrucae* that has broad-spectrum antagonistic activity against fungal pathogens, and can inhibit the growth of various plant pathogenic fungi, especially inhibiting the sclerotium germination and mycelial growth of *Sclerotinia sclerotiorum*. It has good application prospects in the prevention and control of plant diseases such as sclerotinia rot in lettuce.

[0008] The specific technical solution adopted is as follows:

[0009] A strain of Albifimbria sp., classified as JH96, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20231398 and deposited on August 9, 2023.

[0010] The classification and nomenclature of this *Epipremnum aureum* shall be based on the nomenclature stated in the preservation certificate.

[0011] The *Epithecus verrucosum* strain was isolated from soil samples of lettuce fields at the Shanghai Chunchang Vegetable and Fruit Professional Cooperative in Qingpu District, Shanghai. Based on morphological observation and phylogenetic analysis of ITS rDNA, β-tubulin rDNA, and rpb2 rDNA site sequences, the isolate JH96 was identified as *Epithecus verrucosum*.

[0012] The ITS rDNA gene sequence of *Varicospora verrucae* is shown in SEQ ID NO.1, the β-tubulin rDNA gene sequence is shown in SEQ ID NO.2, and the rpb2 rDNA gene sequence is shown in SEQ ID NO.3.

[0013] Experiments have shown that the aforementioned *Lysimachia verrucae* has broad-spectrum antagonistic activity against fungal pathogens and can inhibit the growth of various plant pathogenic fungi.

[0014] The present invention also provides the application of the aforementioned *Lycium verrucosum* in inhibiting plant pathogenic fungi, and its application in preventing and controlling plant diseases caused by plant pathogenic fungi, wherein the plant pathogenic fungi include *Sclerotinias clerotiorum*, *Fusarium oxysporum* (isolated from rice), *Fusarium oxysporum* (isolated from soybean), *Phytophthora capsici*, *Alternaria alstroemeriae*, or *Curvularia subpapendorfii*.

[0015] The plant diseases mentioned include sclerotinia stem rot in lettuce, etc.

[0016] The present invention also provides the application of the aforementioned *Varicospora verrucae* in the preparation of biocontrol agents.

[0017] The present invention also provides a biocontrol agent, the active ingredient of which includes the aforementioned *Varicospora verrucae*.

[0018] Preferably, after inoculating the *Lactobacillus verrucae* into a fermentation medium and culturing it, the fermentation broth is filtered to obtain a filtrate, which is then extracted to obtain the biocontrol agent; or, after inoculating the *Lactobacillus verrucae* into a fermentation medium and culturing it, the fermentation broth is filtered to obtain a filtrate, which is then extracted and rotary evaporated to obtain the biocontrol agent.

[0019] Specifically, the biocontrol agent is diluted with a solvent and sprayed onto the roots or leaves of plants to prevent plant diseases. The solvent is ethyl acetate, methanol, or water, and the plants include lettuce or romaine lettuce, etc.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The *Lycium verrucosum* JH96 screened in this invention can significantly inhibit the growth of *Sclerotium sclerotiorum* hyphae and the germination of its sclerotia. It also has broad-spectrum antagonistic activity against fungal pathogens. It also has good inhibitory effects on plant pathogens such as *Polytrichum heterotrichum*, *Rhizoctonia solani*, *Fusarium oxysporum*, *Phytophthora indicum*, *Alternaria hexandrum*, or *Curvaria*. It can be used to prevent and control plant diseases caused by various plant pathogens and has good application prospects.

[0022] (2) The active ingredient provided by the present invention includes a biocontrol agent of *Lycium verrucosum* JH96, which has a good control effect on sclerotinia rot of detached lettuce leaves, with a control efficacy of 87.88%. Attached Figure Description

[0023] Figure 1This is a colony morphology diagram of *Epithecus verrucae* JH96.

[0024] Figure 2 Phylogenetic tree of *Varicospora var. var.* JH96.

[0025] Figure 3 The figure shows the results of a plate confrontation experiment between *Lysimachia verrucae* JH96 and *Sclerotinia sclerotiorum*, where A is the control group and B is the experimental group.

