A clonostachys rosea and use thereof in preparing a medicine for preventing and treating a plant pathogenic nematode

Abstract

The application belongs to the technical field of biology and specifically relates to a clavate branch of Clonostachys rosea and application thereof in preparation of a medicine for preventing and treating a plant pathogenic nematode, wherein the clavate branch of Clonostachys rosea has a preservation number of CCTCC NO: M20211658. The clavate branch of Clonostachys rosea provided by the application can prevent and treat plant nematode diseases efficiently and promote the growth of plant root height. Meanwhile, the fermentation technology of the clavate branch of Clonostachys rosea is optimized, and a fermentation medium with the highest activity of metabolic products is screened. In addition, the fermentation technology is simple, and the production equipment investment and production cost are greatly reduced. The preparation for preventing and treating nematode diseases provided by the application is a biological preparation, which is safe to use and does not cause drug damage and secondary pollution.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a type of cladoceran fungus and its application in the preparation of agents for controlling plant pathogenic nematodes. Background Technology

[0002] Plant pathogenic nematode infections can lead to decreased plant yield and quality. Globally, nematode damage causes losses of up to $125 billion annually in agricultural and forestry production, seriously impacting agricultural and forestry safety. Currently, chemical nematicides remain a crucial component of nematode control, with global sales of active nematicides reaching 250,000 tons annually, of which the United States and Western Europe account for nearly 84,000 tons and 48,000 tons respectively. However, with the gradual banning or restriction of commonly used nematicides such as aldicarb and methyl bromide, there is an urgent need for more novel and safe nematicides. Natural nematicides derived from plant extracts and bacteria and fungi are increasingly attracting interest and attention. Cladosporium fungi belong to the genus Cladosporium in the family Cladosporaceae of the order Coccidiales in the phylum Ascomycota. Cladosporium cladosporioides, in particular, exhibits good biocontrol effects against pests such as whiteflies, aphids, beet armyworms, and spider mites. Other studies have shown that conidial suspensions of Cladosporium cladosporioides C24G strain have a certain control effect on rice blast. Furthermore, Cladosporium can significantly promote the growth of Salvia miltiorrhiza and also has a significant impact on the effective component of salvianolic acid in field-grown Salvia miltiorrhiza. Hirota et al. (1985) isolated two new compounds, cladospolide A and cladospolide B, from the culture filtrate of Cladosporium um cladosporioides. Experiments showed that cladospolide A could inhibit the root growth of lettuce seedlings, while cladospolide B could promote the root growth of lettuce seedlings.

[0003] Researchers have discovered many novel bioactive substances in the genus Cladosporium, which can be broadly classified according to their chemical structures into: Tetramic acid derivatives, quinazoline derivatives, cyclic peptide derivatives, Cladosporols and their derivatives, Viriditoxins and their derivatives, Cladochromes and their derivatives, naphthoquinone derivatives, anthraquinone derivatives, steroids, benzofurans, coumarins, fatty acids and their derivatives, macrolides, etc. The compounds are classified into 15 major categories, including phenols and ketones, and others. These compounds exhibit outstanding performance in anti-influenza virus activity, antibacterial activity, anti-inflammatory activity, anti-cancer activity, and antioxidant activity. Liu et al. isolated three compounds, cladosporinone (1), viriditoxin (2), and two viriditoxin derivatives (3-4), from the fungus *Cladosporium cladosporioides*. Through cytotoxicity assays on mouse lymphoma cells L5178Y, they showed that viriditoxin (2) was the most cytotoxic compound to this cell line. However, the role of *Cladosporium* in controlling nematodes is rarely reported.

[0004] This invention provides, for the first time, a biocontrol agent with Cladosporium as the main component. This agent can effectively control diseases caused by plant pathogenic nematodes and promote plant growth, showing broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide an isolated Cladosporium strain, the Cladosporium being preserved under the CCT CC NO: M20211658.

[0006] Another object of the present invention is to provide the application of Cladosporium in tomato cultivation.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] Obtaining Cladosporium:

[0009] These strains were isolated from *Metarhizium anisopliae* culture plates contaminated with *Metarhizium anisopliae* in our laboratory. After three generations of continuous culture on PDA plates, colonies were isolated and purified, and the isolated strains were named Ch1, Ch2, Ch3, and Ch4. Morphological and spore characteristics were observed using an inverted optical microscope. Species identification was then performed using 28S r ITS, Actin, GAPDH, and β-tubulin primers for amplification and sequencing comparison. All four strains were identified as *Cladosporium cladosporioides*. The strain with the best nematicidal effect was deposited at the China Center for Type Culture Collection (CCTCC) on December 22, 2021, and is classified as *Cladosporium cladosporioide* Ch 4, accession number: CCTCC NO: M20211658, address: Wuhan University, Wuhan, China.

