Application of fungal virus in prevention and treatment of pear tree canker and apple tree canker

Abstract

The invention discloses application of a fungal virus in prevention and treatment of pear tree canker and apple tree canker. The mycovirus is a positive-sense single-stranded RNA virus, and the cDNA nucleotide sequence of the mycovirus is shown as SEQ ID No. 1. The fungus containing the virus is valsa pear tree rot pathogen HJL1-22 (gamma), the fungus is infected by the fungal virus in the claim 1, the name of the fungus is HJL1-22 (gamma), the classification name of the fungus is Valsa pyri, and the preservation number of the fungus in the China General Microbiological Culture Collection Center is CGMCC No.41449. The virus Vphv1-gamma can be secreted out of cells, so that a culture solution without mycelia can be used as a biocontrol agent for preventing and treating the pear tree canker and the apple tree canker, and pathopoiesis and diffusion of the pear tree canker and the apple tree canker are effectively weakened or inhibited; the application has the prospect of biologically preventing and treating the pear tree canker and the apple tree canker in production practice.

Description

Technical Field

[0001] The invention relates to the field of agricultural biological control, and in particular to application of a fungal virus in preventing and controlling pear tree rot and apple tree rot. Background Art

[0002] Pear rot is the main disease that occurs on pear trees. It is mainly caused by the pathogenic fungus Valsa pyri of the genus Valsa of the Ascomycetes. It occurs to varying degrees in all pear-producing areas in my country. The two pathogenic fungi are highly homologous. According to the infection rules of pear rot pathogens or apple rot pathogens, it is crucial to take necessary and effective prevention and control measures to ensure the sustainable and healthy development of the pear and apple industries. In agricultural production, the commonly used prevention and control methods currently include agricultural prevention, chemical prevention and biological prevention. Among them, agricultural prevention and control controls diseases by comprehensively analyzing the relationship between host plants, pathogens and the environment, but its cycle is long, time-consuming and the effect is not significant enough. Therefore, the main method for field prevention and control of pear rot and apple rot is still chemical prevention. Although chemical prevention is efficient and timely, its large-scale use may cause environmental pollution, ecological imbalance and increased pathogen resistance, posing a threat to the stability of farmland ecosystems. To this end, it is particularly important to explore greener and more efficient biological control measures. At present, biological control mainly focuses on the application of antagonistic microorganisms and their metabolites, while there is still a lack of fungal viruses that can reduce the pathogenicity of pear or apple rot. Early studies have found that low-toxicity viruses present in chestnut blight can significantly reduce the pathogenicity of chestnut blight and have been used in the field. Similarly, the discovery of the attenuated fungal virus VpHV1-γ in this study provides a new idea for the prevention and control of pear rot and apple rot, showing its great potential in reducing the occurrence and spread of diseases. This research provides a new scientific theoretical basis and practical direction for promoting the green and sustainable development of the fragrant pear industry. Summary of the invention

[0003] To solve the above problems, the present invention aims to provide a weakly toxic strain HJL1-22 (γ) with biological control potential. The fungal virus VpHV1-γ carried by the strain can significantly slow down the growth rate of pathogens and weaken their pathogenicity. The supernatant after filtration of the HJL1-22 (γ) liquid culture can be used as a biological control agent for preventing and treating pear rot and apple rot, effectively weakening or inhibiting the pathogenicity and spread of the non-toxic pathogen V.pyri of pear rot and the apple rot V.mali. This method is green, environmentally friendly and easy to operate, and has broad application prospects in agricultural production practice in the future.

[0004] To achieve the above objectives, the specific scheme of the present invention is as follows: a weak fungal strain HJL1-22 (γ) is provided, characterized in that the strain carries the attenuated virus VpHV1-γ. The virus can significantly reduce the pathogenicity of pathogenic fungi, and spraying the culture solution of the HJL1-22 (γ) strain can effectively inhibit the spread of lesions, providing an innovative biological control strategy for the green control of pear rot and apple rot. The specific scheme 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 rot pathogen V. pyri infected by the fungal virus VpHV1-γ also belongs to the protection scope of the present invention.

[0007] The attenuated fungus strain HJL1-22 (γ) of the present invention is named HJL1-22 (γ), classified as Valsapyri, and has a collection number of CGMCC No. 41449 in the General Microbiological Center of the China National Committee for the Collection of Microorganisms.