[0026] Figure 4 The graph shows the inhibitory effect of the biocontrol agent on the mycelial growth of Sclerotinia sclerotiorum. In the graph, A is the control group without the biocontrol agent and B is the experimental group with the biocontrol agent in Example 4 added.

[0027] Figure 5 The graph shows the inhibitory effect of the biocontrol agent on the germination of Sclerotium sclerotia. In the graph, A is the control group without the biocontrol agent and B is the experimental group with the biocontrol agent in Example 4.

[0028] Figure 6 The diagram shows the control effect of biocontrol agents on sclerotinia stem rot in lettuce. In the diagram, A is the control group sprayed with sterile water, and B is the experimental group sprayed with ethyl acetate extract from Example 4.

[0029] Figure 7 This image shows the results of a plate confrontation experiment between *Lysimachia christinae* JH96 and six plant pathogenic fungi. A1 is *Pestalotiopsis microspora*, A2 is *Fusarium oxysporum* (isolated from rice), A3 is *Fusarium oxysporum* (isolated from soybean), A4 is *Phytophthora capsici*, A5 is *Alternaria alstroemeriae*, and A6 is a *Curvularia subpapendorfii* pathogen.

[0030] Figure 8 The diagram shows the inhibitory effects of biocontrol agents on the mycelial growth of six plant pathogenic fungi. Among them, A1 is *Pestalotiopsis microspora*, A2 is *Fusarium oxysporum* (isolated from rice), A3 is *Fusarium oxysporum* (isolated from soybean), A4 is *Phytophthora capsici*, A5 is *Alternaria alstroemeriae*, and A6 is a pathogenic fungus of the genus *Curvularia*. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] The culture media involved in the following examples include:

[0033] Potato glucose agar medium (PDA medium): 200g potato, 20g glucose, 12g agar powder, 1L deionized water, sterilize at 121℃ for 20min.

[0034] Potato glucose culture medium (PDB culture medium): 200g potatoes, 20g glucose, 1L deionized water, sterilized at 121℃ for 20min.

[0035] Example 1: Soil Sample Collection and Isolation of Biocontrol Bacteria

[0036] Soil samples were collected in February 2023 from the lettuce field of Shanghai Chunchang Vegetable and Fruit Professional Cooperative in Qingpu District, Shanghai.

[0037] 1g of collected soil sample was added to 10mL of 0.7mol / L NaCl solution. The solution was shaken for 1min to prepare a soil suspension. The soil suspension was then diluted to 10 mL with 0.7mol / L NaCl solution. -2 10 -3 and 10 -4 Gradients were used to screen antagonistic fungi. 100 μL of diluted soil suspension was evenly spread onto PDA plates containing double the amount of antibiotics, with three plates spread for each gradient. After the PDA plates dried, they were incubated at 25°C. Every 24 hours, the presence of new single colonies was observed. Hyphae from newly grown single colonies were picked and inoculated onto PDA plates for purification (hyphae from the colony edge were picked and subcultured three times). The purified strains obtained after three subcultures were stored at 4°C for later use.

[0038] Using a 6mm inner diameter punch, holes were made at the edge of the obtained purified bacterial colonies. The bacterial discs were then transferred to the edge of a 90mm PDA plate. Activated *Sclerotinia sclerotiorum* bacterial discs of equal diameter were transferred to the other end of the plate. A PDA plate with one side inoculated with *Sclerotinia sclerotiorum* bacterial discs of equal diameter, while the other end remained untreated, served as a control. The experimental and control plates were then incubated at 25℃. After the mycelia in the control group had completely covered the plate, the width of the inhibition zone in the experimental group was observed, and the inhibition rate of different strains on *Sclerotinia sclerotiorum* mycelial growth was calculated. Mycelial growth inhibition rate (%) = [(*Sclerotinia sclerotiorum* colony diameter in the control group - *Sclerotinia sclerotiorum* colony diameter in the experimental group) / *Sclerotinia sclerotiorum* colony diameter in the control group] × 100%. One biocontrol bacterium with good antagonistic effect against *Sclerotinia sclerotiorum* was selected from the above lettuce field soil samples.

