Spiropolyspora pink strain for preventing and treating root knot nematode disease as well as fungicide and application of spiropolyspora pink strain

Abstract

The invention provides a spiropolyspora roseus strain for preventing and treating root knot nematode disease, and the preservation number of the spiropolyspora roseus strain is CGMCC No.41822. The invention also provides a microbial inoculum containing the spiropolyspora pink strain, and the microbial inoculum is prepared by the following steps: inoculating a culture medium with the spiropolyspora pink HLF-14-11, and culturing until spores are produced; spores are eluted to prepare a spore suspension, or a bacterial block is cut to serve as a liquid fermentation inoculant; inoculating the inoculant into a liquid culture medium, and carrying out shake culture; and taking the fermentation liquor to prepare liquid, powder and granules. The spiropolyspora pink strain provided by the invention is separated from root-knot nematode oocysts of diseased tomatoes, can parasitize eggs of root-knot nematodes and inhibit incubation of the eggs, fermentation filtrate of the spiropolyspora pink strain has a relatively strong poisoning effect on second-stage larvae of the root-knot nematodes, and the fatality rate of the second-stage larvae reaches 89.1%. Meanwhile, the strain can produce auxin substances such as indoleacetic acid and the like, and plant growth and root development are promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a pink spiral spore mold strain and an application thereof in preventing and controlling fruit and vegetable diseases. Background Art

[0002] Plant nematodes are a global disease. In my country, nematode diseases have become the second largest disease in crop production. According to FAO (Food and Agriculture Organization of the United Nations), the annual loss of food and fiber crops caused by nematodes worldwide is about 12.3%, and the loss of vegetables, peanuts, tobacco and some fruit trees exceeds 20%, or even reaches 50%. Among them, root-knot nematodes (Meloidogyne spp.) are the most widely distributed and most harmful type of plant pathogenic nematodes. They can infect most food and cash crops. At the same time, the wounds caused by root-knot nematode infection will also aggravate other soil-borne diseases such as wilt and root rot. According to statistics, the global economic losses caused by root-knot nematodes exceed 170 billion US dollars (Gamalero & Glick. The use of plant growth-promoting bacteria to prevent nematode damage to plants [J]. Biology, 2020, 9 (11): 381). In recent years, my country's facility planting area and multiple cropping index have continued to increase. Coupled with the single variety and high water and fertilizer intensity, root-knot nematode disease has become increasingly serious, posing a great threat to crop production.

[0003] Root-knot nematodes have a wide host range, strong environmental adaptability, and rapid reproduction, making them difficult to eradicate. Currently, fumigants such as dazomet and methamphetamine, as well as chemical pesticides such as fumigants such as thiazolin, glyphosate, trifluopyram, and fluopyram, are commonly used in agricultural production to control nematode diseases. However, their extensive use can lead to serious environmental pollution, pesticide residues, and the development of pesticide resistance. In recent years, with growing awareness of ecological protection and food safety, the use of chemical nematicides has gradually decreased, and biological control measures for plant disease control have gained widespread acceptance. Several biocontrol microorganisms, such as Purpureocillium lilacinum, Pochonia chlamydosporia, Bacillus cereus, and Pasteuria penetrans, have demonstrated effective control of root-knot nematodes, making biological control an increasingly important approach to preventing and controlling root-knot nematodes.

[0004] Clonostachys rosea, also known as pink Glime mold, belongs to the Ascomycetes, Hypocreales, and Erythroceraceae families. In 1999, Luangsa-ard et al. classified it in the genus Clonostachys. Clonostachys rosea is a novel biocontrol fungus that can parasitize pathogens such as Fusarium, Rhizoctonia, Sclerotinia, Botrytis cinerea, Verticillium dahliae, and Pythium aphanidermatum. It exhibits broad-spectrum antimicrobial activity and has demonstrated excellent biocontrol efficacy against a variety of fungal and oomycete diseases in greenhouse and field trials. Studies have also shown that Clonostachys rosea can parasitize plant nematodes and exhibit nematicidal activity. Clonostachys rosea has diverse biocontrol mechanisms, rapid growth rate, and strong spore production, suggesting significant potential for biocontrol applications. Exploiting the efficient biocontrol resources of pink spiral spore mold and using pink spiral spore mold and its metabolites to control plant diseases can effectively protect the ecological environment and is of great significance for achieving green disease control, reducing the use of fertilizers and pesticides, and sustainable and healthy development of agriculture. Summary of the Invention

[0005] The present invention aims to provide a pink spiral spore fungus strain for preventing and treating root knot nematode disease;

[0006] The second object of the present invention is to provide a microbial agent containing the pink spiral spore mold strain;

[0007] The third object of the present invention is to propose the application of the strain and the bacterial agent.

[0008] The technical solution for achieving the above-mentioned purpose of the present invention is:

[0009] A pink spiral spore fungus strain for controlling root-knot nematode disease, with a deposit number of CGMCC No. 41822 and HLF-14-11. The deposit was made on March 4, 2025, at the General Microbiology Center of the China Culture Collection Administration.

[0010] A bacterial agent containing the pink spiral polysporon strain is prepared by the following method:

[0011] 1) Strain culture: Inoculate HLF-14-11 on potato dextrose agar (PDA) medium and culture at 25-28°C for 6-10 days until conidia are formed.

[0012] 2) Preparation of liquid fermentation inoculum: eluting spores from the PDA medium to prepare a spore suspension; or cutting 3-6 x 3-6 mm sized bacterial blocks from the cultured plates as inoculum;

[0013] 3) Liquid fermentation culture: Prepare a liquid culture medium, inoculate the spore suspension or bacterial mass, and perform primary culture or secondary culture. The primary culture is a shaking culture on a shaker at 25-28°C. The secondary culture is a scaled-up culture using the product of the primary culture as a seed solution and inoculating it with fresh liquid culture medium.

