Use of a fungal virus in the prevention and treatment of pear tree and apple tree phytophthora diseases

Abstract

The application discloses application of a fungal virus in prevention and treatment of pear tree and apple tree Valsa diseases. The fungal virus is a positive single-stranded RNA virus, and a cDNA nucleotide sequence of the fungal virus is shown as SEQ ID No. 1. A fungus containing the virus is Valsa pyri HJL1-22 (gamma), the fungus is infected by the fungal virus of claim 1, the fungus is named as HJL1-22 (gamma), is classified and named as Valsa pyri, and has a preservation number of CGMCC No. 41449 in China General Microbiological Culture Collection Center. The virus Vphv1-gamma can be secreted to outside of cells, therefore, culture liquid filtered off mycelium can be used as a biocontrol agent for prevention and treatment of pear tree and apple tree Valsa diseases, and can effectively weaken or inhibit pathogenicity and diffusion of Valsa pyri and Valsa mali, and has a prospect for being used for producing and practicing biological prevention and treatment of pear tree and apple tree Valsa diseases.

Description

Technical Field

[0001] This invention relates to the field of agricultural biological control, specifically to the application of a fungal virus in the control of pear tree rot and apple tree rot. Background Technology

[0002] Pear canker is a major disease affecting pear trees, primarily caused by the fungus *Valsa pyri*, belonging to the genus *Valsa* of the Ascomycota phylum. It occurs to varying degrees in all pear-producing areas of my country. These two pathogenic fungi are highly homologous. Based on the infection patterns of pear and apple canker pathogens, taking necessary and effective control measures is crucial to ensuring the sustainable and healthy development of the pear and apple industries. Currently, commonly used control methods in agricultural production include agricultural control, chemical control, and biological control. Agricultural control manages the disease by comprehensively analyzing the interactions between the host plant, pathogen, and environment; however, it is time-consuming, has a long cycle, and its effectiveness is not always significant. Therefore, chemical control remains the main method for controlling pear and apple canker in the field. Although chemical control is highly efficient and timely, its large-scale use may lead to environmental pollution, ecological imbalance, and increased pathogen resistance, threatening the stability of farmland ecosystems. Therefore, exploring greener and more efficient biological control measures is particularly important. Currently, biological control mainly focuses on the application of antagonistic microorganisms and their metabolites, while the application of fungal viruses that weaken the pathogenicity of pear or apple tree canker pathogens remains a blank. Early studies found that low-virulence viruses present in chestnut blight pathogens can significantly reduce the pathogenicity of chestnut blight pathogens and have been applied in the field. Similarly, the discovery of the attenuated fungal virus VpHV1-γ in this study provides a completely new approach to the control of pear and apple tree canker diseases, demonstrating its great potential in reducing disease occurrence and spread. This research provides new scientific theoretical basis and practical direction for promoting the green and sustainable development of the Korla pear industry. Summary of the Invention

[0003] To address the aforementioned problems, this invention aims to provide a virulent strain HJL1-22(γ) with biocontrol potential. The fungal virus VpHV1-γ carried by this strain can significantly slow the growth rate of pathogens and weaken their pathogenicity. The supernatant after filtration of the HJL1-22(γ) liquid culture solution can be used as a biocontrol agent for pear and apple tree canker, effectively reducing or inhibiting the pathogenicity and spread of the non-virulent pathogen *V. pyri* (pear tree canker) and *V. mali* (apple tree canker). This method is environmentally friendly and easy to operate, and has broad application prospects in future agricultural production practices.

[0004] To achieve the above objectives, the specific solution of this invention is as follows: A weakened fungal strain HJL1-22(γ) is provided, characterized in that the strain carries the attenuated virus VpHV1-γ. This virus can significantly weaken the pathogenicity of the fungus. Spraying the culture solution of strain HJL1-22(γ) can effectively inhibit the spread of lesions, providing an innovative biological control strategy for the green control of pear and apple tree canker. The specific solution is as follows:

[0005] The fungal virus VpHV1-γ of the present invention is a positive single-stranded RNA virus, and its nucleotide sequence is shown in SEQ ID No. 1.

[0006] The pear tree rot pathogen V.pyri, which is infected by the fungal virus VpHV1-γ, is also within the scope of protection of this invention.

[0007] The attenuated fungal strain HJL1-22(γ) of this invention is named HJL1-22(γ), classified as Valsapyri, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 41449.

[0008] The present invention also provides a fermentation broth, which is the fermentation broth of the HJL1-22(γ).