[0039] Example 2 Identification of biocontrol bacteria

[0040] I. Morphological Observation

[0041] like Figure 1 As shown, after the selected biocontrol bacteria were cultured at a constant temperature (25℃) for 96 hours on a PDA plate, the colonies were round with neat edges, white hyphae, and black granules were produced at the center edge of the colonies.

[0042] II. Phylogenetic Analysis

[0043] PCR amplification was performed on the ITS, β-tubulin, and rpb2 sites of the fungus using primers (Table 1) to obtain the corresponding sequences. The PCR amplification reaction system was 50 μL: 17.5 μL ddH2O, 25 μL Taq Mix, 2.5 μL ITS-F primer, 2.5 μL ITS-R primer, and 2.5 μL DNA template. The PCR reaction program was 98℃ for 5 min; 98℃ for 30 s, 56℃ for 10 s, and 72℃ for 25 s, for 25 cycles; stored at 4℃. The PCR amplification products were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequence analysis. The ITS rDNA sequence is shown in SEQ ID NO.1, the β-tubulin rDNA sequence is shown in SEQ ID NO.2, and the rpb2 rDNA sequence is shown in SEQ ID NO.3. Homology comparison with known ITS rDNA sequences in GenBank was performed using the Blast program, and the strain was preliminarily identified as belonging to the genus *Varicocephalum*. Then... Homologous sequences were downloaded, with *Fusarium sambucinum* CBS146.95 as the outgroup. The obtained sequences were edited using Clustalx 1.83, and the sequences of each gene were arranged using MAFFT 7.273 software. Blurred regions were removed using Gblocks 0.91b. Finally, a phylogenetic tree was constructed using RaxmlGUI v.1.5 software and the maximum likelihood (ML) method.

[0044] like Figure 2 As shown, a maximum likelihood (ML) tree was constructed by concatenating the rDNA ITS sequence (SEQ ID NO.1), β-tubulin gene sequence (SEQ ID NO.2), and rpb2 gene sequence (SEQ ID NO.3). Based on phylogenetic analysis, strain JH96 was identified as *Albifimbria verrucaria* (named as stated in the preservation certificate). Strain JH96 was deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:M 20231398, on August 9, 2023.

[0045] Table 1 Primer sequences

[0046]

[0047]

[0048] Example 3: Plate confrontation experiment between *Lysimachia verrucae* JH96 and *Sclerotinia sclerotiorum*.

[0049] Using a 6mm inner diameter punch, holes were made at the edge of activated *Lactobacillus verrucae* JH96 colonies. The colony discs were then transferred to the edge of a 90mm PDA plate. At the other end of the plate, activated *Sclerotinia sclerotiorum* colony discs of equal diameter were transferred. A PDA plate with one side inoculated with *Sclerotinia sclerotiorum* colony discs of equal diameter while the other end remained untreated served as a control. Each treatment was repeated three times. The plates were then incubated at 25℃. After the mycelia in the control group had completely covered the plate, the width of the inhibition zone in the experimental group was observed, and the inhibition rate of *Lactobacillus verrucae* JH96 on *Sclerotinia sclerotiorum* mycelial growth was calculated. Mycelial growth inhibition rate (%) = [(*Sclerotinia sclerotiorum* colony diameter in the control group - *Sclerotinia sclerotiorum* colony diameter in the experimental group) / *Sclerotinia sclerotiorum* colony diameter in the control group] × 100%.

[0050] like Figure 3 As shown in A and B, *Leptochloa verrucae* JH96 inhibited the mycelial growth of *Sclerotinia sclerotiorum* by 42.91% in a plate confrontation experiment (4 days of culture).