[0010] The aforementioned Cladosporium is a fungus of the genus Cladosporium. Its conidia are elliptical or rhomboid, growing in clusters or singly, with a distinct protrusion at the tip. The hyphae are septate, and the conidiophores are erect, forming branches in the middle. The growth temperature is 24-25℃.

[0011] The scope of protection of this invention also includes:

[0012] The application of the above-mentioned Cladosporium cladosporioide Ch 4 in the control of tomato root-knot nematode disease.

[0013] The Cladosporium cladosporioide Ch 4 of this invention can also be used to prepare nematode insecticides.

[0014] The Cladosporium cladosporioide Ch 4 of this invention can also be prepared as a tomato growth promoter.

[0015] In the above-described applications, preferably, the mycorrhizal fungus is obtained by fermentation using MEA medium.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) The cladoceran fungus provided by the present invention can effectively prevent and control plant nematode diseases on the one hand, and promote the growth of plant roots and plant height on the other hand.

[0018] (2) This invention optimizes the fermentation technology of Cladosporium and provides a culture medium with the highest activity of its metabolites. In addition, the fermentation technology is simple, which greatly reduces the investment in production equipment and production costs.

[0019] (3) The preparation for preventing and controlling nematode diseases provided by the present invention is a biological agent, which is safe to use and will not cause phytotoxicity or secondary pollution. Attached Figure Description

[0020] Figure 1 The effects of different concentrations of different Cladosporium species in fermentation broth on tomato growth;

[0021] In A, ac represents the effect of different *Cladosporium* species with different fermentation broth concentrations on the aboveground parts of tomato; df represents the effect of different *Cladosporium* species with different fermentation broth concentrations on the roots of tomato.

[0022] In section B: a: Fresh weight of roots and aboveground parts of strain Ch 1 treated with different concentrations of fermentation broth; b: Fresh weight of roots and aboveground parts of strain Ch 2 treated with different concentrations of fermentation broth; c: Fresh weight of roots and aboveground parts of strain Ch 3 treated with different concentrations of fermentation broth; d: Fresh weight of roots and aboveground parts of strain Ch 4 treated with different concentrations of fermentation broth; e: Root length and plant height of strain Ch 1 treated with different concentrations of fermentation broth; f: Root length and plant height of strain Ch 2 treated with different concentrations of fermentation broth; g: Root length and plant height of strain Ch 3 treated with different concentrations of fermentation broth; h: Root length and plant height of strain Ch 4 treated with different concentrations of fermentation broth.

[0023] The same letters on the bar chart indicate no significant difference (LSD, P<0.05), and the scale bar is 5cm.

[0024] Figure 2 The effects of different conidial suspension concentrations of different Cladosporium species on tomato growth;

[0025] In A, ac: the effects of different concentrations of conidial suspensions of different Cladosporium species on the aboveground parts of tomato; df: the effects of different concentrations of conidial suspensions of different Cladosporium species on the roots of tomato.

[0026] In section B: a: Fresh weight of roots and aboveground parts of strain Ch1 treated with different concentrations of conidial suspension; b: Fresh weight of roots and aboveground parts of strain Ch2 treated with different concentrations of conidial suspension; c: Fresh weight of roots and aboveground parts of strain Ch3 treated with different concentrations of conidial suspension; d: Fresh weight of roots and aboveground parts of strain Ch4 treated with different concentrations of conidial suspension; e: Plant height and root length of strain Ch1 treated with different concentrations of conidial suspension; f: Plant height and root length of strain Ch2 treated with different concentrations of conidial suspension; g: Plant height and root length of strain Ch3 treated with different concentrations of conidial suspension; h: Plant height and root length of strain Ch4 treated with different concentrations of conidial suspension. The same letters on the bar chart indicate no significant difference (LSD, P<0.05), and the scale bar is 5 cm.

[0027] Figure 3 This is a schematic diagram illustrating the determination of the lethality of strain PDB fermentation broth against nematodes.

[0028] The scale bar in the figure is 200 μm. The same letters on the bar chart indicate that there is no significant difference between the treatments (P < 0.05).