[0014] 4) Preparation: The fermentation liquid obtained in step 3) is prepared into a bacterial agent, and the dosage form of the bacterial agent is liquid, granule or powder.

[0015] Step 2) eluting the spores can be performed by adding sterile water or 0.05% Tween 80 solution to the culture dish; Step 3) after preparing the liquid culture medium, the liquid culture medium is placed in a conical flask, sterilized by high-temperature wet heat, and when cooled to room temperature, the spore suspension or bacterial block is inoculated for fermentation culture.

[0016] The liquid fermentation culture medium in step 3) comprises the following: 20-40 g of a carbon source, 5-20 g of a nitrogen source, 0.5-2 g of K2HPO4, 0.1-1 g of NaCl, 0.1-1 g of MgSO4·7H2O, 0.01-0.1 g of FeSO4·7H2O, and 0.01-0.1 g of ZnSO4·7H2O. The above ingredients are mixed and water is added to 1000 mL, and the pH is adjusted to 5.5-6.5. The carbon source is selected from one or more of glucose, sucrose, cornstarch, starch, and malt extract. The nitrogen source is selected from one or more of soybean meal, fish meal, yeast extract, soy peptone, urea, and NaNO3, and the amount of nitrogen source added is calculated based on a nitrogen content of 0.3-1.5 g / L. The pH can be adjusted using a 0.1-1 M HCl solution, or other acidic or alkaline solution. After adding water, adjusting the pH, and sterilizing, the fermentation medium is obtained.

[0017] Preferably, the carbon source is a mixture of glucose and corn flour in a mass ratio of 1 to 2:2; the nitrogen source is a mixture of soybean meal powder and urea in a mass ratio of 1 to 3:1.

[0018] Wherein, in step 3), the shaking culture is as follows: the spore suspension inoculation amount is 0.5-5%, or 4-6 pieces of the bacterial blocks are inoculated, the shaking speed is 150-250 r / min, the culture temperature is 25-28° C., and the culture time is 48-96 h.

[0019] At the end of fermentation, the spore content in the fermentation liquid should be ≥1×10 8 pieces / mL.

[0020] After the fermentation liquid is obtained in step 3), it can be used as a seed liquid and then inoculated into a fresh liquid culture medium for secondary amplification culture. The liquid culture medium used has the same formula as the primary fermentation culture medium.

[0021] In step 4), a stabilizer is added to the fermentation broth at a concentration of 0 to 10% by weight of the fermentation broth to obtain a liquid; a carrier and an auxiliary agent are added to the fermentation broth, mixed, granulated, and dried at 30 to 45° C. to obtain a granule; a carrier or a combination of a carrier and an auxiliary agent is added to the fermentation broth, dried at 30 to 45° C., and crushed to obtain a powder; the carrier is one or more of diatomaceous earth, light calcium carbonate, talc, kaolin, attapulgite, bentonite, starch, dextrin, peat, rice husk powder, straw powder, and corn cob powder; and the auxiliary agent is one or more of sodium carboxymethyl cellulose, xanthan gum, amino oligosaccharide, sodium alginate, glycine, and silicate. The amount of the carrier added is 30% to 300% (by weight) of the fermentation broth, and the amount of the auxiliary agent added is 0.1% to 10% (by weight) of the fermentation broth.

[0022] The fermentation broth may be centrifuged and concentrated before the carrier and / or auxiliary agent are added.

[0023] Granules are prepared using a combination of carriers and adjuvants, such as diatomaceous earth, starch, sodium carboxymethyl cellulose, and amino oligosaccharides. Research has found that diatomaceous earth, with its porous structure and large specific surface area, can absorb large amounts of spores and mycelium from the fermentation broth of the HLF-14-11 strain and disperse them evenly. Starch and sodium carboxymethyl cellulose act as a bond to form the granules and help maintain the stability of the formulation. Amino oligosaccharides can stimulate plant immune responses, promote growth, enhance stress resistance, and have a certain preventive and control effect on some plant diseases.

[0024] For example, a combination of a carrier and an auxiliary agent is added to the fermentation broth to prepare granules, wherein the carrier and auxiliary agent combination is a combination of diatomaceous earth, starch, sodium carboxymethyl cellulose and amino oligosaccharide in a mass ratio of 20-30:10:1-2:0.2-0.5.

[0025] The microbial agent is a liquid agent, and the bacterial content in the agent is (1-5)×10 8 CFU / mL; or: the microbial agent is a granule or powder, and the bacterial content in the agent is (1-50)×10 7 CFU / g.

[0026] The pink spiral spore mold strain of the present invention is used in preventing and controlling root knot nematode disease of crops, gray mold of fruits and vegetables, early leaf drop disease of fruit trees, sclerotinia wilt, wilt, verticillium wilt and blight of economic crops.

[0027] The microbial agent of the present invention is used in preventing and treating root knot nematode disease of crops, gray mold of fruits and vegetables, early leaf drop disease of fruit trees, sclerotinia rot, wilt, verticillium wilt and blight of economic crops.

