Catenaria anguillae and culture method and application thereof

Abstract

This invention relates to a strain of *Verticillium chlamydosporium*, its culture method, and its applications, belonging to the field of microbial technology. The *Verticillium chlamydosporium* Vc-GXWFY-01 was deposited on April 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.40400. This strain has a high spore yield and exhibits significant functions in controlling nematodes in agricultural and forestry crops and promoting crop growth. Its control effect on root-knot nematodes reaches 50-87%, increasing crop yield and significantly improving economic benefits. The solid-state fermentation medium of this invention ensures sufficient organic matter for the strain while also increasing aeration within the medium, greatly enhancing the production of *Verticillium chlamydosporium* and the number of chlamydosporin spores, producing up to 9.4 × 10⁻⁶ spores. 7 ~2.2×10 8 The number of conidia is 2.7 × 10⁶ / g. 9 ~2.9×10 9 per g.

Description

Technical Field

[0001] This invention relates to a strain of chlamydosporium verticillata, its culture method and application, and belongs to the field of microbial technology. Background Technology

[0002] Plant nematodes are one of the major diseases causing crop yield reductions, resulting in approximately US$157 billion in losses to global agriculture annually. Methods for controlling plant nematodes mainly include physical control, chemical control, screening and application of resistant varieties, and biological control. Currently, chemical control is the primary method, but chemicals are highly toxic, easily pollute the environment, and leave residues, posing a threat to human and animal health. Physical control is not very effective, and the screening process for resistant varieties is lengthy. Therefore, safe and efficient biological control has become a hot topic in plant nematode control. *Verticillium chlamydosporium* is a multi-trophic fungus that can saprophytically in soil, parasitize nematodes, and be endogenous in plant roots. It controls nematode reproduction by infecting and primarily parasitizing nematode eggs, females, cysts, or oocysts through hyphal infection. It has significant biocontrol potential and growth-promoting effects against plant nematodes, and is mainly used in China to control nematode diseases in agricultural and forestry crops.

[0003] *Verticillium* belongs to the kingdom Fungi, phylum Ascomycota, class Sordariomycetes, subclass Hypocreomycetidae, order Glomerellales, family Plectosphaerellaceae, and genus *Verticillium*. The nematode fungus within this genus has been internationally classified under the genus *Pochonia*. In this invention, except where referenced in the patent, *Verticillium* is used to refer to *Chronicillium*. *Verticillium* produces conidia when its hyphae mature, or through sexual or asexual reproduction when encountering unfavorable growth conditions. Compared to conidia, chlamydospores are more resistant to harsher environments. As a commercial product, chlamydospores have advantages that conidia cannot match: longer shelf life, higher activity, and stronger resistance. Therefore, for factories, high-yield, high-quality thick-walled spores are the basic guarantee for producing efficient microbial agents.

[0004] Chinese patent document CN112315985A discloses a wettable powder of *Puconia thunbergii* (CGMCC No. 19635), its preparation, and its application. According to its patented technology, the freeze-dried bacterial powder produced contains up to 6.8 × 10⁻⁶ spores. 7The Chinese patent document CN107955802A discloses a method for solid-state fermentation of *Prcetinium chlamydosporium* (*Prcetinium chlamydosporium* strain PC152) to produce spores. This method utilizes inexpensive and readily available raw materials such as corn flour, mushroom straw, and sucrose for solid-state fermentation, yielding 1.85 × 10⁻⁶ spores per gram after 3 weeks. 6 1.4 × 10⁶ spores / g. Chinese patent document CN106701655A discloses a method for producing *Przewalski's sinensis* (*Przewalski's sinensis* strain ACCC30601) spores through liquid-solid two-phase fermentation. This method involves first liquid fermentation, followed by fermentation using wheat bran, corn flour, and corn stalk powder as raw materials. After 10 days, 1.4 × 10⁶ spores can be harvested. 6 1 / g thick-walled spores.