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

[0009] The use of the weakly toxic fungal strain or the pear rot pathogen V. pyri or the fermentation liquid in preventing and treating pear rot or apple rot or inhibiting the pathogenicity of pear rot pathogen V. pyri or in preparing a pear rot pathogen V. pyri control agent also falls within the protection scope of the present invention.

[0010] The present invention also provides a pear rot disease prevention and control preparation, comprising the weakly toxic fungal strain and / or the pear rot pathogen V.pyri infected by the fungal virus VpHV1-γ and / or the fermentation liquid.

[0011] Beneficial effects of the present invention:

[0012] The fungal virus VpHV1-γ isolated from the pear rot pathogen HJL1-22(γ) can slow down the growth of V.pyri hyphae and reduce the pathogenicity when infecting the pear rot pathogen V.pyri, and has the potential for biocontrol. The attenuated strain HJL1-22(γ) was inoculated at both ends of the lesions on the branches infected with the pear rot pathogen, or the supernatant of the culture solution of the attenuated strain HJL1-22(γ) after removing the hyphae was sprayed on the lesions on the branches infected with the pear rot pathogen. It can be observed that the lesions are significantly reduced. After testing, the virus can successfully infect non-toxic V.pyri. The supernatant of the culture solution of the attenuated strain HJL1-22(γ) after removing the hyphae was sprayed on apple trees with apple rot in the field, which can inhibit the spread of apple rot pathogens. This method can weaken the pathogenicity of pathogenic fungi and effectively inhibit the spread of lesions. This invention provides a new biological control method for pear tree rot and apple tree rot, and develops a green, environmentally friendly, healthy and sustainable biological control concept for fungal viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The lesion size of the virulent strain HJL1-22(γ) and the non-virulent strain V.pyri after inoculation of detached pear tree leaves for 3, 7, and 14 days

[0014] Figure 2 . Biological control with the virulent strain HJL1-22(γ); inoculating the virulent strain HJL1-22(γ) on both sides of the colony of the non-toxic strain V.pyri slowed down the spread of the branch lesions

[0015] Figure 3 .Spraying the culture supernatant of the virulent strain HJL1-22(γ) onto the detached branches of pear trees inoculated with the non-virulent strain of pear rot pathogen can reduce the pathogenicity of the non-virulent strain.

[0016] Figure 4 . Phenotype of the avirulent strain V.pyri isolated from the junction of diseased and healthy tissues and RT-PCR detection of the virus VpHV1-γ

[0017] Figure 5 Field experiment of spraying the supernatant of the culture solution of the virus-carrying strain HJL1-22(γ) on apple tree branches

[0018] Figure 6 Statistical data of field experiment on spraying apple tree branches with supernatant of culture solution of virus-carrying strain HJL1-22(γ)

[0019] Figure 7 . RT-PCR detection of virus VpHV1-γ contained in strains isolated from apple tree branch tissues in field experiments

[0020] Figure 8Phenotype of strains carrying VpHV1-γ isolated from apple tree branch tissues in field experiments Biological material deposits

[0021] Name: HJL1-22(γ);

[0022] Classification and nomenclature: Valsa pyri;

[0023] Deposit date: September 4, 2024;

[0024] Depository: General Microbiology Center of China Microbiological Culture Collection Administration (Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing);

[0025] Deposit number: CGMCC No.41449. DETAILED DESCRIPTION

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

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

[0028] The specific method for separating pathogenic fungal tissue is as follows: the diseased and healthy junction tissue of the obtained sample material is cut into 0.5 cm 2 The tissue fragments were disinfected with 75% alcohol twice, then rinsed with sterile water twice, and the tissue fragments were drained on sterile filter paper and placed on a PDA plate for room temperature culture. When hyphae grew around the tissue, the new hyphae were picked and transferred to a new PDA plate for morphological observation and DNA extraction. Fungal classification was achieved through ITS identification. The DNA obtained above was amplified by PCR using ITS primers (ITS1, ITS4), and the resulting PCR products were sent to the company for sequencing. The sequencing results were compared and classified by NCBI.