[0029] Figure 4 This is a schematic diagram illustrating the determination of the lethality of nematodes by the fermentation broth of strain SDA;

[0030] The scale bar in the figure is 200 μm. The same letters on the bar chart indicate that there is no significant difference between the treatments (P < 0.05).

[0031] Figure 5 This is a schematic diagram illustrating the determination of the lethality of nematodes by the fermentation broth of strain SDAY.

[0032] The scale bar in the figure is 200 μm. The same letters on the bar chart indicate that there is no significant difference between the treatments (P < 0.05).

[0033] Figure 6 This is a schematic diagram illustrating the determination of the lethality of nematodes by the fermentation broth of strain OA.

[0034] The scale bar in the figure is 200 μm. The same letters on the bar chart indicate that there is no significant difference between the treatments (P < 0.05).

[0035] Figure 7 This is a schematic diagram illustrating the determination of the lethality of nematodes by the fermentation broth of strain MEA.

[0036] The scale bar in the figure is 200 μm. The same letters on the bar chart indicate that there is no significant difference between the treatments (P < 0.05). Detailed Implementation

[0037] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0038] Example 1:

[0039] Obtaining Cladosporium:

[0040] These strains were isolated from *Metarhizium anisopliae* culture plates contaminated with *Metarhizium anisopliae* in our laboratory. After three generations of continuous culture on PDA plates, colonies were isolated and purified, and the isolated strains were named Ch1, Ch2, Ch3, and Ch4. Morphological and spore characteristics were observed using an inverted optical microscope. Species identification was then performed by amplification and sequencing using 28S rDNAITS, Actin, GAPDH, and β-tubulin primers. All four strains were identified as *Cladosporium cladosporioides*. The strain with the best nematode resistance was deposited at the China Center for Type Culture Collection (CCTCC) on December 22, 2021, with the classification name: *Cladosporium cladosporioide* Ch4, accession number: CCTCC NO: M2 0211658, address: Wuhan University, Wuhan, China.

[0041] The aforementioned Cladosporium is a fungus of the genus Cladosporium. Its conidia are elliptical or rhomboid, growing in clusters or singly, with a distinct protrusion at the tip. The hyphae are septate, and the conidiophores are erect, forming branches in the middle. The growth temperature is 24-25℃.

[0042] Example 2:

[0043] Culture of Cladosporium cladosporioides:

[0044] Cladosporium cladosporioide (Ch4) mycelial blocks grown for 7 days were punched and inoculated into 100 mL of PDB medium. The mixture was incubated at 28°C for 3 days using a shaker at 180 rpm. The resulting fermentation broth was used as the seed culture for the strain. 5 mL of this seed culture was inoculated into 100 mL of MEA medium and incubated at 28°C for 14 days using a shaker at 180 rpm to obtain the Cladosporium cladosporioide fermentation filtrate. The fermentation broth was filtered through four layers of sterile lens paper in a clean bench to remove mycelia, yielding a spore solution. This solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain the Cladosporium cladosporioide fermentation filtrate (hereinafter referred to as Ch4 fermentation broth).

[0045] Conidia of *Cladosporium* were washed off from PDA medium grown for 7 days with dd H2O and prepared with dd H2O to a concentration of 1×10⁻⁶. 3 CFU / mL, 1×10 6 CFU / mL, 1×10 8 A spore suspension of CFU / mL.

[0046] Other selected herbaceous spore fungi Ch 1-3 were also prepared into spore suspensions using the same method described above.

[0047] Example 3:

[0048] Application of Cladosporium in the control of tomato root-knot nematode disease:

[0049] 1.1 Cultivation and transplanting of tomato seedlings and nematode inoculation

[0050] Select healthy, plump tomato seeds. Rinse them twice with sterile water, then soak them in 75% ethanol for 2 minutes, and rinse them three times with sterile water. Germinate the sterilized tomato seeds in a petri dish. Sow the tomato seeds in sterilized nutrient soil and cultivate them at 28℃ under light for 21 days. When the tomatoes have grown 4 true leaves, transplant them into pots containing 500mL of soil. After the tomatoes have established themselves, inoculate each pot with approximately 800 Southern Root-Knot Nematodes J2.