[0028] The beneficial effects of the present invention are:

[0029] The pink spiral nematode HLF-14-11 strain proposed in this invention is isolated from root-knot nematode oocysts found in diseased tomatoes. This strain has high cell wall-degrading enzyme activity, enabling it to parasitize root-knot nematode eggs and inhibit their hatching. Its fermentation filtrate is highly toxic to second-instar larvae of root-knot nematodes, achieving a mortality rate of 89.1%. HLF-14-11 also produces auxins such as indoleacetic acid, promoting plant growth and root development. Furthermore, this strain exhibits broad-spectrum antimicrobial activity, inhibiting plant pathogens such as Sclerotinia sclerotiorum, Botrytis cinerea, apple early leaf drop pathogen, and pepper wilt pathogen.

[0030] Compared to other strains, the pink spiral polypore strain proposed in this invention has significant disease prevention and growth-promoting effects. In potted plant experiments, the tomato roots in the control group were severely damaged, with deformed root tissue and numerous root knots. However, after root irrigation with the HLF-14-11 fermentation liquid, the roots were relatively robust, with only a few small root knots formed. The control group achieved an 86.2% efficacy against root-knot nematodes, and tomato plant height, stem diameter, chlorophyll content, and aboveground fresh weight increased by 27.4%, 48.6%, 37.5%, and 64.6%, respectively. In greenhouse field experiments, continuous use of the HLF-14-11 agent achieved a 74.3% efficacy against tomato root-knot nematodes. Furthermore, it significantly promoted tomato growth, with stem diameter increasing by 25.3% and leaf area by 49.3%, thereby promoting photosynthesis and improving quality and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the colony morphology of the pink spiral mycelium HLF-14-11 strain on PDA medium.

[0032] Figure 2 This is the homology phylogenetic tree of the ITS rDNA sequence of the pink spiral fungus HLF-14-11 strain.

[0033] Figure 3 These are the eggs of the root-knot nematode parasitized by the pink spiral fungus HLF-14-11 strain.

[0034] Figure 4 The toxic effect of the pink spiral fungus HLF-14-11 strain on root-knot nematode larvae.

[0035] Figure 5 To determine the antagonistic ability of the pink spiral spore fungus HLF-14-11 strain against tomato gray mold and apple early leaf drop pathogen.

[0036] Figure 6 The method is to produce indoleacetic acid during the liquid culture of the pink spiral fungus HLF-14-11 strain.

[0037] Figure 7The study is about the control effect of the fermentation liquid of the pink spiral polycystic ovary strain on tomato root knot nematode disease and its rooting promotion effect under greenhouse pot conditions. DETAILED DESCRIPTION

[0038] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0039] Unless otherwise specified, the test materials and testing instruments used in the instructions can be purchased commercially.

[0040] Unless otherwise specified, all parts in the examples are parts by mass and all percentages are by mass.

[0041] The pink spiral spore mold used in this invention was isolated by our research team from root-knot nematode oocysts in diseased tomatoes grown in the Chuizhuang greenhouse in Jiuzhou Town, Guangyang District, Langfang City, Hebei Province. The strain is designated HLF-14-11. Its culture deposit number is CGMCC No. 41822, dated March 4, 2025, and is deposited at the General Microbiology Center of the China Culture Collection Administration.

[0042] Example 1 Separation of root-knot nematode eggs and parasitic bacteria on female nematodes

[0043] Diseased roots of tomato plants infected with root-knot nematodes were collected from greenhouses in Langfang, Hebei Province. The soil was rinsed off with tap water, and oocysts were removed with a dissecting needle. The oocysts were placed in a 1% NaClO solution and stirred with a glass rod for 3 minutes. The nematode suspension was passed through sterile 200- and 500-mesh sieves, repeatedly rinsed with sterile water to remove residual NaClO. The nematode eggs on the 500-mesh sieve were collected and spread onto polydimethylsiloxane (PDA) plates supplemented with streptomycin sulfate and ampicillin. Meanwhile, female nematodes were isolated from rotting root tissue, disinfected with 0.5% NaClO solution, rinsed with sterile water, crushed, and spot-seeded onto PDA plates supplemented with antibiotics. After incubation at 28°C for 3–5 days, individual colonies were streaked onto PDA plates for purification, transferred, and the isolated root-knot nematode parasites were stored at 4°C.

[0044] Example 2 Identification of the root-knot nematode egg parasite HLF-14-11 strain

[0045] A total of 73 strains of parasitic fungi on root-knot nematode eggs and female nematodes were isolated through Example 1. Each strain was inoculated into PDA medium and cultured at 26°C for 7 days. A small amount of hyphae and spores were picked up with a dissecting needle, placed on a slide with sterile water, covered with a cover glass, and the morphology of hyphae and conidia was observed under a microscope. Among them, 14 strains had relatively consistent morphology. Based on the colony morphology and microstructural observation, it was preliminarily determined that they might be pink spiral polycystic fungi. Among them, the colony morphology of HLF-14-11 is as follows: Figure 1 As shown, a is the front morphology of the colony of HLF-14-11 strain, and b is the back morphology of the colony.

[0046] The ITS sequences of these 14 fungal strains were then compared with those of known fungi for molecular identification. DNA from the fungal strains was extracted using the CTAB method, and PCR amplification was performed using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). A 25 μL reaction system consisted of 22 μL of 2× Taq PCR Mix, 1 μL of each primer, and 1 μL of DNA template. The amplification procedure was as follows: 95°C pre-denaturation for 5 min; 94°C denaturation for 1 min; 30 cycles of annealing at 56°C for 1 min, extension at 72°C for 2 min, and 72°C extension for 5 min, followed by storage at 4°C. PCR products were analyzed by electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The resulting sequences were aligned using BLAST in the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method. The results showed that the ITS sequences of 12 fungi including strain HLF-14-11 were consistent and had 100% homology with the pink spiral fungus. Taking the HLF-14-11 strain as an example, the homology comparison phylogenetic tree is as follows: Figure 2 shown.