[0009] The use of the aforementioned weakly toxic fungal strains, or the aforementioned pear rot pathogen V. pyri, or the aforementioned fermentation broth in the prevention and control of pear rot or apple rot, or in inhibiting the pathogenicity of pear rot pathogen V. pyri, or in the preparation of pear rot pathogen V. pyri control agents, also falls within the scope of protection of this invention.

[0010] The present invention also provides a pear tree rot disease control agent, comprising the aforementioned attenuated fungal strain and / or the pear tree rot pathogen V.pyri infected by the fungal virus VpHV1-γ and / or the aforementioned fermentation broth.

[0011] Beneficial effects of the present invention:

[0012] The fungal virus VpHV1-γ, isolated from the pear canker pathogen HJL1-22(γ), slows down the mycelial growth and reduces the pathogenicity of V. pyri, demonstrating biocontrol potential. Inoculating the ends of lesions on branches infected with the attenuated strain HJL1-22(γ), or spraying the supernatant of the culture broth after removing mycelia onto the lesions, significantly reduced the size of the lesions. Testing confirmed that the virus can successfully infect non-virulent V. pyri. Spraying the supernatant of the culture broth after removing mycelia from apple trees infected with apple canker in the field inhibited the spread of the pathogen. This method weakens the pathogenicity of the fungus and effectively suppresses the spread of lesions. This invention provides a novel biological control method for pear tree rot and apple tree rot, and develops a green, environmentally friendly, healthy and sustainable biological control concept for fungi and viruses. Attached Figure Description

[0013] Figure 1 The size of lesions after inoculating detached pear leaves with the virus-carrying strain HJL1-22(γ) and the non-virus-carrying strain V. pyri for 3, 7, and 14 days were compared.

[0014] Figure 2 Biological control using the virus-carrying strain HJL1-22(γ): Inoculating the sides of the colony of the virus-free strain V. pyri with blocks of the virus-carrying strain HJL1-22(γ) slowed the spread of lesions on branches.

[0015] Figure 3 Spraying the supernatant of the culture solution of the virus-carrying strain HJL1-22(γ) onto detached pear branches inoculated with a virus-free strain of pear rot pathogen can reduce the pathogenicity of the virus-free strain.

[0016] Figure 4 Phenotypic analysis of avirulent strain V. pyri isolated from the disease-health site and RT-PCR detection of virus VpHV1-γ.

[0017] Figure 5 Field experiment using supernatant from culture medium of virus-carrying strain HJL1-22(γ) sprayed on apple tree branches.

[0018] Figure 6 Statistical data from a field experiment involving spraying apple tree branches with the supernatant of culture medium containing the virus strain HJL1-22(γ)

[0019] Figure 7 RT-PCR detection of virus VpHV1-γ in apple tree branch tissue isolates from field experiments

[0020] Figure 8Phenotypic diagram of strains carrying VpHV1-γ isolated from apple tree branch tissue in field experiments; biological material preservation.

[0021] Name: HJL1-22(γ);

[0022] Category naming: Valsa pyri;

[0023] Date of preservation: September 4, 2024;

[0024] Preservation Institution: China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing);

[0025] Accession number: CGMCC No.41449. Detailed Implementation

[0026] Example 1. Acquisition and identification of fungal virus and infected fungus HJL1-22(γ)

[0027] The pear tree rot samples used in this study were provided by Professor Luo Ming's laboratory at Xinjiang Agricultural University.

[0028] The specific method for isolating pathogenic fungi from tissue is as follows: The tissue at the junction of diseased and healthy tissue in the obtained sample material is cut into 0.5cm sections. 2 Tissue fragments of varying sizes were disinfected twice with 75% alcohol, followed by rinsing twice with sterile water. After draining the water from the fragments on sterile filter paper, they were placed on PDA plates and incubated at room temperature. Once hyphae appeared around the tissue, the newly formed hyphae were picked and transferred to new PDA plates for morphological observation and DNA extraction. Fungal classification was achieved using ITS identification. The obtained DNA was amplified by PCR using ITS primers (ITS1, ITS4). The resulting PCR products were sent to the company for sequencing, and the sequencing results were compared and classified using NCBI.

