A strain of volatile gas-producing fungus that antagonizes mycotic wilt of morel

Abstract

This invention discloses a strain of *Pseudomonas aeruginosa* that produces volatile gases and antagonizes *Morchella esculenta*, the pathogen causing *Fusarium wilt*. Pseudomonas chlororaphis subsp. aurantiaca YX3, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 26192, is a strain of this invention that produces volatile gases on NA solid medium that antagonize morel wilt fungi. It significantly inhibits the mycelial growth and spore germination of morel wilt fungi and can be used to prepare fumigants for inhibiting morel wilt fungi. It is safe, efficient, and easy to operate, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology and biological control, specifically involving a strain of morel fungi that antagonizes wilt caused by volatile gases and its application. Background Technology

[0002] Morel mushrooms are a prized edible and medicinal fungus, prized for their delicious flavor and enjoyed internationally for a long time. They contain approximately 28% protein, over 18 kinds of amino acids, and are rich in unsaturated fatty acids. Morel mushrooms possess medicinal properties such as boosting immunity, anti-fatigue, antioxidant, anti-cancer, anti-tumor, antibacterial, anti-inflammatory, lipid-lowering, and liver-protecting and stomach-strengthening effects, earning them the title of "King of Fungi." Since successful artificial cultivation of morel mushrooms in China in 2012, their cultivation area has been continuously expanding. During the ascocarp development season, high temperatures and humidity can easily lead to outbreaks of fungal diseases, such as wilt. This disease causes nearly 70% of growers to lose stable income each year.

[0003] The pathogen causing morel wilt is *Lysimachia longissima*. (Longispora) primarily affects the caps of morel ascocarps, with lesions exhibiting white, downy mycelium on the surface, ultimately leading to wilting, rotting, and deformed fruiting bodies. Currently, agricultural control measures primarily focus on agricultural practices, such as pre-planting tillage and sun exposure of the land, application of quicklime; timely removal of external nutrient bags; crop rotation or land replacement; prompt harvesting of diseased ascocarps; and prevention of high temperature and humidity conditions in greenhouses. To find safe and effective methods for controlling fungal wilt, biocontrol microorganisms are an effective means of prevention and control.

[0004] Microorganisms can produce unique odors—volatile organic compounds—which can rapidly volatilize into the gas phase under conditions of 20℃ and 0.01kPa, exhibiting good cell membrane permeability and high-efficiency diffusion capabilities in air and soil pores. In recent years, reports on the use of Pseudomonas in the biological control of plant diseases have gradually increased. For example, patent publication number CN 111944728A discloses that *Pseudomonas aeruginosa* Zm-1 has a good inhibitory effect on peanut white mold; patent publication number CN112899205A discloses that *Pseudomonas aeruginosa* MN225969 has a good antagonistic effect on *Fusarium graminearum*, *Rhizoctonia solani*, *Citrus canker*, and *Rhizoctonia solani*; and patent CN 108148782A discloses the application of volatile gases produced by *Pseudomonas aeruginosa* subspecies SPS-41 in the control of postharvest black spot disease in sweet potatoes. There are few reports on the application of volatile antibacterial gases produced by microorganisms in the control of morel wilt fungi, and finding biocontrol strains that have a preventive effect on this disease has important practical application value. Summary of the Invention

[0005] In view of the above situation, in order to overcome the shortcomings and deficiencies of existing technologies in the prevention and control of morel wilt, the technical problem to be solved by the present invention is to provide a strain of morel wilt fungus that produces volatile gases to antagonize it, and the application of the volatile gases produced by the fungus in the prevention and control of morel wilt.

[0006] The first aspect of the present invention is the method for obtaining the antagonistic bacteria described above:

[0007] (1) Take healthy, mature morel fruiting bodies, clean the surface impurities, strictly disinfect the surface, add sterile water to the tissue block and grind it into a paste, take the supernatant and spread it on NA medium for culture.

[0008] (2) After the colonies grow on the above NA medium, isolate and purify the colonies to obtain pure strains.

