Esteria nematode wettable powder as well as preparation method and application thereof

Abstract

The invention provides Esteria nematode wettable powder as well as a preparation method and application thereof, and belongs to the technical field of biopesticide preparations. The Esteria nematode wettable powder is prepared from the following components in parts by weight: 20 to 30 parts of Esteria nematode freeze-dried spore powder, 2 to 6 parts of sodium lignin sulfonate, 4 to 8 parts of sodium alkyl naphthalene sulfonate and 50 to 70 parts of corn starch. According to the invention, the variety and the optimal concentration of the cryoprotectant with a good protection effect on the Esteria nematode spores are screened out, and the live spore rate of the freeze-dried spore powder is improved to the maximum extent. A carrier and an auxiliary agent which have good biocompatibility to the Esteria nematode are further screened out, the formula of the Esteria nematode wettable powder is determined, and the produced Esteria nematode wettable powder not only can keep the activity of strains, but also is convenient to store and transport. Good application prospects and popularization values are realized on prevention and control of pine wood nematode diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological pesticide preparations, and particularly relates to a wettable powder of Esteya vermicola and its preparation method and application. Background Art

[0002] Bursaphelenchus xylophilus is a major quarantine pest globally. Currently, the main control measures for Bursaphelenchus xylophilus disease mainly focus on clearing diseased wood, such as pulverizing, burning, and fumigating the diseased wood. Secondly, chemical agents are sprayed to kill vector insects, emamectin benzoate is injected into the trunk to control Bursaphelenchus xylophilus, and resistant tree species are selected and bred. Although certain results have been achieved, there are problems such as chemical agents polluting the environment, the trunk injection method being not suitable for large-scale application, the slow progress of resistant tree species breeding, and the poor emergency response of vector insect control.

[0003] Esteya vermicola is the first reported endoparasitic fungus of Bursaphelenchus xylophilus in the world. It shows high infectivity and parasitism rate to Bursaphelenchus xylophilus and has also been proven to have a certain preventive effect in the forest. It has the prospect and potential as a biological control agent for Bursaphelenchus xylophilus disease.

[0004] Currently, the main problems restricting the large-scale application of Esteya vermicola are that the liquid dosage form occupies space during storage and transportation, and it needs to be refrigerated at 4°C during storage and requires cold chain transportation during long-distance transportation. Otherwise, the spores are easily inactivated and contaminated. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a wettable powder of Esteya vermicola and its preparation method and application to solve the problems of difficult storage and transportation of Esteya vermicola.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a wettable powder of Esteya vermicola, which includes the following components in parts by weight: 20 - 30 parts of freeze-dried spore powder of Esteya vermicola, 2 - 6 parts of sodium lignosulfonate, 4 - 8 parts of alkylnaphthalene sulfonate, and 50 - 70 parts of corn starch.

[0008] The present invention also provides a preparation method of the wettable powder of Esteya vermicola, which includes the following steps:

[0009] 1) After activating Esteya vermicola, inoculate it into a liquid medium to culture a seed liquid, and ferment the seed liquid to obtain a spore suspension of Esteya vermicola;

[0010] 2) Centrifuge the spore suspension of Esteya vermicola to obtain a spore paste of Esteya vermicola, mix the spore paste of Esteya vermicola with a freeze-drying protectant, and freeze-dry to obtain the freeze-dried spore powder of Esteya vermicola;

[0011] 3) Mix the freeze-dried spore powder of Paecilomyces nematophagus, sodium lignosulfonate, alkylnaphthalene sulfonate and corn starch to obtain the wettable powder of Paecilomyces nematophagus.

[0012] Preferably, the cryoprotectant in step 2) is a sucrose solution, and the concentration of the sucrose solution is 15-20%.

[0013] Preferably, the volume ratio of the spore slurry of Paecilomyces nematophagus to the cryoprotectant in step 2) is 1:1-3.

[0014] Preferably, pre-freezing is carried out before freeze-drying in step 2), the temperature of pre-freezing is -40 to -60 °C, and the time of pre-freezing is 1-3 h;

[0015] The temperature of freeze-drying is -40 to -60 °C, and the time of freeze-drying is 20-40 h.

[0016] Preferably, the activation medium in step 1) is a potato dextrose agar medium, the activation temperature is 24-26 °C, and the activation time is 5-7 d.