[0005] As can be seen from the above reports and studies, the production of chlamydospores of *P. chlamydosporium* on solid and liquid culture media using existing patented technologies generally results in low yields, and some fermentation cycles are too long to meet industrial production needs. Furthermore, the widely used shallow-tray fermentation method has limited production scale; the temperature and humidity of parallel fermentation trays are difficult to control uniformly, leading to large and unstable variations in sporulation yields and low overall production. Therefore, it is essential to screen for *Verticillium chlamydosporium* strains with high sporulation yields, especially high chlamydosporium spore yields, and to explore fermentation methods with stable yields. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a strain of *Verticillium chlamydosporium*, its culture method, and its applications.

[0007] The technical solution of the present invention is as follows:

[0008] A strain of Verticillium chlamydosporium Vc-GXWFY-01 was deposited on April 12, 2023, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.40400.

[0009] The solid-state fermentation method for the above-mentioned Verticillium chlamydosporium Vc-GXWFY-01 comprises the following steps:

[0010] (1) Take the chlamydomonas verticillata Vc-GXWFY-01 and inoculate it into Bengal red medium or PDA solid medium. Under the conditions of 25℃~28℃, activate and culture for 3~4 days to obtain the activated strain.

[0011] (2) Take the activated strain from step (1) and inoculate it into the seed culture medium. Under the conditions of 28℃~30℃ and 150~170r / min, shake and culture for 2~4 days to obtain the first-grade seed liquid.

[0012] (3) Inoculate the primary seed culture from step (2) into the secondary seed culture medium at a volume percentage of 4-6%, and culture it under shaking conditions of 28℃-30℃ and 150-170r / min for 2-3 days to obtain the secondary seed culture.

[0013] (4) Inoculate the secondary seed liquid from step (3) into a solid fermentation medium at a mass percentage of 10-20%, and culture at 25℃-32℃ for 7-15 days. Dry at low temperature to obtain the fermentation product of Chlamydomonas verticillata.

[0014] According to a preferred embodiment of the present invention, in step (1), the formula of the Bengal red culture medium is: 5g peptone, 10g glucose, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 20g agar, 100mL 1 / 3000 Bengal red solution, and distilled water to a final volume of 1000mL; the formula of the PDA solid culture medium is: 200g potato, 20g glucose, 20g agar, distilled water to a final volume of 1000mL, with a natural pH.

[0015] According to a preferred embodiment of the present invention, in step (2), the seed culture medium is PDA liquid culture medium with the following formula: 200g potato, 20g glucose, and distilled water to a final volume of 1000mL, with a natural pH.

[0016] According to a preferred embodiment of the present invention, in step (3), the secondary seed culture medium formula is as follows: 20 g / L sucrose, 10 g / L corn flour, 10 g / L urea, 1.15 g / L sodium nitrate, 0.8 g / L potassium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate heptahydrate, 0.3 g / L potassium chloride, 0.2 g / L sodium chloride, 0.3 g / L potassium dihydrogen phosphate, and pH 7.

[0017] According to a preferred embodiment of the present invention, in step (4), the solid fermentation culture medium contains the following raw materials in parts by weight: 20-35 parts wheat bran, 20-35 parts rice husk powder, 5-15 parts corn flour, 5-10 parts rice flour, 20-40 parts peanut shell powder, and 55-110 parts inorganic salt nutrient solution.

[0018] More preferably, the inorganic salt nutrient solution contains 5-15 g / L sucrose, 5-15 g / L ammonium sulfate, 10-20 g / L sodium nitrate, 0.8 g / L potassium nitrate, 0.5-1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate heptahydrate, and 5 g / L potassium dihydrogen phosphate.

[0019] According to a preferred embodiment of the present invention, the solid fermentation medium is prepared by the following method:

[0020] Weigh out fresh wheat bran, rice husk powder, corn flour, rice flour and peanut shell powder by weight ratio, mix them evenly to obtain solid material;

[0021] Add sucrose, ammonium sulfate, sodium nitrate, potassium nitrate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, and potassium dihydrogen phosphate to water in the specified weight ratio to obtain an inorganic salt nutrient solution.

[0022] Add the solid material to the inorganic salt nutrient solution, stir evenly, sterilize by high temperature and high pressure steam at 121℃ for 30 minutes, and it can be used as a solid fermentation culture medium after cooling.

[0023] According to a preferred embodiment of the present invention, in step (4), the temperature is lowered by cooling water and pressure pulsation is performed during the expansion culture process. The pressure pulsation is mainly used to supply oxygen for microbial growth. The pulsation is performed once every 6 hours during the lag phase, once every 2 hours during the logarithmic phase, and once every 3 to 4 hours during the stationary phase.