[0029] Mycovirus is a type of virus that parasitizes fungal cells and can replicate stably. According to the genome type, it is divided into double-stranded RNA virus, positive single-stranded RNA virus, negative single-stranded RNA virus and circular single-stranded DNA virus. There are 7 families of dsRNA viruses, 5 families of positive single-stranded RNA viruses, and only 1 family of negative single-stranded RNA. Circular single-stranded DNA viruses and many new fungal viruses discovered have not yet been divided into families. Because RNA viruses produce double-stranded RNA (dsRNA) intermediates during replication, in order to determine whether the isolated fungi carry fungal viruses, dsRNA is first extracted from fungal isolates, and some double-stranded RNA samples are used for agarose gel electrophoresis detection for preliminary screening. Strains containing double-stranded RNA can be considered to contain fungal viruses. Further confirmation work includes sending the extracted double-stranded RNA to the company for high-throughput sequencing, combining the sequencing results and annotation information, and predicting the virus-encoded proteins through NCBI ORF Finder. The predicted proteins were compared for homology using NCBI blastP, NCBICD Search, and MAFFT software to analyze their sequences, genetic characteristics, and evolutionary relationships. It was ultimately determined that the virus belonged to the genus B attenuated virus of the family Hypoviridae, and the virus was subjected to rapid amplification of cDNA ends (RACE) to obtain its full-length sequence. The 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) was deposited in the China General Microbiological Culture Collection Center (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with a deposit date of September 4, 2024 and a deposit number of CGMCC No.41449.

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

[0032] 1. Acquisition of the avirulent strain V.pyri

[0033] HJL1-22(γ) was cultured until asexual spores (conidia) were produced, and then single spores were isolated as follows: conidia were collected and graded diluted in sterile water, and then evenly spread on PDA plates. Once spores germinated and hyphae were observed, the new hyphae were transferred to new PDA plates for subculture in preparation for RNA extraction and RT-PCR detection.

[0034] 2. Pathogenicity detection of the virus-carrying strain HJL1-22(γ)

[0035] The virulent strain HJL1-22 (γ) and the non-virulent strain V.pyri were placed on PDA plates and cultured at 25°C for 3-4 days. Fresh mycelium blocks with a diameter of 6 mm were taken and inoculated into detached pear tree leaves. During the culture process, a constant temperature of 25°C was maintained, and the leaf petiole was wrapped with wet cotton to maintain humidity. The extension length of the leaf lesions was measured on the 3rd, 7th, and 14th days after inoculation, and the measured data were analyzed for significant differences (Student's t-test, ** indicates p < 0.01). The experimental results are shown in Figure 2. Figure 1 As shown, the length of pear tree leaf lesions caused by the virulent strain HJL1-22(γ) was significantly lower than that of the avirulent strain of V.pyri, indicating that the attenuated virus VpHV1-γ can significantly reduce the pathogenicity of the avirulent strain of V.pyri.

[0036] 3. Effect of the virus-carrying strain HJL1-22(γ) on the prevention and treatment of pear rot

[0037] The virulent strain HJL1-22 (γ) and the non-toxic strain Valsa pyri were placed on PDA plates and cultured at 25°C for 3-4 days. Fresh mycelium blocks with a diameter of 6 mm were taken and preferentially inoculated on the detached pear tree branches. During the culture process, a constant temperature of 25°C was maintained, and both ends of the branches were wrapped with wet cotton to maintain humidity. After 2 days of inoculation, the virulent strain HJL1-22 (γ) blocks of the same size were planted 1 cm on both sides of the colony of the non-toxic strain V.pyri, and the inoculated PDA medium was used as a blank control. The length of the lesions on the pear tree branches was measured 3, 5, 7, and 14 days after inoculation, and the experimental results were analyzed for significant differences (Student's t-test, ** indicates p < 0.01). The results showed that the virulent strain HJL1-22 (γ) could significantly inhibit the expansion of lesions starting from the 5th day after infection ( Figure 2 ).

[0038] IV. The effect of the fermentation liquid of the virulent strain HJL1-22(γ) on the prevention and treatment of pear rot

[0039] In vitro branch spraying method: inoculate fresh mycelium of the virulent strain HJL1-22 (γ) in 50 mL PDB, culture at 25°C for 7 days, filter out the mycelium, and collect the culture solution. Spray the culture solution on the diseased part of the in vitro branch inoculated with the colony of the non-toxic strain Valsa pyri once a week. After one month, change to spraying twice a month. After 14 days of treatment, it was found that the lesions on the branches sprayed with the culture solution of the virulent strain HJL1-22 (γ) were significantly smaller than those in the control group (the control group was sprayed with PDB culture solution). The tissues at the junction of the diseased and healthy were isolated, cultured and tested, and a total of 52 strains were isolated, including 10 strains of Valsa pyri, of which 8 strains carrying the virus VpHV1-γ ( Figure 4 ), with an infection efficiency of 80% (Table 1), and no VpHV1-γ was detected in other strains. This indicates that the virus VpHV1-γ can successfully infect the avirulent strain Valsa pyri and significantly reduce the pathogenicity of the pathogenic fungus V. pyri ( Figure 3 , Figure 4 ), showing a certain potential for biological control.