[0009] (3) In a two-part culture dish, take the bacteria from (2) and streak them on one side of the NA medium, and inoculate the Fusarium wilt pathogen on the other side of the PDA medium. The control group does not inoculate bacteria on the NA side. Seal and culture.

[0010] (4) Measure the diameter of the wilt pathogen after culture. If the diameter of the treatment group is smaller than that of the control group, the bacteria can produce volatile gases to antagonize the wilt pathogen.

[0011] The second aspect of the present invention is the antagonistic bacteria that can produce volatile gases, identified as *Pseudomonas chlororaphis* subsp. *aurantiaca* YX3, which was deposited on December 14, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26192, and classified as *Pseudomonas chlororaphis* subsp. *aurantiaca*. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0012] A third aspect of the invention is to provide conditions for the production of volatile gases by strain Pseudomonas aeruginosa subsp. citrinum YX3: beef extract medium (NA).

[0013] Furthermore, the culture medium formula is as follows: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, and 20 g / L agar powder.

[0014] Furthermore, the culture conditions for strain YX3 are: temperature 28-32℃, pH 7.

[0015] In a fourth aspect, the present invention provides the application of the volatile gas produced by the strain YX3 in morel fungi wilt pathogens.

[0016] Furthermore, the volatile gases produced by strain YX3 have an inhibitory effect on the mycelial growth of Fusarium wilt.

[0017] Furthermore, the volatile gases produced by strain YX3 inhibit the germination of Fusarium wilt conidia.

[0018] Furthermore, the application of the volatile gases produced by strain YX3 in the fumigation control of morel fruiting body wilt disease.

[0019] The beneficial effects of this invention are as follows: The antagonistic bacteria of this invention are derived from endophytic bacteria of morel fruiting bodies, which can produce volatile antibacterial gases that can effectively inhibit the mycelial growth and spore germination of Fusarium wilt. The biocontrol strain works by producing volatile gases, and the agent does not need to come into contact with the pathogen or the mushroom body. It is a biological control method that can achieve long-distance control, and it is pollution-free, has no toxin residues, and does not pose a threat to humans, animals, or the environment, and has a very good application prospect. Attached Figure Description

[0020] Figure 1 This is a bipartite plot showing the inhibition of morel fungal wilt pathogens by the volatile gas produced by strain YX3 described in Example 1 of the present invention. Plot A is the blank group, and plot B is the treatment group.

[0021] Figure 2 This is a photograph of the colony morphology of strain YX3 in Example 1 of the present invention after 48 hours of culture in NA medium;

[0022] Figure 3 This is the phylogenetic tree of strain YX3 constructed based on the gyr B gene as described in Example 1 of this invention;

[0023] Figure 4 The images show the inhibition of fusarium wilt conidia germination by the volatile gas produced by strain YX3 in this invention for 24 hours. Image A is the blank group, and image B is the treatment group.

[0024] Figure 5 These are photographs showing the inhibitory effect of the volatile gas produced by strain YX3 of this invention on the wilt disease of morel fruiting bodies. In the photograph, A is the blank group and B is the treatment group. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1

[0027] Isolation and screening of strains that produce volatile gases and antagonize morel fungi causing wilt.

[0028] (1) NA medium composition: 10g peptone, 3g beef extract, 5g sodium chloride, 20g agar powder, 1000mL water, pH 7.

[0029] PDA medium composition: 200g potato, 20g glucose, 20g agar, 1000mL distilled water.

[0030] (2) Strain isolation and purification: Fresh, healthy morel fruiting bodies were collected from Neixiang County, Nanyang City. Surface impurities were rinsed with sterile water, and the samples were cut into pieces. On a clean bench, the pieces were sequentially immersed in 75% ethanol for 1 minute, rinsed three times with sterile water, then immersed in 5% sodium hypochlorite for 3 minutes, immersed in 75% ethanol for 30 seconds, and rinsed five times with sterile water. (0.1 mL of the final rinse solution was spread on NA medium; if no microbial growth was observed after 48 hours, the surface was considered clean). 1 g of tissue was weighed into a sterile mortar, 10 mL of sterile water was added, and the mixture was ground and allowed to stand for 5 minutes. The supernatant was serially diluted 10-fold with sterile water. 100 μL of both the original solution and the diluted solution were spread onto NA medium plates. After incubation at 28℃ for 2-3 days, colonies of different sizes, colors, and morphologies were picked and streaked onto purification medium for purification. The purified strains were then preserved using the glycerol preservation method and stored in an ultra-low temperature freezer for later use.