[0024] According to a preferred embodiment of the present invention, in step (4), the number of chlamydospores in the chlamydospore fermentation product is mostly concentrated at 9.4 × 10⁻⁶. 7 ~2.2×10 8 The number of conidia is concentrated at 2.7 × 10⁶ / g. 9 ~2.9×10 9 The number of cells / g can reach up to 3.2 × 10⁻⁶. 9 per g.

[0025] All the ingredients described in this invention are commercially available products. For any omissions, existing technologies can be used.

[0026] The beneficial effects of this invention are as follows:

[0027] 1. This invention provides a strain of Verticillium chlamydosporium Vc-GXWFY-01, which has a high spore yield and significant functions in controlling nematodes in agricultural and forestry crops and promoting crop growth. The control effect on root-knot nematodes reaches 50-87%, which can increase crop yield and greatly improve economic benefits.

[0028] 2. The solid-state fermentation method provided by this invention uses wheat bran, rice husk powder, corn flour, rice flour, and peanut shell powder as a solid culture medium. This ensures sufficient organic matter for the bacterial strain while increasing the aeration rate within the solid-state fermentation medium, significantly improving the production of *Verticillium chlamydosporium* and the number of chlamydosporins, achieving a yield of 9.4 × 10⁻⁶ chlamydosporins. 7 ~2.2×10 8 The number of conidia per gram of solid fermentation medium was 2.7 × 10⁶. 9 ~2.9×10 9 Solid fermentation medium (units / g).

[0029] 3. This invention utilizes a gas-phase dual-dynamic solid-state fermentation reactor for effective scale-up cultivation, achieving the requirements for pure culture and large-scale industrialization, and realizing the large-scale production of Cercospora thunbergii. Attached image description:

[0030] Figure 1 The colony morphology and hyphal microstructure of strain Vc-GXWFY-01;

[0031] In the figure, A shows the colony morphology of strain Vc-GXWFY-01 on Bengal Red medium or PDA solid medium; B shows the hyphal microstructure of strain Vc-GXWFY-01 after two days of growth (hyphae under 40x and 100x objectives, respectively).

[0032] Figure 2 This indicates the position of strain Vc-GXWFY-01 in the maximum likelihood phylogenetic tree.

[0033] Figure 3 A visual representation of the fermentation product of *Verticillium chrysogenum*.

[0034] Figure 4 Images of chlamydospores observed under 10×10 and 40×10 magnification microscopes of the fermentation product of *Verticillium chlamydosporum*.

[0035] Figure 5 The image shows the effects of Verticillium chrysanthum on the control of root-knot nematodes in tomatoes and on promoting plant growth.

[0036] In the figure, CK represents the control, and T represents the treatment with Verticillium chlamydosporium solid fermentation. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, specific embodiments are provided below to further explain the invention, but these embodiments do not constitute a limitation on the invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods; and unless otherwise specified, all percentage contents in the following embodiments are weight percentages.

[0038] Example 1

[0039] From diseased tomato fields in Linyi City, Shandong Province, approximately 20 root-knot nematode egg masses were collected from the roots of infected tomatoes using tweezers. The egg masses were rinsed three times with sterile water, and the contents of the eggs were extracted using sterile tweezers and spread onto PDA solid medium containing 300 mg of streptomycin sulfate. The medium was incubated at 28°C for 5 days. After isolation and purification, a fungus was obtained and named Vc-GXWFY-01. Strain Vc-GXWFY-01 was inoculated onto Bengal red agar and PDA medium, respectively, and incubated at 28°C for approximately 2 to 5 days. Colony morphology was observed, and colony growth radius was measured.

[0040] Depend on Figure 1 It can be seen that after 2 days of culture, strain Vc-GXWFY-01 showed white hair-like structures with regular edges on both Bengal Red medium and PDA solid medium, with a morphology similar to... Figure 1 As shown, after about 5 days of growth on the PDA, the colony radius increased by 10-13 mm.