[0029] Mycoviruses are a class of viruses that parasitize fungal cells and can replicate stably. Based on genome type, they are classified into double-stranded RNA viruses, positive single-stranded RNA viruses, negative single-stranded RNA viruses, and circular single-stranded DNA viruses. There are 7 families of dsRNA viruses, 5 families of positive single-stranded RNA viruses, and only 1 family of negative single-stranded RNA viruses. Circular single-stranded DNA viruses and many newly discovered mycoviruses are not yet classified. Because RNA viruses produce double-stranded RNA (dsRNA) intermediates during replication, to determine whether isolated fungi carry mycoviruses, dsRNA is first extracted from the fungal isolates. A portion of the double-stranded RNA sample is used for agarose gel electrophoresis for preliminary screening. Strains containing double-stranded RNA are considered to contain mycoviruses. Further confirmation involves sending the extracted double-stranded RNA to a company for high-throughput sequencing. Combining the sequencing results with annotation information, viral protein prediction is performed using NCBI ORF Finder. Homology comparisons of the predicted proteins were performed using software such as NCBI blastP, NCBICD Search, and MAFFT to analyze their sequences, genetic characteristics, and evolutionary relationships. Ultimately, the virus was determined to belong to the genus beta-attenuated virus of the family Hypoviridae. The full-length sequence of the virus was obtained by rapid amplification of cDNA ends (RACE), and its cDNA nucleotide sequence is shown in SEQ ID No. 1. Based on the analysis results, the obtained virus was named VpHV1-γ, and the fungal strain carrying the virus was named HJL1-22(γ).

[0030] The strain HJL1-22(γ) is deposited at the China General Microbiological Culture Collection Center (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​September 4, 2024, with accession number CGMCC No. 41449.

[0031] Example 2. Identification of the attenuated virulence characteristics of strain HJL1-22(γ)

[0032] I. Obtaining the non-virulent strain V. pyri

[0033] HJL1-22(γ) was cultured until asexual spores (conidia) were produced, followed by single-spore isolation. The specific method was as follows: conidia were collected and serially diluted in sterile water, and then evenly spread on PDA plates. Once the spores germinated and produced hyphae, the newly formed hyphae were transferred to new PDA plates for subculture in preparation for RNA extraction and RT-PCR detection.

[0034] II. Pathogenicity detection of the infected strain HJL1-22(γ)

[0035] Virus-carrying strain HJL1-22(γ) and virus-free strain V. pyri were cultured separately on PDA plates at 25℃ for 3-4 days. Fresh mycelial blocks with a diameter of 6 mm were then inoculated onto detached pear leaves. During the culture process, a constant temperature of 25℃ was maintained, and the leaf petioles were wrapped with moist cotton to maintain humidity. The length of leaf lesions was measured on days 3, 7, and 14 after inoculation. Significance analysis of the measurement data was performed (Student's t-test, ** indicates p < 0.01). The experimental results are as follows: Figure 1 As shown, the leaf spot length caused by the virus-carrying strain HJL1-22(γ) was significantly shorter than that caused by the non-virus V. pyri, indicating that the attenuated virus VpHV1-γ can significantly reduce the pathogenicity of the non-virus V. pyri.

[0036] III. The Control Effect of Virus-Infected Strain HJL1-22(γ) on Pear Tree Rot Disease

[0037] Virus-carrying strain HJL1-22(γ) and the non-virulent strain Valsa pyri were cultured separately on PDA plates at 25℃ for 3-4 days. Fresh mycelial blocks with a diameter of 6 mm were then preferentially inoculated onto detached pear tree branches. During the culture process, a constant temperature of 25℃ was maintained, and the ends of the branches were wrapped with moist cotton to maintain humidity. Two days after inoculation, virus-carrying strain HJL1-22(γ) blocks of the same size were inoculated 1 cm to both sides of the non-virulent strain V. pyri colony. PDA medium was used as a blank control. The length of lesions on pear tree branches was measured on days 3, 5, 7, and 14 after inoculation. The results were analyzed for statistical significance (Student's t-test, ** indicates p < 0.01). The results showed that virus-carrying strain HJL1-22(γ) could significantly inhibit lesion expansion starting from day 5 after infection. Figure 2 ).

[0038] IV. The effect of fermentation broth of the virus-carrying strain HJL1-22(γ) on the prevention and control of pear tree canker.

[0039] Method of spraying detached branches: Fresh mycelia of the virus-carrying strain HJL1-22(γ) were inoculated into 50 mL of PDB and cultured at 25°C for 7 days. After filtration to remove the mycelia, the culture solution was collected. The culture solution was sprayed onto the diseased parts of detached branches inoculated with the virus-free strain Valsa pyri once a week for one month, then twice a month. After 14 days of treatment, it was found that the lesions on branches sprayed with the culture solution of the virus-carrying strain HJL1-22(γ) were significantly smaller than those in the control group (the control group was sprayed with PDB culture solution). Tissue at the disease-health boundary was isolated, cultured, and tested. A total of 52 strains were isolated, including 10 Valsa pyri strains, of which 8 were found to carry the virus VpHV1-γ (VJL1-22(γ)). Figure 4 The infection efficiency reached 80% (Table 1), and VpHV1-γ was not detected in other strains. This indicates that the virus VpHV1-γ can successfully infect the avirulent strain Valsa pyri and significantly weaken the pathogenicity of the fungus V. pyri. Figure 3 , Figure 4 This demonstrates a certain potential for biological control.