[0031] (3) Pour PDA medium onto one side of the septate plate and inoculate with 5 mm diameter Fusarium wilt pathogens. Pour NA medium onto the other side and streak the isolated bacterial strains. Use an uninoculated septate plate as a control. Seal with sealing film. Repeat each treatment three times. Incubate the septate plates upside down at 28°C for 7 days. Measure the diameter of Fusarium wilt colonies using the cross-hatching method and calculate the inhibition rate according to the following formula:

[0032]

[0033] An endophytic bacterium, designated YX3, was obtained from morel fruiting bodies using the screening method described above. Figure 1 As shown, after 7 days of cultivation, the colony diameter of the wilt pathogen in the control group was 34.55 mm, while the colony diameter in the YX3 treatment group was 8.51 mm, with a mycelial growth inhibition rate of 88.1%. This demonstrates that the volatile gas produced by strain YX3 has a good antibacterial effect on morel wilt pathogens.

[0034] Identification of strain YX3

[0035] Morphological characteristics of strain YX3: such as Figure 2As shown, strain YX3 was cultured on NA medium at 28°C for 48 hours. The colonies were round, milky white, opaque, and viscous with neat edges. The bacteria were short rod-shaped, non-spore-forming, and Gram-negative.

[0036] Bacterial cells were collected by centrifugation at 7000 rpm after 48 h of NA liquid culture in shake flasks at 28℃ and 150 rpm. Genomic DNA was extracted using a bacterial DNA extraction kit. PCR amplification of the 16S rRNA and gyrB genes was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-AAGGAGGTGATCCAGCCGCA-3'); primers F (5'-GACAAGCTGGTGTCTTCCGA-3') and R (5'-CATCTCGCCCAGACCTTTGT-3). Sequencing was performed after agarose gel electrophoresis. The sequences were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing results were compared with NCBI, and Mega 5.0 software was used for sequence alignment and analysis. Finally, a phylogenetic tree was constructed using the neighbor-joining (NJ) method, and phylogenetic analysis was performed. Figure 3 As shown, sequence alignment revealed that this strain had the highest sequence similarity to effective species of the genus *Pseudomonas*, and a 97.79% similarity to *Pseudomonas chlororaphis* subsp. *aurantiaca* CP009290.1. Therefore, strain YX3 was named *Pseudomonas chlororaphis* subsp. *aurantiaca*, and its gyrB gene sequence is shown in SEQ ID NO.1, while its 16S rRNA gene sequence is shown in SEQ ID NO.2.

[0037] Inhibitory activity of volatile gases from *Pseudomonas aeruginosa* against fungal wilt pathogens.

[0038] (1) Inhibitory effect of volatile gases from strain YX3 on spore germination of fungal wilt pathogens.

[0039] Preparation of spore suspension of *Fusarium wilt* spores from *Morchella esculenta*: *Fusarium wilt* spores from *Morchella esculenta* were cultured on PDA medium at 25°C for 14 days. The spores were then rinsed with sterile water in the plates to prepare a spore suspension with a concentration of 10. 5 Species / mL. Using a 90*15mm bipartite culture dish, pour NA medium into one side, streak-inoculate with *Pseudomonas aeruginosa* strain YX3, and incubate at 28℃ for 48 h. Then, place a sterile glass slide on the other side and inoculate with a suspension of morel wilt fungal spores at a concentration of 10. 5Spores / mL, 20 μL per tablet, 4 replicates, control without inoculation with strain YX3. Incubate at 25℃ for 6 h. When the spore germination rate of the control is greater than 85%, observe the spore germination under an optical microscope. Examine 100 spores per treatment, and define germination as germ tube length greater than the short radius of the spore. Calculate the spore germination rate. Figure 4 The image shows the inhibitory effect of the volatile gases from strain YX3 on the germination of morel wilt fungal spores.