[0051] 1.2 Investigation of tomato morphological indicators and root knot index

[0052] After 60 days of treatment, the biological yield (plant height and fresh weight) and underground (root weight, root length, and root knot index) growth of tomatoes were measured. Plant height refers to the total length from the cotyledon node to the top of the plant; aboveground fresh weight refers to the weight of the plant after removing the roots. Results are as follows: Figure 1-2 As shown, compared to other mycorrhizal fungi, the mycorrhizal fungus Ch4 of the present invention has a significant growth-promoting effect.

[0053] The root knot index of tomatoes is graded as follows: Grade 0: No root knots; Grade 1: A few root knots, accounting for 1%-25% of the total root knots; Grade 2: A moderate number of root knots, accounting for 26%-50% of the total root system; Grade 3: A large number of root knots, accounting for 51%-75% of the total root system; Grade 4: A large number of root knots, accounting for 76%-100% of the total root system (Benjamn et al 1987).

[0054]

[0055]

[0056] 1.3 Survey of the number of root-knot nematodes J2 and eggs in tomato roots

[0057] The number of tomato root-knot nematode J2 and its eggs was mainly investigated using the maceration-flocculation-flotation method (Hooper 1990). The separation procedure is as follows:

[0058] (1) After washing the roots of each batch, cut them into small sections of 1-2cm and mix them thoroughly.

[0059] (2) Put the root segments into a juicer and add 0.5% sodium hypochlorite solution until it just submerges the tomato root segments;

[0060] (3) Turn on the juicer to stir the root tissue. Stir for 40 seconds, then rest for 10 seconds. Repeat this process twice.

[0061] (4) Pass the crushed root tissue through 200 mesh and 500 mesh screens in sequence. After collecting the residue in the 500 mesh screen, transfer it to a beaker. Then, take the collected suspension and observe and count it under a microscope. Take 1 mL each time and place it in a biconcave slide. Count the number of root-knot nematodes J2 and eggs respectively. Calculate the average value after observing 3 times.

[0062] Formula for calculating the root-knot nematode J2 in tomatoes:

[0063]

[0064] Formula for calculating root-knot nematode eggs in tomato roots

[0065]

[0066] 1.4 Survey of the number of root-knot nematodes J2 in the soil

[0067] The number of root-knot nematodes J2 in the soil was investigated after 60 days of growth of tomatoes in each treatment. Soil samples were taken from 5-8 cm below the stem of the tomato plants in each treatment. After the soil samples of each replicate were thoroughly mixed, 100 mL of soil sample was taken and the number of root-knot nematodes J2 was counted using the modified shallow dish method (Whitehead and Hemming 1965).

[0068] The steps of the improved shallow dish method are as follows:

[0069] (1) Place the soil sample into a 5000mL measuring cup, add 3000mL of water, break up the soil clods and stir thoroughly with a glass rod for 5 minutes. The root-knot nematode J2 will then be suspended in the supernatant.

[0070] (2) Pass the supernatant through 100 mesh, 200 mesh and 500 mesh screens in sequence, and collect the residual root tissue on the 100 mesh screen and the soil residue on the 500 mesh screen.

[0071] (3) Place the collected residue on an Oxford cup with nematode filter paper (or tissue paper) and a mesh support;

[0072] (4) Submerge the residue with water and replenish the evaporated water;

[0073] (5) Place the apparatus at room temperature and collect root-knot nematodes J2 after 24-48 hours. Aspirate the collected liquid and observe it under a stereomicroscope. Aspirate 1 mL each time and place it in a self-made double-layered glass slide. Count the number of root-knot nematodes J2. Calculate the average value after observing 3 times.

[0074] Formula for calculating root-knot nematode J2 in soil:

[0075]

[0076] 1.5 Experimental Design

[0077] This experiment included 13 treatments, each repeated 3 times, with 5 pots of tomatoes planted in each replicate and arranged randomly.

[0078] The treatments are shown in the table below:

[0079]

[0080]

[0081] Effects of Cladosporium conidia on tomato root-knot nematodes

[0082] The experimental procedure is the same as above.

[0083] Experimental Design

[0084] This experiment included 13 treatments, each repeated 3 times, with 5 pots of tomatoes planted in each replicate and arranged randomly.

[0085] The treatments are shown in the table below:

[0086]

[0087] Table 1-1 Relative efficacy of Cladosporium fermentation broth against tomato root-knot nematode disease

[0088]

[0089]

[0090] Table 1-2 Relative control efficacy of Cladosporium conidium suspension treatment against tomato root-knot nematode disease.