[0041] After the strain was cultured on a PDA plate, its 18S rDNA was sent for external detection. Sequencing yielded a fragment length of 589 bp, as shown in SEQ ID NO.1. The sequence of a standard reference strain was obtained from the NCBI database. The 18S rDNA sequences of the isolated strain and the reference strain were analyzed using MEGA-6, and a construct was built as follows: Figure 2 The maximum likelihood phylogenetic tree of the isolated strain and the reference strain is shown.

[0042] Depend on Figure 2It is evident that the 18S rDNA gene sequence of strain Vc-GXWFY-01 isolated in this invention has extremely high similarity to the 18S rDNA gene sequence of *Pochonia chlamydosporia*. As seen in the phylogenetic tree, the distance bar is very small, at 0.0005, which aligns with Gams W. and Zara R's (A revision of *Verticillium sect. Prostrata. III. Generic classification*) classification of nematode fungi in *Verticillium* to the genus *Pochonia* in 2001. Therefore, strain Vc-GXWFY-01 is identified as *Verticillium chlamydosporium* and named *Verticillium chlamydosporium* Vc-GXWFY-01. This strain was deposited on April 12, 2023, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.40400.

[0043] Example 2

[0044] A solid-state fermentation method for *Verticillium chlamydosporium*, comprising the following steps:

[0045] (1) Inoculate Verticillium chrysogenum Vc-GXWFY-01 into Bengal Red medium or PDA solid medium, and culture it in an incubator at 28℃ for 3 days to obtain the activated strain;

[0046] The formula for the Bengal Red culture medium is as follows: 5g peptone, 10g glucose, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 20g agar, 100mL 1 / 3000 Bengal Red solution, and distilled water to a final volume of 1000mL.

[0047] The formula for the PDA solid culture medium is: 200g potato, 20g glucose, 20g agar, and distilled water to a final volume of 1000mL, with a natural pH.

[0048] (2) Use a punch to take a fungal cake from the edge of the colony as an inoculum. Inoculate 5 fungal cakes per 100ml of PDA liquid medium. Inoculate the activated strain from step (1) into PDA liquid medium and culture it for 2 days at 28℃ and 160rpm to obtain the first-grade seed liquid.

[0049] The formula for the PDA liquid culture medium is as follows: 200g potato, 20g glucose, and distilled water to a final volume of 1000mL, with a natural pH.

[0050] (3) Inoculate the primary seed culture from step (2) into the secondary seed culture medium at a volume percentage of 5%, and culture it under shaking conditions at 28℃ and 160 r / min for 3 days to obtain the secondary seed culture.

[0051] The secondary seed culture medium formula is as follows: 20 g / L sucrose, 10 g / L corn flour, 10 g / L urea, 1.15 g / L sodium nitrate, 0.8 g / L potassium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate heptahydrate, 0.3 g / L potassium chloride, 0.2 g / L sodium chloride, 0.3 g / L potassium dihydrogen phosphate, with a pH of 7.

[0052] (4) The secondary seed liquid from step (3) was inoculated into the solid fermentation medium at a mass percentage of 15%. After stirring evenly, it was placed into a fermentation tank and cultured at 28°C for 8 days. After low-temperature drying, the fermentation product of Chlamydomonas verticillata was obtained.

[0053] The solid fermentation medium is prepared by mixing 5 parts corn flour, 5 parts rice flour, 30 parts wheat bran, 25 parts rice husk powder, and 35 parts peanut shell powder evenly, then adding 82 parts inorganic salt nutrient solution and stirring evenly to achieve a moisture content of approximately 45% and a natural pH; then fermenting at 121℃ and a pressure of 2 kg / cm². 3 Under the conditions of high pressure steam sterilization in an autoclave for 30 minutes, the material is then pumped into a double cone tank for cooling to obtain a solid fermentation medium.

[0054] The inorganic salt nutrient solution contains 10 g / L sucrose, 10 g / L ammonium sulfate, 15 g / L sodium nitrate, 0.8 g / L potassium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, and a natural pH.

[0055] During the expanded culture process, the temperature is lowered by cooling water and pressure pulsation is performed. The pressure pulsation is mainly used to supply oxygen for microbial growth. The pulsation is performed once every 6 hours during the lag phase, once every 2 hours during the logarithmic phase, and once every 3 hours during the stationary phase.