[0040] Table 1. VpHV1-γ Infection Efficiency

[0041]

[0042] V. Field spraying method for controlling apple rot disease using the virus-carrying strain HJL1-22(γ)

[0043] Fresh mycelia of the virus-carrying strain were inoculated into 400 mL of PDB and cultured at 25°C for 7 days. The supernatant was collected by filtration and sprayed onto the diseased parts of apple tree branches in the field twice a month. After three months of treatment, the lesions on branches sprayed with the culture solution of the virus-carrying strain HJL1-22(γ) were significantly smaller than those in the control group (which was sprayed with PDB culture solution). This indicates that the virus VpHV1-γ can successfully inhibit the spread of canker lesions on apple trees in the field. Figure 5 , Figure 6 Tissues at the disease-healthy junction were isolated, cultured, and tested, resulting in the isolation of 108 strains, of which 54 strains were found to carry VpHV1-γ. Figure 7 , Figure 8 The virus carrier rate was 50%. Among the 108 isolates, 61 strains of the pathogenic fungus *V. mali* were detected, of which 35 strains carried VpHV1-γ, a carrier rate as high as 57.37%; the remaining 47 strains carried VpHV1-γ, with 19 strains carrying VpHV1-γ, a carrier rate of 40.43% (Table 2). This indicates that the virus VpHV1-γ can successfully infect not only the non-virulent strain *V. mali*, but also other fungi in nature, and can significantly inhibit the spread of lesions in the field. Figure 5This demonstrates a certain potential for biological control.

[0044] Table 2. Field infection efficiency of VpHV1-γ

[0045]

[0046] The above experiments demonstrate that the Valsa pyri strain carrying VpHV1-γ and its liquid culture supernatant can significantly weaken the pathogenicity of the pathogenic fungi V. pyri and V. mali, effectively inhibiting the spread of lesions. Simultaneously, it verifies that VpHV1-γ can invade natural strains V. pyri, V. mali, and other fungi. This research provides a novel biological control strategy for the prevention and control of pear and apple tree canker diseases. Its advantages are its green and safe nature, avoiding the ecological imbalance, environmental pollution, and drug resistance development that may result from chemical control methods, thus possessing significant application value.

[0047] The embodiments described above are merely illustrative of several implementations of the present invention, and are described in detail, but are not intended to limit the scope of the invention. Those skilled in the art will recognize that many modifications, variations, and improvements can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations, and improvements fall within the protection scope of the present invention.

Claims

1. A weakly virulent fungal strain containing fungal virus VpHV1-γ, named HJL1-22(γ), and classified as follows: Valsa pyri The accession number at the China General Microbiological Culture Collection Center is CGMCC No. 41449; the fungal virus VpHV1-γ is a positive single-stranded RNA virus, and its nucleotide sequence is shown in SEQ ID No.

1.

2. A fermentation broth, characterized in that: The fermentation broth contains the weakly toxic fungal strain HJL1-22(γ) as described in claim 1.

3. The application of the attenuated fungal strain HJL1-22(γ) according to claim 1 or the fermentation broth according to claim 2 in the prevention and control of pear tree rot and apple tree rot, wherein the pathogen of pear tree rot is... Valsa pyri The pathogen causing the apple tree rot disease is... Valsa mali .

4. The attenuated fungal strain HJL1-22(γ) according to claim 1 or the fermentation broth according to claim 2 in inhibiting pear tree rot pathogens. Valsa pyri Applications in pathogenicity.

5. The application of the attenuated fungal strain HJL1-22(γ) according to claim 1 or the fermentation broth according to claim 2 in the preparation of control agents for pear tree rot and apple tree rot, wherein the pathogen of pear tree rot is... Valsa pyri The pathogen causing the apple tree rot disease is... Valsa mali .

6. A pear tree rot control agent, comprising the attenuated fungal strain HJL1-22(γ) as described in claim 1 and / or the fermentation broth as described in claim 2; wherein the pathogen of the pear tree rot is... Valsa pyri .

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