[0047] Example 3 Screening of highly effective pink spiral polycystis strains for preventing and treating root-knot nematodes

[0048] 22 strains of pink spiral spore mold isolated from root-knot nematode eggs and female nematodes in different regions and 35 strains of pink spiral spore mold isolated and preserved in the laboratory in the early stage were subjected to indoor biological testing to screen for highly effective biocontrol strains.

[0049] (1) Parasitic effect on root-knot nematode eggs

[0050] Tomatoes were grown in a greenhouse to propagate southern root-knot nematodes. Diseased roots were collected, soil clods were washed off with tap water, and oocysts were picked with a dissecting needle. These oocysts were placed in a 0.5% NaClO solution and stirred with a glass rod for 3 minutes. The nematode suspension was passed through sterile 200- and 500-mesh sieves, rinsed repeatedly with sterile water, and eggs collected from the 500-mesh sieve. The eggs were transferred to a 50 mL centrifuge tube and the egg suspension concentration was adjusted to 1000 eggs / mL. The suspension was then set aside.

[0051] 57 strains of Polysporum roseum were inoculated on PDA medium and cultured for 7 days. 0.05% Tween 80 solution was added to elute the spores and 10 6 cfu / mL of spore suspension for later use.

[0052] 1 mL of spore suspension and 100 μL of nematode egg suspension were added to a 24-well tissue culture plate, mixed evenly, and allowed to stand at 26°C for 3 days. The parasitism of pink spiral polycystis strains on root-knot nematode eggs was measured under an inverted microscope (CK2, Olympus, Japan). Sterile water was used as a control, and 3 replicates were performed. The test found that 86% of the pink spiral polycystis strains (49 strains) could parasitize the eggs of root-knot nematodes. After the nematode eggs were cultured in contact with pink spiral polycystis for 3 days, fungal hyphae grew on the body wall of the nematodes. The parasitism rate of most pink spiral polycystis on root-knot nematodes is concentrated in the range of 30% to 60%, while the parasitism rate of strains BD-2-1, NYC-16-6, and HLF-14-11 on root-knot nematode eggs reached more than 60%. The situation of root-knot nematode eggs being parasitized by the HLF-14-11 strain is shown in the figure. Figure 3 Figures (a) show a healthy root-knot nematode egg with an intact body wall; (b) shows fungal hyphae penetrating the nematode's body wall, with the egg developing abnormally; and (c) shows the fungus utilizing the nematode's nutrients to reproduce in large numbers within the nematode, with hyphae extending beyond the eggshell and the egg wall beginning to dissolve.

[0053] (2) Toxic effect on second-instar larvae of root-knot nematodes

[0054] Place a sterilized support in a sterile culture dish and cover it with sterile filter paper. Transfer the sterilized root-knot nematode eggs onto the filter paper, add water to keep them moist, and incubate them at 25°C for 3 days. Collect the second-instar larvae and prepare a 1000 larvae / mL suspension for later use.

[0055] Fifty-seven strains of the pink spiral spore fungus were inoculated on polystyrene acetate (PDA) and cultured for 7 days. Approximately 5 mm thick pieces of culture were excised and inoculated into 500 mL Erlenmeyer flasks containing 100 mL of fermentation broth. The culture medium contained 25 g of glucose, 10 g of yeast extract, 1.0 g of potassium phosphate (K₂HPO₄), 0.5 g of sodium chloride (NaCl), 0.5 g of magnesium sulfate (MgSO₄·7H₂O), 0.05 g of fesulfate (FeSO₄·7H₂O), and 0.05 g of zinc sulfate (ZnSO₄·7H₂O) per liter, and the pH was adjusted to 6.0. The culture was shaken at 28°C and 180 rpm for 4 days to obtain the pink spiral spore fungus broth. The fermentation broth was centrifuged at 6000 rpm for 15 minutes, and the supernatant was filtered through a 0.22 μm microporous membrane to remove bacterial cells, obtaining the fermentation filtrate for later use.

[0056] To a 24-well tissue culture plate, add 0.1 mL of root-knot nematode larvae suspension and 1 mL of the fermentation filtrate of the pink spiral fungus strain, respectively, and mix. An equal volume of sterile water was used as a blank control. Incubate at 25°C for 2 days. The number of live and dead nematodes was recorded under an inverted microscope to calculate the corrected mortality rate. Repeat three times. Larvae that were active and moving in a curved manner were considered live, while those that were rigid and remained motionless after stimulation with NaOH solution were considered dead. The results showed that 52.6% of the tested strains (30 strains) had a mortality rate of less than 20% on second-instar larvae, 36.8% of the strains (21 strains) had a lethal effect on nematodes between 20% and 60%, and 6 strains (10.5%) had a mortality rate greater than 60%. Among them, the mortality rates of HLF-14-8-2 and HLD-1 strains reached 81.0% and 72.6% respectively. The HLF-14-11 strain had the strongest toxic effect, with a larval mortality rate of 89.1% and an adjusted mortality rate of 86.5%, which was significantly different from other strains (P < 0.01). Figure 4 Figure 2 shows the survival of second-instar larvae of the root-knot nematode (M. spp.) treated with the fermentation filtrate of the HLF-14-11 strain. (a) A healthy larva; (b) The breakdown of substances in the larvae's body after treatment with the fermentation filtrate of the HLF-14-11 strain; (c) The body wall of the larvae disintegrates, the contents leak out, and the nematode dies after treatment with the fermentation filtrate of the strain.