[0040]

[0041] The results showed that the volatile gas produced by strain YX3 after 48 hours of growth could completely inhibit the germination of conidia of the fungal wilt pathogen after 6 hours of treatment, and could still completely inhibit spore germination after 24 hours.

[0042] (2) Biocontrol effect of volatile gases from strain YX3 on fungal wilt pathogens affecting morel fruiting bodies

[0043] The preparation of the morel wilt fungal spore suspension was the same as above. Strain YX3 was streaked onto NA agar plates and incubated at 28°C for 48 hours. Fresh, healthy morel fruiting bodies were collected and placed in sealed, partitioned containers. Four culture dishes containing strain YX3 were placed in the lower layer, and six morel fruiting bodies were placed in the upper layer. Each treatment was repeated four times, with culture dishes not inoculated with strain YX3 serving as a control. The inoculum concentration per fruiting body was 10. 5 10 μL of a suspension of spores of the fungal wilt pathogen per mL was incubated at 20 °C. After 3 days of incubation, the disease index was investigated and the biocontrol effect was calculated. The results are shown in Table 1.

[0044] Disease investigation methods: Grade 0: No symptoms; Grade 1: Slight lesions at the inoculation point, necrotic area less than 5%; Grade 3: Necrotic area 5%-10%; Grade 5: Necrotic area 10%-25%; Grade 7: Necrotic area 25%-50%; Grade 9: Necrotic area exceeding 50%. Statistical calculation methods for disease index and biocontrol effect: Disease index (%) = ∑(Disease grade × Number of fruits at that disease grade) / (Highest disease grade × Total number of fruits) × 100; Biocontrol effect (%) = (Control disease index - Treatment group disease index) / Control disease index × 100.

[0045] The results showed that the volatile gases produced by strain YX3 had a good biocontrol effect on fungal wilt of morel fruiting bodies, with a biocontrol effect of 57.7% after 3 days of treatment.

[0046] Table 1. Biocontrol effects of volatile gases from strain YX3 on *Fusarium wilt*, the fungal causal agent of postharvest wilt of morel fruiting bodies.

[0047] Disease severity index (%) 24.13±1.89 57.04±2.45 Preventive efficacy (%) 57.70 --

[0048] The biocontrol strain YX3 of this invention is an endophytic bacterium of morel mushrooms. This bacterium has a significant antagonistic effect on the fungal wilt pathogen of morel mushrooms. Its volatile gases have a strong inhibitory effect on the mycelial growth and spore germination of the fungal wilt pathogen of morel mushrooms. It has a good biocontrol effect on fungal wilt of morel mushrooms and has good prospects for development and application.

[0049] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A strain of fungus that produces volatile gases and antagonizes morel-borne fungal wilt pathogens, characterized in that, The strain is *Pseudomonas aeruginosa* subsp. *orange*. Pseudomonas chlororaphis subsp. orange ca)YX3, is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCCNO:26192.

2. The application of the volatile gas produced by the strain that produces volatile gases to antagonize morel wilt fungi as described in claim 1 in inhibiting the growth of morel wilt fungi, wherein the pathogen of wilt is *Lysimachia longissima* (…). Diploospora longispor a).

3. The application of the volatile gas produced by the strain of claim 1, which antagonizes morel fungi and fusarium wilt pathogens, in inhibiting the mycelial growth of fusarium wilt pathogens, wherein the pathogen of fusarium wilt is *Lysimachia foetida* (…). Diploospora longispora ).

4. The application of the volatile gas produced by the strain of claim 1, which antagonizes morel fungi and fusarium wilt pathogens, in inhibiting the germination of conidia of fusarium wilt pathogens, wherein the pathogen of fusarium wilt is *Lysimachia foetida* (…). Diploospora longispora ).

5. The application of the volatile gas produced by the strain of claim 1, which antagonizes the fungal wilt pathogen of morel fungi, in the prevention and control of morel fungal wilt, wherein the pathogen of wilt is *Lysimachia longissima* (…). Diploospora longispora ).

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