[0017] Preferably, the liquid medium in step 1) comprises the following components: glucose 1-3%, yeast extract powder 0.3-0.7%, peptone 0.05-0.15%, calcium chloride 0.3-0.8%, dipotassium hydrogen phosphate 0.005-0.015%, magnesium sulfate 0.03-0.08%, ferrous sulfate 0.005-0.015% and the balance of water.

[0018] Preferably, the culture temperature in step 1) is 24-26 °C, the culture rotation speed is 180-220 rpm, and the culture time is 5-7 d.

[0019] Preferably, the fermentation temperature is 24-26 °C, and the fermentation time is 3-4 d.

[0020] The present invention also provides the application of the wettable powder of Paecilomyces nematophagus or the wettable powder of Paecilomyces nematophagus prepared by the preparation method in the prevention and control of pine wilt disease.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention screens out cryoprotectants with better protective effects on the spores of *Esteya nematophila* and the optimal concentration, maximizing the viable spore rate of the freeze-dried spore powder. Furthermore, carriers and auxiliaries with good biocompatibility with *Esteya nematophila* are screened out, and the formulation of the wettable powder of *Esteya nematophila* is determined. The produced wettable powder of *Esteya nematophila* can not only maintain the activity of the strain, but also facilitate storage and transportation, and has good application prospects and promotion value for the prevention and control of pine wilt disease. Brief Description of the Drawings

[0023] Figure 1 is the comparison of the freeze-drying protective effects of different cryoprotectants on *Esteya nematophila*;

[0024] Figure 2 is the comparison of the freeze-drying protective effects of sucrose solutions with different concentrations on *Esteya nematophila*;

[0025] Figure 3 is the influence of different carriers on the colony growth rate of *Esteya nematophila*;

[0026] Figure 4 is the influence of different types of dispersants on the spore activity of *Esteya nematophila*;

[0027] Figure 5 is the influence of different types of wetting agents on the spore activity of *Esteya nematophila*. Detailed Embodiments

[0028] The present invention provides a wettable powder of *Esteya nematophila*, which comprises the following components in parts by weight: 20-30 parts of freeze-dried spore powder of *Esteya nematophila*, 2-6 parts of sodium lignosulfonate, 4-8 parts of alkylnaphthalene sulfonate, and 50-70 parts of corn starch. The number of viable spores in the wettable powder of *Esteya nematophila* is 1-3×10 8 cfu / g, the wetting time is 100 s, the suspension rate is 90%, and the pH is 6.5.

[0029] In the present invention, the dosage of the freeze-dried spore powder of *Esteya nematophila* is preferably 25-30 parts; the dosage of the sodium lignosulfonate is preferably 3-5 parts; the dosage of the alkylnaphthalene sulfonate is preferably 5-7 parts; and the dosage of the corn starch is preferably 55-65 parts.

[0030] The present invention also provides a preparation method of the wettable powder of *Esteya nematophila*, which comprises the following steps:

[0031] 1) After activating *Esteya nematophila*, inoculate it into a liquid medium to culture a seed solution, and ferment the seed solution to obtain a spore suspension of *Esteya nematophila*;

[0032] 2) Centrifuge the spore suspension of Paecilomyces nematophilus to obtain the spore paste of Paecilomyces nematophilus, mix the spore paste of Paecilomyces nematophilus with a cryoprotectant, and lyophilize to obtain the lyophilized spore powder of Paecilomyces nematophilus;

[0033] 3) Mix the lyophilized spore powder of Paecilomyces nematophilus, sodium lignosulfonate, alkylnaphthalene sulfonate and corn starch to obtain the wettable powder of Paecilomyces nematophilus.

[0034] In the present invention, after activating Paecilomyces nematophilus, inoculate it into a liquid medium for culture to obtain a seed solution, and ferment the seed solution to obtain a spore suspension of Paecilomyces nematophilus. The activation medium is preferably a potato dextrose agar medium, the activation temperature is preferably 24-26 °C, more preferably 25 °C; the activation time is preferably 5-7 d, more preferably 6 d; the liquid medium includes the following components: glucose 1-3%, yeast extract powder 0.3-0.7%, peptone 0.05-0.15%, calcium chloride 0.3-0.8%, dipotassium hydrogen phosphate 0.005-0.015%, magnesium sulfate 0.03-0.08%, ferrous sulfate 0.005-0.015% and the balance of water; the dosage of glucose is preferably 1.5-2.5%; the dosage of yeast extract powder is preferably 0.4-0.6%; the dosage of peptone is preferably 0.07-0.12%; the dosage of calcium chloride is preferably 0.4-0.7%; the dosage of dipotassium hydrogen phosphate is preferably 0.007-0.012%; the dosage of magnesium sulfate is preferably 0.04-0.07%; the dosage of ferrous sulfate is preferably 0.008-0.012%; the culture temperature is preferably 24-26 °C, more preferably 25 °C; the culture method is preferably dark culture, the culture rotation speed is preferably 180-220 rpm, more preferably 190-210 rpm; the culture time is preferably 5-7 d, more preferably 6 d; the fermentation temperature is preferably 24-26 °C, more preferably 25 °C; the fermentation time is preferably 3-4 d, and the spore concentration in the spore suspension of Paecilomyces nematophilus after fermentation exceeds 3*10 9 cfu / mL.