[0056] The fermentation product of *Verticillium chlamydosporum* obtained in this embodiment is as follows: Figure 3 As shown, the chlamydospores of the *Verticillium chlamydosporum* fermentation product under 10×10 and 40×10 magnification microscopes are as follows: Figure 4 As shown. By Figure 3 and Figure 4 It is known that the present invention has successfully obtained the fermentation product of *Verticillium chlamydosporum*, and the fermentation product of *Verticillium chlamydosporum* contains a large number of spores.

[0057] Then, the sporulation rate of the fermentation product of *Verticillium chlamydosporium* prepared in this embodiment was determined, and the specific method is as follows:

[0058] 10g of the fermentation product of *Verticillium chrysogenum* obtained in this example was placed in an Erlenmeyer flask containing glass beads. 100mL of 0.15% Tween-80 solution was added, and the flask was shaken thoroughly on a shaker for 30min at a speed of 170r / min to obtain a spore suspension. After serial dilution by 10-fold, 15μL of the spore suspension was pipetted onto a glass slide, covered with a coverslip, and counted under an optical microscope. The results were repeated 3 times and the average was taken. The results are shown in Table 1.

[0059] Table 1

[0060] Thick-walled spores <![CDATA[1.9×10 8 [Units / g solid fermentation medium] Conidia <![CDATA[2.9×10 9 [Units / g solid fermentation medium]

[0061] Example 3

[0062] The solid fermentation method of a strain of Chlamydomonas verticillata is the same as that described in Example 1, except that step (4) is: the secondary seed liquid is inoculated into the solid fermentation culture medium at a mass percentage of 20%, stirred evenly, and then placed into a fermentation tank. It is cultured at 26°C for 10 days and then dried at low temperature to obtain the fermentation product of Chlamydomonas verticillata.

[0063] The solid fermentation medium is prepared by uniformly mixing 15 parts corn flour, 10 parts rice flour, 20 parts wheat bran, 35 parts rice husk powder, and 20 parts peanut shell powder, then adding 82 parts inorganic salt nutrient solution and stirring until the water content is about 45% and the pH is natural; then it is subjected to 121℃ and 2Kg / cm pressure. 3 Under the conditions of high pressure steam sterilization in an autoclave for 30 minutes, the material is then pumped into a double cone tank for cooling to obtain a solid fermentation medium.

[0064] The inorganic salt nutrient solution contains 15 g / L sucrose, 15 g / L ammonium sulfate, 11.5 g / L sodium nitrate, 0.8 g / L potassium nitrate, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, and a natural pH.

[0065] The number of chlamydospores and conidia in the fermentation product of *Verticillium chlamydosporum* in this example was determined according to the method described in Example 2, and the results are shown in Table 2.

[0066] Table 2

[0067] Thick-walled spores <![CDATA[9.4×10 7 [Units / g solid fermentation medium] Conidia <![CDATA[2.9×10 9 [Units / g solid fermentation medium]

[0068] Example 4

[0069] The solid fermentation method of a strain of Chlamydomonas verticillata is the same as that described in Example 1, except that step (4) is: the secondary seed liquid is inoculated into the solid fermentation culture medium at a mass percentage of 10%, stirred evenly, and then placed into a fermentation tank. It is cultured at 26°C for 9 days and then dried at low temperature to obtain the fermentation product of Chlamydomonas verticillata.

[0070] The solid fermentation medium is prepared by mixing 5 parts corn flour, 5 parts rice flour, 20 parts wheat bran, 35 parts rice husk powder, and 35 parts peanut shell powder evenly, then adding 82 parts inorganic salt nutrient solution and stirring evenly to achieve a moisture content of approximately 45% and a natural pH; and then fermenting at 121℃ and a pressure of 2 kg / cm². 3 Under the conditions of high pressure steam sterilization in an autoclave for 30 minutes, the material is then pumped into a double cone tank for cooling to obtain a solid fermentation medium.

[0071] The inorganic salt nutrient solution contains 5 g / L sucrose, 5 g / L ammonium sulfate, 20 g / L sodium nitrate, 0.8 g / L potassium nitrate, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, and a natural pH.