[0051] Example 4: Preparation of biocontrol agents

[0052] PDB culture medium was selected as the fermentation culture medium for *Lactobacillus verrucae* JH96. Holes were punched at the edges of activated *Lactobacillus verrucae* colonies using a 6mm inner diameter punch, yielding four mycelial cakes. These cakes were inoculated into Erlenmeyer flasks containing 800mL of PDB culture medium and cultured at 25℃ and 180rpm for 7 days to obtain the fermentation broth. The fermentation broth was filtered through three layers of gauze to obtain the filtrate (containing secondary metabolites of *Lactobacillus verrucae* JH96). The filtrate was extracted twice with ethyl acetate at a 1:1 volume ratio, retaining the ethyl acetate layer. The filtrate was then concentrated using a rotary evaporator to obtain the ethyl acetate extract, which was the crude fermentation extract. The crude extract was dissolved in 3mL of methanol. Any crude extract adhering to the flask wall could be dissolved by ultrasonic shaking. The extract was then evaporated to dryness in an ultra-low temperature rotary evaporator to obtain the biocontrol agent. This biocontrol agent solution was prepared by dissolving it in methanol to a specific concentration.

[0053] Example 5: Effect of biocontrol agents on the mycelial growth of Sclerotinia sclerotiorum.

[0054] The biocontrol agent solution prepared in Example 4 was added to 50 mL of PDA medium to achieve a final concentration of 1 mg / mL. The solution was poured evenly into three 90 mm Petri dishes and allowed to cool and air-dry. Holes were punched at the edges of the activated *Sclerotinia sclerotiorum* colonies using a 6 mm inner diameter punch, and the resulting bacterial culture was inoculated into the center of the PDA plate. A PDA plate prepared with an equal volume of methanol was used as a control. The plates were incubated at 25°C. After the mycelia of the control group had completely covered the plate, the colony diameter of each plate was observed and recorded, and the growth inhibition rate of the biocontrol agent on *Sclerotinia sclerotiorum* mycelia was calculated.

[0055] The results are as follows Figure 4 As shown in A and B, the experimental results indicate that the biocontrol agent has a significant inhibitory effect on the mycelial growth of Sclerotinia sclerotiorum, with a mycelial inhibition rate of 100%.

[0056] Example 6: Effect of biocontrol agents on sclerotium germination of Sclerotium sclerotia

[0057] Preparation of Sclerotinia sclerotiorum var. sclerotiorum: Sclerotinia sclerotiorum was inoculated into the center of a PDA plate and incubated at 25°C for 10 days. Mature sclerotia were collected from the plate and treated with 75% alcohol for 5 minutes, followed by rinsing with sterile water. Subsequently, the plate was disinfected with 1% sodium hypochlorite solution for 5 minutes, and then rinsed three times with sterile water, each time for 1 minute. PDA plates containing 1 mg / mL biocontrol agent were prepared according to the method in Example 5. The treated sclerotia were placed on air-dried plates, with 4 sclerotia inoculated into each plate. The experiment was repeated three times, with PDA plates prepared by adding an equal volume of methanol as a control. The plates were incubated at 25°C for 7 days. After the sclerotia on the plates without biocontrol agent germinated and grew into mycelia, the germination status was observed and recorded, and the sclerotia germination rate was calculated.

[0058] Sclerotium germination rate (%) = [(Average number of germinating sclerotia in the control group - Average number of germinating sclerotia in the experimental group) / Average number of germinating sclerotia in the control group] × 100%.

[0059] like Figure 5 As shown in Figures A and B, in the control group without biocontrol agents, the sclerotium germination rate was 100%, while in the experimental group with a biocontrol agent concentration of 1 mg / mL, the sclerotium germination rate was 37.5%, and the sclerotium germination inhibition rate was 62.5%. These experimental results indicate that the fermentation product of *Russula verrucosum* JH96 can effectively inhibit the sclerotium germination of *Sclerotinia sclerotiorum*.

[0060] Example 7: In vitro control experiment using detached lettuce leaves

[0061] Fresh lettuce leaves with uniform growth vigor were taken and rinsed thoroughly with sterile water. A 6mm diameter *Sclerotinia sclerotiorum* spore patch was inoculated onto each leaf, and the petiole was covered with a moistened cotton ball. In the control group, each leaf was sprayed with 2mL of sterile water, while in the experimental group, each leaf was sprayed with 2mL of a biocontrol agent (ethyl acetate extract from Example 4, maintaining a spore concentration of 1×10⁻⁶). 7 ~1×10 8 (cfu / mL), 10 replicates per group, and the experiment was repeated three times. The lettuce was incubated at 25℃ for 2 days, and the disease incidence on the lettuce leaves was observed and recorded. The incidence rate, disease index, and control effect were calculated.