[0035] In the present invention, the spores suspension of Esteya nematophila is centrifuged to obtain the spore paste of Esteya nematophila, and the spore paste of Esteya nematophila is mixed with a cryoprotectant and freeze-dried to obtain the freeze-dried spore powder of Esteya nematophila. The rotation speed of the centrifugation is preferably 3000 - 5000 rpm, more preferably 3500 - 4500 rpm; the time of the centrifugation is preferably 5 - 15 min, more preferably 8 - 12 min; the cryoprotectant is preferably a sucrose solution, and the concentration of the sucrose solution is preferably 15 - 20%, more preferably 16 - 18%; the volume ratio of the spore paste of Esteya nematophila to the cryoprotectant is preferably 1:1 - 3, more preferably 1:1.5 - 2.5; pre-freezing is carried out before freeze-drying, and the temperature of the pre-freezing is preferably -40 - -60 °C, more preferably -45 - -55 °C; the time of the pre-freezing is preferably 1 - 3 h, more preferably 1.5 - 2.5 h; the temperature of the freeze-drying is -40 - -60 °C, more preferably -45 - -55 °C; the time of the freeze-drying is preferably 20 - 40 h, more preferably 25 - 35 h; the viable count in the freeze-dried spore powder of Esteya nematophila is 1*10 9 ~5*10 9 cfu / g.

[0036] In the present invention, the freeze-dried spore powder of Esteya nematophila, sodium lignosulfonate, alkylnaphthalene sulfonate and corn starch are mixed to obtain the wettable powder of Esteya nematophila.

[0037] The present invention also provides the application of the wettable powder of Esteya nematophila or the wettable powder of Esteya nematophila prepared by the preparation method in controlling pine wilt disease.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] The strain number of Esteya nematophila is Fxy121, and the deposit number is CGMCC No. 20215 (see specifically CN112574894A - An Entomopathogenic Fungus against Nematodes and Its Application)

[0040] Example 1

[0041] A wettable powder of Esteya nematophila is composed of the following components in parts by weight: 30 parts of the freeze-dried spore powder of Esteya nematophila, 4 parts of sodium lignosulfonate, 6 parts of alkylnaphthalene sulfonate and 60 parts of corn starch.

[0042] The preparation method of the wettable powder of Esteya nematophila is as follows:

[0043] Add 20 g of glucose, 5 g of yeast extract powder, 1 g of peptone, 5 g of calcium chloride, 0.1 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, and 0.1 g of ferrous sulfate to pure water to prepare 1 L of liquid fermentation medium; divide the liquid fermentation medium into 1 L Erlenmeyer flasks, with 400 mL of liquid in each flask, sterilize at 121 °C and 105 KPa for 20 min, and cool to room temperature for standby.

[0044] Inoculate the preserved nematode Esteria Fxy121 strain on potato dextrose agar medium and activate it at 25 °C for 6 d to obtain a bacterial cake of this strain. Inoculate the bacterial cake into the liquid fermentation medium and culture it at 25 °C and 200 rpm for 6 d to obtain a seed solution. Inoculate the seed solution into a fermenter and expand the culture at 25 °C for 4 d to obtain a nematode Esteria spore suspension.

[0045] After centrifuging the nematode Esteria spore suspension at a low speed for 10 min at 4000 rpm, obtain the nematode Esteria spore paste. Mix the nematode Esteria spore paste with a 15% sucrose solution, and the volume ratio of the 15% sucrose solution to the nematode Esteria spore paste is 2:1. Equilibrate for 30 min, pre-freeze at -50 °C for 2 h, and freeze-dry at -50 °C for 30 h to obtain the freeze-dried nematode Esteria spore powder.

[0046] Fully mix the freeze-dried nematode Esteria spore powder, sodium lignosulfonate, alkylnaphthalene sulfonate, and corn starch with a stirrer to obtain a wettable powder of nematode Esteria.