[0091]

[0092] Example 4:

[0093] The insecticidal effects of Cladosporium Ch4 cultured in different fermentation broths on nematodes:

[0094] 1. Mortality rate of PDB fermentation broth against pine wood nematode

[0095] Cladosporium Ch 4 mycelial blocks grown for 7 days were punched and inoculated into 100 mL of PDB medium. The mixture was incubated at 28℃ for 3 days using a shaker at 180 rpm. The resulting fermentation broth was used as the seed culture for the strain. 5 mL of this seed culture was inoculated into 100 mL of PDB medium and incubated at 28℃ for 14 days using a shaker at 180 rpm to obtain the Cladosporium fermentation filtrate. The fermentation broth was filtered through four layers of sterile lens paper in a clean bench to remove mycelia, yielding a spore solution. This solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain the Cladosporium fermentation filtrate. The fermentation broth was used to treat pine wood nematodes at a final concentration of 50% (v / v) to determine the effect of the Cladosporium fermentation filtrate on the survival rate of the pine wood nematodes. Pine wood nematodes were collected from Botrytis cinerea plates and sterile water was added to make the nematode suspension concentration 1000 nematodes / mL. 100 μL of the nematode suspension and 100 μL of Cladosporium fermentation filtrate were added to 96-well plates, along with 0.2% streptomycin to prevent bacterial contamination. (This part of the original manuscript contained an error - note by Xiao Yannong) The control group consisted of nematode suspension and sterile water, and was incubated at 20°C. After 48 hours, the nematodes were observed under a microscope, and their survival was recorded. The corrected mortality rate was calculated. The results are as follows: Figure 3 As shown, the corrected mortality rates of pine wood nematodes for Ch 1-Ch 4 after 48 hours of treatment were 36.76%, 38.09%, 50.6%, and 72.17%, respectively.

[0096] 2. Preparation method of SDA fermentation broth and nematode mortality rate

[0097] Cladosporium Ch 4 mycelial blocks grown for 7 days were punched and inoculated into 100 mL of PDB medium. The mixture was incubated at 28℃ for 3 days using a shaker at 180 rpm. The resulting fermentation broth was used as the seed culture for the strain. 5 mL of this seed culture was inoculated into 100 mL of SDA medium and incubated at 28℃ for 14 days using a shaker at 180 rpm to obtain the Cladosporium fermentation filtrate. The fermentation broth was filtered through four layers of sterile lens paper in a clean bench to remove mycelia, yielding a spore solution. This solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain the Cladosporium fermentation filtrate. The fermentation broth was used to treat pine wood nematodes at a final concentration of 50% (v / v) to determine the effect of the Cladosporium fermentation filtrate on the survival rate of the pine wood nematodes. Pine wood nematodes were collected from Botrytis cinerea plates and sterile water was added to make a nematode suspension concentration of 1000 nematodes / mL. 100 μL of the nematode suspension and 100 μL of Cladosporium fermentation filtrate were added to each well of a 96-well plate, along with 0.2% streptomycin to prevent bacterial contamination. The control group consisted of nematode suspension and sterile water, and the plates were incubated at 20°C. After 48 hours, the nematodes were observed and their survival was recorded under a microscope, and the corrected mortality rate was calculated. Results are as follows: Figure 4 As shown, the corrected mortality rates of pine wood nematodes for Ch 1-Ch 4 after 48 hours of treatment were 36.71%, 38.2%, 47.55%, and 71.23%, respectively.

[0098] 3. Mortality rate of SDAY fermentation broth against pine wood nematode

[0099] Cladosporium Ch 4 mycelial blocks grown for 7 days were punched and inoculated into 100 mL of PDB medium. The mixture was incubated at 28℃ for 3 days using a shaker at 180 rpm. The resulting fermentation broth was used as the seed culture for the strain. 5 mL of this seed culture was inoculated into 100 mL of SDAY medium and incubated at 28℃ for 14 days using a shaker at 180 rpm to obtain the Cladosporium fermentation filtrate. The fermentation broth was filtered through four layers of sterile lens paper in a clean bench to remove mycelia, yielding a spore solution. This solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain the Cladosporium fermentation filtrate. The fermentation broth was used to treat pine wood nematodes at a final concentration of 50% (v / v) to determine the effect of the Cladosporium fermentation filtrate on the survival rate of the pine wood nematodes. Pine wood nematodes were collected from Botrytis cinerea plates and sterile water was added to make a nematode suspension concentration of 1000 nematodes / mL. 100 μL of the nematode suspension and 100 μL of Cladosporium fermentation filtrate were added to each well of a 96-well plate, along with 0.2% streptomycin to prevent bacterial contamination. The control group consisted of nematode suspension and sterile water, and the plates were incubated at 20°C. After 48 hours, the nematodes were observed and their survival was recorded under a microscope, and the corrected mortality rate was calculated. Results are as follows: Figure 5 As shown, the corrected mortality rates of pine wood nematodes for Ch 1-Ch 4 after 48 hours of treatment were 33.88%, 39.7%, 44.98%, and 71.68%, respectively.