[0040] Table 1. VpHV1-γ infection efficiency

[0041]

[0042] 5. Field spraying method of the virus-carrying strain HJL1-22(γ) to control apple rot

[0043] The fresh mycelium of the virus-carrying strain was inoculated into 400 mL PDB, cultured at 25°C for 7 days, and the supernatant culture solution was collected by filtration and sprayed on the diseased parts of apple tree branches in the field, twice a month. After three months of treatment, it was found that the lesions on the 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), indicating that the virus VpHV1-γ can successfully inhibit the spread of rot lesions on apple trees in the field ( Figure 5 , Figure 6 ). The tissues at the junction of the diseased and healthy areas were isolated, cultured and tested, and a total of 108 strains were isolated, of which 54 strains were found to carry VpHV1-γ ( Figure 7 , Figure 8 ), with a virus rate of 50%. A total of 61 pathogenic fungi V.mali were detected in 108 isolated bacteria, of which 35 carried VpHV1-γ, with a virus rate of up to 57.37%; 47 other strains, 19 carried VpHV1-γ, with a virus rate of 40.43% (Table 2). This shows that the virus VpHV1-γ can not only successfully infect the non-toxic strain V.mali, but also infect other fungi in nature, and can significantly inhibit the spread of lesions in the field ( Figure 5), showing a certain potential for biological control.

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

[0045]

[0046] The above experiments show that the Valsa pyri strain carrying VpHV1-γ and its liquid culture supernatant can significantly reduce the pathogenicity of pathogenic fungi V.pyri and V.mali, effectively inhibit the spread of lesions, and verify that VpHV1-γ can invade natural strains of V.pyri, V.mali and other fungi. This study provides a new biological control strategy for the prevention and control of pear rot and apple rot, which is green and safe, and will not cause ecological imbalance, environmental pollution, and pathogen resistance caused by chemical control methods. It has important application value.

[0047] The above-described embodiments only illustrate several implementation methods of the present invention, and the descriptions thereof are relatively specific, but are only illustrative and not restrictive of the present invention. For those skilled in the art, many modifications, variations and improvements may be made without departing from the spirit and scope defined by the appended claims, and all of these belong to the protection scope of the present invention.

Claims

1. A fungal virus, characterized in that: It is a positive single-stranded RNA virus, and its nucleotide sequence is shown in SEQ ID No.

1.

2. A pear rot pathogen (Valsa pyri), characterized in that: The fungus is infected by the fungal virus VpHV1-γ according to claim 1.

3. A weakly toxic fungal strain containing the fungal virus according to claim 1, whose name is HJL1-22(γ), classified as Valsa pyri, and whose deposit number in the General Microbiological Center of China Microorganism Culture Collection Administration is CGMCC No.41449.

4. A fermentation liquid, characterized in that: The fermentation broth of HJL1-22(γ) according to claim 3.

5. Use of the weakly toxic fungal strain according to claim 3, the V.pyri infected by the virus VpHV1-γ according to claim 2, or the fermentation liquid according to claim 4 in preventing and controlling pear rot and apple rot.

6. Use of the weakly toxic fungal strain according to claim 3, the V. pyri infected by the virus VpHV1-γ according to claim 2, or the fermentation broth according to claim 4 in inhibiting the pathogenicity of V. pyri.

7. Use of the weakly toxic fungal strain according to claim 3, the V.pyri infected by the virus VpHV1-γ according to claim 2, or the fermentation liquid according to claim 4 in the preparation of a control agent for pear rot and apple rot.

8. A pear rot control agent, comprising the weakly toxic fungal strain according to claim 3 and / or the V.pyri infected by the virus VpHV1-γ according to claim 2 and / or the fermentation liquid according to claim 4.

Images