[0072] The number of chlamydospores and conidia in the fermentation product of *Verticillium chlamydosporum* in this example was determined according to the method described in Example 2, and the results are shown in Table 3.

[0073] Table 3

[0074] Thick-walled spores <![CDATA[2.2×10 8 [Units / g solid fermentation medium] Conidia <![CDATA[2.7×10 9 [Units / g solid fermentation medium]

[0075] As shown in Tables 1-3 of Examples 2-4, the *Verticillium chlamydosporum* Vc-GXWFY-01 strain provided by this invention exhibits high spore yield. Furthermore, the solid-state fermentation method uses wheat bran, rice husk powder, corn flour, rice flour, and peanut shell powder as the solid culture medium. This ensures sufficient organic matter for the strain while also increasing the aeration rate within the solid-state fermentation medium, thereby increasing the production of *Verticillium chlamydosporum* and the number of chlamydosporinus spores, achieving a yield of 9.4 × 10⁻⁶ spores. 7 ~2.2×10 8 The number of conidia per gram of solid fermentation medium was 2.7 × 10⁶. 9 ~2.9×10 9 Solid fermentation medium (units / g).

[0076] Test case

[0077] Methods for grading diseased plants:

[0078]

[0079] Disease index (%) = ∑(Number of diseased plants at each level × Relative level value) / (Total number of plants surveyed × 4) × 100

[0080] Relative prevention and control effect (%) = (Disease index of blank control - Disease index of treatment) / Disease index of blank control × 100

[0081] 1. Experiment on the control of cucumber root-knot nematodes

[0082] Experimental location: Xihaizi Village, Linshu County, Shandong Province. Experimental treatment: When transplanting cucumbers, the experimental group was mixed with the fermentation product of *Puconia thunbergii* from Example 2 at a rate of 1 g / plant with the soil around the cucumber roots. The control group was treated with water. After 40 days of treatment, the plants were harvested. Four plants from each treatment and control were taken to test the root nematode disease, and the disease index and control effect were calculated. The results are shown in Table 4.

[0083] Table 4

[0084] control group 100.0％ / experimental group 23.7％ 76.3％

[0085] Thirty-five days after transplanting, the control group showed severe root damage, with leaves turning yellow and wilting, while the treatment with *Verticillium chlamydosporum* fermentation product showed no significant damage. Forty days after transplanting, the control cucumbers died, with roots rotting and turning brown, while the cucumbers treated with *Verticillium chlamydosporum* fermentation product grew normally. Table 4 shows that *Verticillium chlamydosporum* fermentation product has a significant inhibitory and control effect on root-knot nematodes during cucumber cultivation, with a control efficacy exceeding 75%. Simultaneously, the aboveground biomass increased by 63.9% (control 535 g / plant, treatment 876.6 g / plant).

[0086] 2. Experiment on the control of tomato root-knot nematodes

[0087] Experimental location: Potted plants in Jinzhengda greenhouse, Linshu, Shandong Province. Experimental treatment: At the time of tomato transplanting, the experimental group (T) mixed 1g / plant of the fermented *Verticillium chlamydosporium* from Example 2 with the soil around the tomato roots. The control group (CK) was treated with water. After 30 days of treatment, the plants were harvested. Four plants from each group were taken to test for root nematode disease, and the disease index and control effect were calculated. The results are shown in Table 5. A visual representation of the plants harvested after treatment in this example is also shown. Figure 5 As shown.

[0088] Table 5

[0089] control group 93.8％ / experimental group 43.8％ 53.3％

[0090] From Table 5 and Figure 5 It is known that the fermentation product of *Verticillium chrysogenum* has a control effect of more than 50% on root-knot nematodes in the early stage of tomato growth, and promotes the growth of aboveground biomass of tomato plants.

[0091] In summary, this invention provides a strain of Verticillium chlamydosporum Vc-GXWFY-01 with high spore yield. This strain also has significant functions in controlling nematodes in agricultural and forestry crops and promoting crop growth. In the early stage of crop growth, the control effect on root-knot nematodes reaches 50-87%, while increasing crop yield and improving economic benefits.

[0092] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Chlamydosporium Verticillium ( Verticillium chlamydosporium Vc-GXWFY-01 was deposited on April 12, 2023, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.40400.