[0057] Based on the results of indoor bioassays, the pink spiral fungus HLF-14-11 strain was screened out, which can strongly parasitize root-knot nematode eggs and kill second-instar larvae.

[0058] Example 4 Effect of the Fermentation Filtrate of the Pink Helicoverpa HLF-14-11 Strain on the Hatching of Nematode Eggs

[0059] To a 24-well cell culture plate, 2 mL of HLF-14-11 fermentation filtrate was added, followed by 100 μL of a root-knot nematode egg suspension (approximately 100 eggs). An equal volume of sterile water was added as a blank control. The plates were incubated at 28°C. After 7 days, the strain's effect on root-knot nematode egg hatching was examined microscopically. The relative inhibition rate (RI) was calculated using clear water as the control. The RI = (egg hatching rate - hatching rate in clear water) / hatching rate in clear water × 100%. Three replicates were performed. The results showed that the RI of root-knot nematode eggs treated with HLF-14-11 fermentation filtrate was only 11.3%, resulting in a relative inhibition rate of 84%, demonstrating that HLF-14-11 fermentation filtrate effectively inhibits nematode egg hatching.

[0060] Example 5 Determination of the antagonistic ability of the pink spiral spore mold HLF-14-11 strain against several pathogenic fungi

[0061] The pink spiral polypore HLF-14-11 strain was inoculated and activated on a PDA plate. After it was covered with fresh mycelium, a 5mm cake was taken and inoculated 2cm away from the edge of the PDA plate (diameter 9cm). After culturing at 26℃ for 5 days, the cakes of tomato gray mold and apple early leaf fall pathogen were inoculated at the same distance on the opposite end of the plate. After culturing at 26℃ for 3-5 days, the distance that HLF-14-11 expanded to the pathogen was measured. Each experiment was repeated 3 times. The results are as follows Figure 5 As shown, the pink spiral fungus HLF-14-11 strain can produce broad-spectrum antimicrobial substances, effectively inhibiting the growth and expansion of hyphae of various pathogenic fungi. (a) The antagonistic effect of HLF-14-11 strain against tomato gray mold; (b) The antagonistic effect of HLF-14-11 strain against apple early leaf drop pathogen.

[0062] Example 6 Effect of the Fermentation Filtrate of the Pink Helicoverpa HLF-14-11 Strain on Tomato Seed Germination and Seedling Growth

[0063] Take the pink spiral polycystis HLF-14-11 strain fermentation filtrate prepared in Example 4, add an equal amount of sterile water to dilute it, and set aside. Take plump tomato seeds (Zhongza No. 9, purchased from Zhongshu Seed Technology Co., Ltd.), disinfect them with 2% NaClO for 3 minutes, rinse them repeatedly with sterile water, and after drying, immerse the seeds in a 2-fold diluted HLF-14-11 strain fermentation filtrate. After 15 minutes, take them out, dry them, transfer them to a 9 cm sterile culture dish with two layers of filter paper on the bottom, and place them in an incubator at 28°C for cultivation. Place 10 seeds in each culture dish, and use sterile water and PD culture medium as controls. After 24 hours, start counting the number of germinated seeds, calculate the germination index after 7 days, and measure the length of the shoots and roots. The results show that pink spiral polycystis can promote tomato seed germination and the growth of young shoots and roots. The germination index of tomato seeds treated with HLF-14-11 fermentation filtrate was significantly better than that of the water and PD culture medium controls, and the seed sprout length and root length increased by 47.5% and 55.4% respectively compared with the water control.

[0064] Example 7 Detection of indoleacetic acid (IAA) in metabolites of Polyporus roseus HLF-14-11 strain

[0065] The pink spiral polycytobacter HLF-14-11 strain was inoculated into PD medium containing 1% L-tryptophan, cultured at 28°C and 180r / min for 7 days, the fermentation broth was centrifuged at 6000r / min for 15min, filtered through a 0.22μm microporous filter membrane to remove hyphae and spores, and then the sterile fermentation filtrate was centrifuged at 12000r / min at 4°C for 10min to obtain the supernatant. 2mL of supernatant was added to the test tube, followed by 4mL of Salkowaski reagent and 2 drops of orthophosphoric acid, and shaken to mix. The mixture was incubated at 28°C for 2h. If the mixture turned red, it means that the metabolites of pink spiral polycytobacter contain IAA. The OD value of the mixture was measured at 530nm using a UV spectrophotometer, and compared with the IAA standard curve to calculate the concentration of IAA in the metabolites of pink spiral polycytobacter. Figure 6 Figure 1 shows the results after adding the detection reagent to PD medium containing 1% L-tryptophan, showing no red reaction. Figure 2 shows a distinct red reaction after adding the detection reagent to the fermentation filtrate of the pink spiral fungus HLF-14-11 strain, indicating that it produces a large amount of IAA during metabolism. Compared to the standard curve, the IAA content in the sample was 96.5 mg / L.

[0066] Example 8 Screening of liquid fermentation medium for the pink spiral polypore HLF-14-11 strain

[0067] In order to obtain a microbial agent suitable for production and field application, a single factor experiment was conducted to screen and optimize the fermentation medium of the HLF-14-11 strain.

[0068] The pink spiral polyporus with the above-mentioned deposit number CGMCC No. 41822 was inoculated from the deposited slant into PDA medium, cultured at 28° C. for 7 days, and sterile water was added to wash away the spores to prepare a spore suspension for later use.