[0062] The grading criteria for lesions are shown in Table 2:

[0063] Table 2. Grading Criteria for Lesions

[0064]

[0065] Disease index = ∑(number of diseased leaves at each level × relative level) / (total number of leaves × highest level) × 100;

[0066] Efficacy (%) = [(Average disease index of sterile water control group - Average disease index of experimental group) / Average disease index of sterile water control group] × 100%.

[0067] like Figure 6 As shown in A and B in Table 3, this biocontrol agent can effectively prevent the occurrence of sclerotinia rot on detached lettuce leaves. The disease index of the control group was 82.5, while that of the experimental group was 10, indicating that the biocontrol agent achieved a control efficacy of 87.88%. The experimental results demonstrate that the JH96 biocontrol agent for *Russula versicolor* can effectively inhibit the infection of *Sclerotinia rotundifolia* on lettuce leaves.

[0068] Table 3. Control efficacy of JH96 biocontrol agent against Sclerotinia sclerotinia rot on detached lettuce leaves.

[0069]

[0070] Example 8: Antagonistic Effect of *Lysimachia verrucae* JH96 on Six Plant Pathogenic Fungi

[0071] Following the experimental method in Example 3, *Lysimachia christinae* JH96 was subjected to a confrontation experiment with *Pestalotiopsis microspora*, *Fusarium oxysporum* (isolated from rice), *Fusarium oxysporum* (isolated from soybean), *Phytophthora capsici*, *Alternaria alstroemeriae*, and *Curvularia subpapendorfii*. The strains were incubated at 25°C for 7 days, and their antibacterial effect was observed. The experimental results are as follows: Figure 7 As shown in A1-A6, compared with the control group where plant pathogenic fungi were cultured alone, the mycelial growth of the six pathogenic fungi in the confrontation test group was inhibited, and obvious inhibition zones appeared in the groups of *Polytrichum heterotrichum*, *Rhizoctonia solani*, *Phytophthora nicotine*, *Alternaria hexandra*, and *Curvaria* pathogenic fungi.

[0072] Referring to the experimental method of Example 5, the effect of the biocontrol agent of Example 4 on the mycelial growth of six plant pathogenic fungi was further tested.

[0073] The test results are as follows Figure 8 As shown in A1-A6, compared with the control group, the experimental group with added biocontrol agent showed slow growth of six plant pathogenic fungi. The biocontrol agent had a significant inhibitory effect on the mycelial growth of pathogenic fungi such as *Polytrichum heterotrichum*, *Rhizoctonia solani*, *Fusarium oxysporum*, *Phytophthora nicotineum*, *Alternaria hexandracea*, and *Curvaria*. After 7 days of culture, the mycelial inhibition rates were 63.18%, 76.19%, 40.38%, 76.00%, 40.88%, and 57.99%, respectively.

[0074] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of *Lysimachia verrucae* in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi are *Sclerotinia sclerotiorum*, *Rhizoctonia solani*, or *Fusarium oxysporum*. The aforementioned Myrothecium verrucaria is classified and named Myrothecium verrucaria, strain number JH96, and is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20231398.

2. The application of *Lysimachia verrucae* in the control of plant diseases caused by pathogenic fungi, characterized in that... The plant pathogenic fungi mentioned are Sclerotinia sclerotiorum, Rhizoctonia solani, or Fusarium oxysporum. The aforementioned Myrothecium verrucaria is classified and named Myrothecium verrucaria, strain number JH96, and is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20231398. The plant diseases mentioned include sclerotinia stem rot in lettuce.

3. The application of *Lysimachia verrucae* in the preparation of biocontrol agents for sclerotinia stem rot, characterized in that, The aforementioned Myrothecium verrucaria is classified and named Myrothecium verrucaria, strain number JH96, and is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20231398.

4. A biocontrol agent for sclerotinia stem rot, characterized in that, The active ingredient includes Myrothecium verrucaria, which is classified as Myrothecium verrucaria, strain number JH96, and is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20231398.

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