[0047] Example 2

[0048] A wettable powder of nematode Esteria is composed of the following components in parts by weight: 25 parts of freeze-dried nematode Esteria spore powder, 5 parts of sodium lignosulfonate, 5 parts of alkylnaphthalene sulfonate, and 65 parts of corn starch.

[0049] The preparation method of the wettable powder of nematode Esteria is as follows:

[0050] Add 10 g of glucose, 3 g of yeast extract powder, 0.5 g of peptone, 3 g of calcium chloride, 0.05 g of dipotassium hydrogen phosphate, 0.3 g of magnesium sulfate, and 0.05 g of ferrous sulfate to pure water to prepare 1 L of liquid fermentation medium; divide the liquid fermentation medium into 1 L Erlenmeyer flasks, with 400 mL of liquid in each flask, sterilize at 121 °C and 105 KPa for 20 min, and cool to room temperature for standby.

[0051] The preserved strain of Esteya nematophila Fxy121 was inoculated on potato dextrose agar medium and activated at 24 °C for 5 days to obtain a bacterial cake of this strain. The bacterial cake was inoculated into a liquid fermentation medium and cultured at 24 °C and 180 rpm for 5 days to obtain a seed solution. The seed solution was inoculated into a fermenter and cultured at 24 °C for 3 days to expand the culture to obtain a spore suspension of Esteya nematophila.

[0052] The spore suspension of Esteya nematophila was centrifuged at a low speed for 5 minutes to obtain a spore paste of Esteya nematophila, with a rotation speed of 5000 rpm. The spore paste of Esteya nematophila was mixed evenly with a 20% sucrose solution. The volume ratio of the 20% sucrose solution to the spore paste of Esteya nematophila was 1:1, balanced for 20 minutes, pre-frozen at -60 °C for 1 hour, and freeze-dried at -60 °C for 20 hours to obtain freeze-dried spore powder of Esteya nematophila.

[0053] The freeze-dried spore powder of Esteya nematophila, sodium lignosulfonate, alkylnaphthalene sulfonate, and corn starch were fully mixed by a stirrer to obtain a wettable powder of Esteya nematophila.

[0054] Example 3

[0055] A wettable powder of Esteya nematophila is composed of the following components in parts by weight: 25 parts of freeze-dried spore powder of Esteya nematophila, 3 parts of sodium lignosulfonate, 7 parts of alkylnaphthalene sulfonate, and 65 parts of corn starch.

[0056] The preparation method of the wettable powder of Esteya nematophila is as follows:

[0057] 30 g of glucose, 7 g of yeast extract powder, 1.5 g of peptone, 8 g of calcium chloride, 0.15 g of dipotassium hydrogen phosphate, 0.8 g of magnesium sulfate, and 0.15 g of ferrous sulfate were added to pure water to prepare 1 L of liquid fermentation medium; the liquid fermentation medium was dispensed into 1 L Erlenmeyer flasks, with 400 mL of liquid in each flask, sterilized at 121 °C and 105 KPa for 20 minutes, and cooled to room temperature for standby.

[0058] The preserved strain of Esteya nematophila Fxy121 was inoculated on potato dextrose agar medium and activated at 26 °C for 7 days to obtain a bacterial cake of this strain. The bacterial cake was inoculated into a liquid fermentation medium and cultured at 26 °C and 220 rpm for 7 days to obtain a seed solution. The seed solution was inoculated into a fermenter and cultured at 26 °C for 4 days to expand the culture to obtain a spore suspension of Esteya nematophila.

[0059] The spores of Esteya nematophila were centrifuged at a low speed for 15 min to obtain the spore paste of Esteya nematophila at a rotation speed of 3000 rpm. The spore paste of Esteya nematophila was mixed evenly with 20% sucrose solution. The volume ratio of 20% sucrose solution to the spore paste of Esteya nematophila was 3:1. After equilibration for 40 min, it was pre-frozen at -40 °C for 2 h and freeze-dried at -40 °C for 40 h to obtain the freeze-dried spore powder of Esteya nematophila.

[0060] The freeze-dried spore powder of Esteya nematophila, sodium lignosulfonate, alkylnaphthalene sulfonate and corn starch were fully mixed by a stirrer to obtain the wettable powder of Esteya nematophila.