[0100] 4. Mortality rate of OA fermentation broth against pine wood nematode

[0101] Cladosporium Ch 4 mycelial blocks grown for 7 days were punched and inoculated into 100 mL of PDB medium. The mixture was incubated at 28℃ for 3 days using a shaker at 180 rpm. The resulting fermentation broth was used as the seed culture for the strain. 5 mL of this seed culture was inoculated into 100 mL of OA medium and incubated at 28℃ for 14 days using a shaker at 180 rpm to obtain the Cladosporium fermentation filtrate. The fermentation broth was filtered through four layers of sterile lens paper in a clean bench to remove mycelia, yielding a spore solution. This solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain the Cladosporium fermentation filtrate. The fermentation broth was used to treat pine wood nematodes at a final concentration of 50% (v / v) to determine the effect of the Cladosporium fermentation filtrate on the survival rate of the pine wood nematodes. Pine wood nematodes were collected from Botrytis cinerea plates and sterile water was added to make a nematode suspension concentration of 1000 nematodes / mL. 100 μL of the nematode suspension and 100 μL of Cladosporium fermentation filtrate were added to each well of a 96-well plate, along with 0.2% streptomycin to prevent bacterial contamination. The control group consisted of nematode suspension and sterile water, and the plates were incubated at 20°C. After 48 hours, the nematodes were observed and their survival was recorded under a microscope, and the corrected mortality rate was calculated. Results are as follows: Figure 6 As shown, the corrected mortality rates of pine wood nematodes for Ch 1-Ch 4 after 48 hours of treatment were 32.22%, 34.65%, 41.91%, and 79.11%, respectively.

[0102] 5. Mortality rate of MEA fermentation broth against pine wood nematode

[0103] Cladosporium Ch 4 mycelial blocks grown for 7 days were punched and inoculated into 100 mL of PDB medium. The mixture was incubated at 28℃ for 3 days using a shaker at 180 rpm. The resulting fermentation broth was used as the seed culture for the strain. 5 mL of this seed culture was inoculated into 100 mL of MEA medium and incubated at 28℃ for 14 days using a shaker at 180 rpm to obtain the Cladosporium fermentation filtrate. The fermentation broth was filtered through four layers of sterile lens paper in a clean bench to remove mycelia, yielding a spore solution. This solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain the Cladosporium fermentation filtrate. The fermentation broth was used to treat pine wood nematodes at a final concentration of 50% (v / v) to determine the effect of the Cladosporium fermentation filtrate on the survival rate of the pine wood nematodes. Pine wood nematodes were collected from Botrytis cinerea plates and sterile water was added to make a nematode suspension concentration of 1000 nematodes / mL. 100 μL of the nematode suspension and 100 μL of Cladosporium fermentation filtrate were added to each well of a 96-well plate, along with 0.2% streptomycin to prevent bacterial contamination. The control group consisted of nematode suspension and sterile water, and the plates were incubated at 20°C. After 48 hours, the nematodes were observed and their survival was recorded under a microscope, and the corrected mortality rate was calculated. Results are as follows: Figure 7 As shown, the corrected mortality rates of pine wood nematodes for Ch1-Ch4 after 48 hours of treatment were 61.66%, 76.52%, 77.05%, and 84.04%, respectively.

Claims

1. An isolated Cladosporium ( Cladosporium cladosporioides The preservation number of the mycorrhizal fungus is CCTCC NO: M20211658.

2. The application of the Cladosporium of claim 1 in the prevention and control of root-knot nematode disease in tomatoes.

3. The use of the Cladosporium of claim 1 in the preparation of nematode insecticides.

4. The application of the Cladosporium of claim 1 in the preparation of a tomato growth promoter.

5. In any one of the applications according to claims 2 to 4, the mycorrhizal fungus is obtained by fermentation using MEA medium.

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