2. The solid-state fermentation method for Verticillium chlamydosporium Vc-GXWFY-01 according to claim 1, characterized in that, The steps are as follows: (1) Take the chlamydomonas verticillata Vc-GXWFY-01 and inoculate it into Bengal red medium or PDA solid medium. Under the conditions of 25℃~28℃, activate and culture for 3~4 days to obtain the activated strain. (2) Take the activated strain from step (1) and inoculate it into the seed culture medium. Under the conditions of 28℃~30℃ and 150~170r / min, shake and culture for 2~4 days to obtain the first-grade seed liquid. (3) Inoculate the primary seed culture from step (2) into the secondary seed culture medium at a volume percentage of 4-6%, and culture it under shaking conditions of 28℃-30℃ and 150-170r / min for 2-3 days to obtain the secondary seed culture. (4) Inoculate the secondary seed liquid from step (3) into a solid fermentation medium at a mass percentage of 10-20%, and culture at 25℃-32℃ for 7-15 days. Dry at low temperature to obtain the fermentation product of Chlamydomonas verticillata.

3. The solid-state fermentation method as described in claim 2, characterized in that, In step (1), the formula for the Bengal Red culture medium is: 5g peptone, 10g glucose, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 20g agar, 100mL 1 / 3000 Bengal Red solution, and distilled water to a final volume of 1000mL; the formula for the PDA solid culture medium is: 200g potato, 20g glucose, 20g agar, distilled water to a final volume of 1000mL, with a natural pH.

4. The solid-state fermentation method as described in claim 2, characterized in that, In step (2), the seed culture medium is PDA liquid culture medium with the following formula: 200g potato, 20g glucose, and distilled water to a final volume of 1000mL, with a natural pH.

5. The solid-state fermentation method as described in claim 2, characterized in that, In step (3), the secondary seed culture medium formula is as follows: 20 g / L sucrose, 10 g / L corn flour, 10 g / L urea, 1.15 g / L sodium nitrate, 0.8 g / L potassium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate heptahydrate, 0.3 g / L potassium chloride, 0.2 g / L sodium chloride, 0.3 g / L potassium dihydrogen phosphate, and pH 7.

6. The solid-state fermentation method as described in claim 2, characterized in that, The solid fermentation culture medium described in step (4) contains the following raw materials in parts by weight: 20-35 parts wheat bran, 20-35 parts rice husk powder, 5-15 parts corn flour, 5-10 parts rice flour, 20-40 parts peanut shell powder, and 55-110 parts inorganic salt nutrient solution.

7. The solid-state fermentation method as described in claim 6, characterized in that, The inorganic salt nutrient solution contains 5-15 g / L sucrose, 5-15 g / L ammonium sulfate, 10-20 g / L sodium nitrate, 0.8 g / L potassium nitrate, 0.5-1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate heptahydrate, and 5 g / L potassium dihydrogen phosphate.

8. The solid-state fermentation method as described in claim 6, characterized in that, The solid fermentation medium is prepared by the following method: Weigh out fresh wheat bran, rice husk powder, corn flour, rice flour and peanut shell powder by weight ratio, mix them evenly to obtain solid material; Add sucrose, ammonium sulfate, sodium nitrate, potassium nitrate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, and potassium dihydrogen phosphate to water in the specified weight ratio to obtain an inorganic salt nutrient solution. Add the solid material to the inorganic salt nutrient solution, stir evenly, sterilize by high temperature and high pressure steam at 121℃ for 30 minutes, and it can be used as a solid fermentation culture medium after cooling.

9. The solid-state fermentation method as described in claim 2, characterized in that, In step (4), the expanded culture process is cooled by cooling water and pressure pulsation is performed. The pressure pulsation is mainly used to supply oxygen for microbial growth. The strain pulsation is performed once every 6 hours during the lag phase, once every 2 hours during the logarithmic phase, and once every 3 to 4 hours during the stationary phase.

10. The solid-state fermentation method as described in claim 2, characterized in that, In step (4), the number of chlamydospores in the chlamydospore fermentation product is concentrated at 9.4 × 10⁻⁶. 7 ~2.2×10 8 The number of conidia is concentrated at 2.7 × 10⁶ / g. 9 ~2.9×10 9 per g.

Images