[0069] Optimization of carbon source:

[0070] Liquid culture media containing different carbon sources were prepared at a dosage of 30 g / L to screen for a suitable carbon source for the conidia formation of pink spiral fungus HLF-14-11. Six treatments were set up: (1) glucose, (2) corn flour, (3) sucrose, (4) starch, (5) malt extract, and (6) glucose / corn flour = 1:2 (mass ratio). The other ingredients in the culture medium were yeast extract 7 g, K2HPO4 1.0 g, NaCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.05 g, and ZnSO4·7H2O 0.05 g. Water was added to 1000 mL and the pH was adjusted to 6.2. 100 mL of liquid culture medium was added to a 500 mL conical flask and sterilized by high-temperature wet heat. When cooled to room temperature, the spore suspension was inoculated into the liquid culture medium at a 1% inoculum rate and cultured at 26°C with shaking at 180 r / min for 72 h to obtain a fermentation broth. The spore content in different fermentation broths was determined by hemocytometer counting, with 3 replicates for each treatment. The results are shown in Table 1. When glucose and corn flour were used as a combined carbon source, HLF-14-11 had the highest spore production, reaching 2.93×10 8 / mL, which was significantly different from other treatments (P<0.05).

[0071] Table 1 Effects of different carbon sources on conidia production of Polycystis rosea HLF-14-11

[0072]

[0073] Optimization of nitrogen source:

[0074] Liquid culture media containing different nitrogen sources were prepared, and shake flask cultures were used to screen for suitable nitrogen sources for conidia formation in the pink spiral fungus HLF-14-11. A total of eight treatments were set up: (1) soybean meal, (2) fish meal, (3) yeast extract powder, (4) soy peptone, (5) urea, (6) NaNO3, (7) soybean meal / urea (nitrogen mass ratio 1:1), and (8) yeast extract powder / urea (nitrogen mass ratio 1:1). Different types of nitrogen sources were used in the experiment, including organic nitrogen, inorganic nitrogen, and a combination of organic and inorganic nitrogen, to achieve higher fermentation levels and minimize costs.

[0075] The amount of each nitrogen source added to the culture medium was calculated based on a nitrogen content of 0.6 g / L. The other ingredients were glucose 25 g, K₂HPO₄ 1.0 g, NaCl 0.5 g, MgSO₄·7H₂O 0.5 g, FeSO₄·7H₂O 0.05 g, and ZnSO₄·7H₂O 0.05 g. Water was added to 1000 mL, and the pH was adjusted to 6.2. 100 mL of liquid culture medium was added to a 500 mL Erlenmeyer flask and sterilized with high-temperature moist heat. After cooling to room temperature, the spore suspension was inoculated into the liquid culture medium at a 1% inoculum rate. The culture was shaken at 26°C and 180 rpm for 72 h to obtain a fermentation broth. Spore counts were performed on a hemocytometer, and spore counts were determined in the different fermentation broths. Three replicates were performed for each treatment. The results are shown in Table 2. Significant differences were observed among the different nitrogen source treatments (P < 0.05). When organic nitrogen soybean meal, fish meal, yeast extract powder or inorganic nitrogen urea was used as nitrogen source, the fermentation level of HLF-14-11 strain could reach 10 8 Spores / mL. Inorganic nitrogen quickly provides nitrogen, promoting biomass growth and reducing raw material costs; organic nitrogen facilitates microbial growth and metabolism. Combining organic and inorganic nitrogen, urea in the culture medium as a fast-acting nitrogen source, enables rapid germination and growth of biocontrol bacteria, while soybean meal powder or yeast extract powder, as a persistent nitrogen source, provides long-lasting effects, promoting bacterial growth and sporulation. When soybean meal powder / urea is used as a combined nitrogen source, spore production at the end of fermentation increases by over 50% compared to using a single nitrogen source.

[0076] Table 2 Effects of different nitrogen sources on conidia production of Polycystis rosea HLF-14-11

[0077]

[0078] Example 9 Liquid fermentation of pink spiral polyporus HLF-14-11 strain

[0079] The pink spiral polysporus HLF-14-11 strain was inoculated into PDA culture medium and cultured at 28°C for 7 days. Sterile water was added to wash off the spores and prepare a spore suspension. 100 mL of liquid culture medium was added to a 500 mL conical flask and sterilized by high-temperature wet heat. When cooled to room temperature, the spore suspension was inoculated into the liquid culture medium at a 1% inoculation rate and cultured at 26°C and 180 r / min for 64 hours to prepare a seed liquid. The seed liquid was then inoculated into a 2000 mL conical flask containing 500 mL of fermentation medium at a 2% inoculation rate and cultured at 28°C and 200 r / min for 4 days to obtain the HLF-14-11 strain fermentation liquid. The spore content of the fermentation liquid was measured by hemocytometer counting and was 2.2×10 8 pieces / mL.

[0080] The liquid culture medium is formulated as follows: 10.0 g glucose, 20.0 g corn flour, 8.0 g soybean meal, 0.6 g urea, 1.0 g K₂HPO₄, 0.5 g NaCl, 0.5 g MgSO₄·7H₂O, 0.05 g FeSO₄·7H₂O, and 0.05 g ZnSO₄·7H₂O per 1000 mL of water. Adjust the pH to 6.0 with 1 M hydrochloric acid and sterilize with moist heat at 121°C to obtain the fermentation medium.

[0081] Example 10: Bacterial agent containing pink spiral polyporus strain

[0082] Preparation of pink spiral polyporus HLF-14-11 powder:

[0083] A carrier consisting of diatomaceous earth and rice husk powder (mass ratio 1:1) was added to the fermentation broth of the HLF-14-11 strain obtained in Example 9. The mixture was uniformly mixed, dried at 35°C, and pulverized to prepare an HLF-14-11 powder. This preparation is primarily used as a seedling medium and for hole application during transplanting.

[0084] Weigh 1 g of powder, add sterile water to dilute it serially, spread it on PDA culture medium, and determine the viable bacterial content of the powder to be 1.4×10 8 CFU / g.

[0085] Preparation of pink spiral polyporus HLF-14-11 granules:

[0086] The fermentation broth of the HLF-14-11 strain obtained in Example 9 was added with a combination of a carrier and an auxiliary agent in a mass ratio of 1:1.1, specifically a combination of diatomaceous earth, starch, sodium carboxymethyl cellulose and amino oligosaccharides (mass ratio of 25:10:1.5:0.3), mixed, granulated with a granulator, and dried at 35°C to obtain granules.

[0087] Weigh 1 g of granules, add sterile water to dilute serially, spread on PDA culture medium, and determine the viable bacterial content of the granules to be 1.5×10 8 CFU / g.

[0088] The preparation is soluble in water, so it can be applied in holes, furrows, or root irrigation when transplanting seedlings, or it can be drip-irrigated or flushed during the growing season.

[0089] Example 11 Potted Plant Experiment Verification of the Pink Spirospora Strain for Controlling Tomato Root-knot Nematode Disease

[0090] Four pink spiral spore mold strains (HLF-14-11, HLF-14-8-2, HLD-1 and BD-2-1) with good results in the laboratory initial screening were inoculated on PDA plates and cultured for 7 days. 0.05% Tween 80 solution was added to elute the spores, and the spores were inoculated into fermentation medium at a 2% inoculum rate. The culture was shaken at 28°C and 180 r / min for 3 days to obtain the fermentation broth of the four pink spiral spore mold strains.

[0091] The pot experiment was conducted in the greenhouse of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing. The tomato variety used was Zhongza No. 9. Full-grained tomato seeds were selected, soaked in 2% NaClO solution for 3 minutes for disinfection, rinsed repeatedly with sterile water, placed in a culture dish lined with sterile filter paper, and kept moisturized at 28°C for germination. After germination, the seeds were sown in a sterile substrate seedling tray. When they grew to 4 true leaves, seedlings with uniform growth were selected and transplanted into 10×10 cm plastic pots, with one tomato seedling transplanted per pot. Seven days after transplanting, a hole was punched within 2 cm around the base of each seedling stem, and 10 mL of the strain fermentation liquid was poured in. At the same time, a suspension of second-instar larvae of the root-knot nematode (about 500) was inoculated. A total of six treatments were set up in the experiment, namely (1) HLF-14-11, (2) BD-2-1, (3) HLD-1, (4) HLF-14-8-2, (5) CK1 (clear water control), and (6) CK2 (fermentation medium control). Five plants were treated per treatment, with three replicates, and treatment groups were randomly arranged. The greenhouse temperature was 25°C / 22°C (day / night), with a photoperiod of 16 hours / 8 hours (day / night). After 30 days, tomato plant height, stem diameter, chlorophyll content, and aboveground and underground fresh weight were measured. Disease was graded according to a 5-level scale: Level 0: No root knots; Level 1: Only a few root knots, accounting for 1% to 25% of the total root system; Level 3: Moderate number of root knots, accounting for 26% to 50% of the total root system; Level 5: Numerous root knots, accounting for 51% to 75% of the total root system; Level 7: Extremely numerous root knots, accounting for 76% to 100% of the total root system. The disease index and relative control efficacy were calculated using the following formula.

[0092] The investigation found that the pink spiral fungus liquid had a good effect on promoting tomato growth and root development and preventing and controlling root knot nematode disease after root irrigation. Among them, the HLF-14-11 strain had the best effect. After treatment, the height, stem diameter, chlorophyll content and aboveground fresh weight of tomatoes increased by 27.4%, 48.6%, 37.5% and 64.6% respectively. Figure 7 As shown, the tomato roots in the control group were severely damaged, with deformed root tissue and numerous root knots. However, the roots of the tomatoes treated with HLF-14-11 solution only developed a few small root knots, achieving an 86.2% efficacy against root-knot nematodes (Table 3). Furthermore, the roots of the plants treated with HLF-14-11 solution were robust, with root weight increasing by 2.4 times compared to the water control group.

[0093] Table 3 Effect of pink spiral polysporus liquid on disease prevention and growth promotion of tomato potted plants

[0094]

[0095]

[0096] Example 12: Effect of the pink spiral spore mold HLF-14-11 on the prevention and treatment of tomato root knot nematode disease in greenhouses

[0097] The experiment was carried out in August 2024 in a solar greenhouse at the experimental base of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Langfang, Hebei Province, for 90 days. The ridge length of the greenhouse was 6m and the interval was 0.75m. Before planting, the base number of nematodes in the soil was determined by the Kaber sieving method to be 161 heads / 100g soil. The tomato seedlings that had been cultivated for 4 weeks were transplanted, and the pink spiral polysporus HLF-14-11 granules (prepared in Example 10) were applied in holes at a dosage of 1kg per mu, and the root was irrigated again after 30 days to apply the fungus. The field disease prevention effect of pink spiral polysporus was evaluated using no fungus treatment and the chemical pesticide trifluoperazine as controls. The growth of tomatoes was measured 30 days after planting, and the plant height and stem thickness were measured with a tape measure and a vernier caliper. Three leaves were picked from the upper, middle and lower parts of the plant, and the leaf area was calculated using the grid method. After 90 days, the incidence of tomato root-knot nematodes was investigated, and the disease index and prevention effect were calculated. The number of root-knot nematodes in the soil was measured 30, 60, and 90 days after tomato planting. 200 g of soil samples were collected using the Kappa sieve separation method and counted under an inverted microscope to determine the effect of the pink spiral spore inoculation on the root-knot nematode population density in the soil. Conventional greenhouse management was used throughout the experiment, with normal watering and fertilization for all treatments. Three rows, 15 seedlings per row, and three replicates were used for each treatment.

[0098] Tomato growth indicators were measured 30 days after application. Results showed that continuous application of HLF-14-11 achieved a 74.3% efficacy against tomato root-knot nematodes, slightly lower than the chemical pesticide trifluoperazine. However, it significantly promoted tomato growth, with stem diameter increasing by 25.3% and leaf area by 49.3%, thereby boosting photosynthesis and improving quality and yield. Nematode abundance in soil at different planting times was measured. After 90 days of HLF-14-11 application, the root-knot nematode count dropped to 75 per 100g of soil, representing a decrease of over 50% from the initial count. In untreated soil, the nematode count rose to 409 per 100g, a 154% increase. This indicates that the pink spiral polycystic ovary syndrome (HLP) agent significantly reduced nematode infestation and effectively inhibited root infection.

[0099] Although the present invention has been described above through the embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from the spirit and essence of the present invention should fall within the scope of protection of the present invention.

Claims

1. A pink spiral nematode strain for preventing and treating root-knot nematodes, characterized in that: The deposit number is CGMCC No.41822.

2. A bacterial agent containing the pink spiral spore mold strain according to claim 1, characterized in that: The bacterial agent is prepared by the following method: 1) Strain culture: Inoculate HLF-14-11 on potato dextrose agar and culture at 25-28°C for 6-10 days until conidia are formed. 2) Liquid fermentation inoculum preparation: Elute the spores and prepare a spore suspension, or cut 3-6 x 3-6 mm pieces of bacteria from the cultured plates to use as the inoculum; 3) Liquid fermentation culture: Prepare a liquid culture medium, inoculate the spore suspension or bacterial mass, and perform primary culture or secondary culture. The primary culture is a shaking culture on a shaker at 25-28°C. The secondary culture is a scaled-up culture using the product of the primary culture as a seed solution and inoculating it with fresh liquid culture medium. 4) Preparation: The fermentation liquid obtained in step 3) is prepared into a bacterial agent, and the dosage form of the bacterial agent is liquid, granule or powder.

3. The microbial agent according to claim 2, characterized in that The formula of the culture medium for liquid fermentation culture in step 3) is: 20-40g of carbon source, 5-20g of nitrogen source, 0.5-2g of K2HPO4, 0.1-1g of NaCl, 0.1-1g of MgSO4·7H2O, 0.01-0.1g of FeSO4·7H2O, and 0.01-0.1g of ZnSO4·7H2O; the above ingredients are mixed and water is added to 1000mL, and the pH is adjusted to 5.5-6.5; the carbon source is selected from one or more of glucose, sucrose, corn flour, starch and malt extract; the nitrogen source is selected from one or more of soybean cake powder, fish meal, yeast extract, soy peptone, urea and NaNO3, and the amount of nitrogen source added is calculated based on the nitrogen content of 0.3-1.5g / L.

4. The microbial agent according to claim 3, characterized in that The carbon source is a mixture of glucose and corn flour in a mass ratio of 1 to 2:2; the nitrogen source is a mixture of soybean cake powder and urea in a nitrogen element mass ratio of 1 to 3:

1.

5. The microbial agent according to claim 2, characterized in that In step 3), the shaking culture is as follows: the spore suspension inoculation amount is 0.5-5%, or 4-6 pieces of the bacterial blocks are inoculated; The shaking speed is 150-250 r / min, the culture temperature is 25-28°C, and the culture time is 48-96 h.

6. The microbial agent according to any one of claims 2 to 5, characterized in that In step 4), a stabilizer is added to the fermentation broth in an amount of 0 to 10% by weight of the fermentation broth to obtain a liquid; a carrier and an auxiliary agent are added to the fermentation broth, mixed, granulated, and dried at 30 to 45° C. to obtain a granule; a carrier or a combination of a carrier and an auxiliary agent is added to the fermentation broth, dried at 30 to 45° C., and crushed to obtain a powder; the carrier is one or more of diatomaceous earth, light calcium carbonate, talc, kaolin, attapulgite, bentonite, starch, dextrin, peat, rice husk powder, straw powder, and corn cob powder; the auxiliary agent is one or more of sodium carboxymethyl cellulose, xanthan gum, amino oligosaccharide, sodium alginate, glycine, and silicate; the amount of the carrier added is 30% to 300% by weight of the fermentation broth, and the amount of the auxiliary agent added is 0.1% to 10% by weight of the fermentation broth.

7. The microbial agent according to any one of claims 2 to 5, characterized in that The bacterial agent is a liquid agent, and the bacterial content in the bacterial agent is (1-5)×10 8 CFU / mL; or: the microbial agent is a granule or powder, and the bacterial content in the agent is (1-50)×10 7 CFU / g.

8. Use of the pink spiral spore mold strain according to claim 1 in preventing and controlling root knot nematode disease of crops, gray mold of fruits and vegetables, early leaf drop disease of fruit trees, sclerotinia wilt, wilt, verticillium wilt, and blight of economic crops.

9. Use of the microbial agent according to any one of claims 2 to 7 in preventing and controlling root-knot nematode disease of crops, gray mold of fruits and vegetables, early leaf drop disease of fruit trees, sclerotinia rot, wilt, verticillium wilt, and blight of economic crops.

Images