[0061] Experimental Example 1

[0062] Screening of cryoprotectants for freeze-dried spores of Esteya nematophila

[0063] (1) Liquid fermentation medium

[0064] 20 g of glucose, 5 g of yeast extract powder, 1 g of peptone, 5 g of calcium chloride, 0.1 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate and 0.1 g of ferrous sulfate were added to pure water to prepare 1 L of liquid fermentation medium; the liquid medium was dispensed into 1-L Erlenmeyer flasks, with 400 mL of liquid in each flask, sterilized at 121 °C and 105 KPa for 20 min, and cooled to room temperature for standby.

[0065] (2) Preparation of spore powder

[0066] The preserved Fxy121 strain was inoculated on potato dextrose agar medium for activation culture to obtain the bacterial cake of this strain. The bacterial cake was inoculated into the liquid fermentation medium and cultured at 25 °C and 200 rpm / min for 6 d to obtain a spore suspension. The spore suspension was centrifuged at a low speed to obtain the spore paste at a rotation speed of 4000 rpm / min. The spore paste was mixed evenly with the cryoprotectant solution. The volume ratio of the cryoprotectant to the spore paste was 2:1. After equilibration for 30 min, it was pre-frozen at -50 °C for 2 h and freeze-dried for 24 h to obtain the spore powder of Paecilomyces lilacinus. The cryoprotectant solutions were 5% sucrose, maltose, trehalose, lactose, xylose, gelatin, sorbitol, skim milk powder, and glycerol aqueous solution respectively, and this was repeated 3 times.

[0067] (3) Determination of spore powder activity

[0068] 0.1 g of spore powder was dissolved in 10 mL of sterile water and then serially diluted. 100 μL of the diluted solution was spread on a PDA plate and cultured in an incubator at 25 °C for 4 d. The number of colonies on the plate was counted, with the absence of a cryoprotectant as the control.

[0069] Experimental results: As Figure 1 shown. From Figure 1It can be seen that sucrose has the best freeze-drying protection effect on nematode Estrella.

[0070] The protection effects of sucrose solutions with different concentrations (5%, 10%, 15%, 20%, 30%, 40%) were further determined.

[0071] Experimental results: As Figure 2 shown. From Figure 2 it can be seen that 15 - 20% sucrose solution can significantly improve spore survival rate.

[0072] Experimental Example 2

[0073] Screening of carriers and auxiliaries

[0074] Each test carrier (kaolin, silica, corn starch, ammonium sulfate, calcium hydrogen carbonate) was added to PDA medium at a ratio of 5% (w / v), sterilized, shaken well and poured into petri dishes to make plates. In a sterile environment, a mycelial block with vigorous growth at the edge of the Fxy121 strain cultured for 14 days was taken with a 5 mm diameter puncher, inoculated onto each medium, and cultured in a constant temperature incubator at 25°C for 10 days, then the diameter of each colony was measured to determine the effect of each carrier on the growth of the Fxy121 strain. A PDA plate without a carrier was set as a control, and each treatment had 3 replicates.

[0075] Each dispersant (sodium lignosulfonate, sodium dodecylbenzenesulfonate, sodium carboxymethylcellulose, sodium polyacrylate, polyacrylamide) was added to the sample containing 30% spore powder at a ratio of 4%, and the rest was supplemented with the carrier, and mixed well. Take 0.1 g of the sample, add 10 mL of sterile water to dissolve it, take 100 μL of the diluted solution after gradient dilution and coat it on a PDA plate, and culture it in a 25°C incubator for 4 days, and count the number of colonies on the plate, using the sample without a dispersant as a control.

[0076] Each wetting agent (sodium dodecyl sulfate, alkylbenzene sulfonate, sodium methylene bisnaphthalenesulfonate, alkylbenzene sulfonate) was added to the sample containing 4% sodium lignosulfonate and 30% spore powder at a ratio of 6%, and the rest was supplemented with the carrier, and mixed well. Take 0.1 g of the sample, add 10 mL of sterile water to dissolve it, take 100 μL of the diluted solution after gradient dilution and coat it on a PDA plate, and culture it in a 25°C incubator for 4 days, and count the number of colonies on the plate, using the sample without a wetting agent as a control.

[0077] Experimental results: As Figures 3 - 5 shown. From Figure 3 it can be seen that corn starch has the least effect on the colony growth of the Fxy121 strain, and there is no significant difference from the control. The other 4 carriers all inhibit the growth of this strain.

[0078] From Figure 4It can be seen that different dispersants have different effects on the growth of strain Fxy121. Among them, sodium lignosulfonate has no significant difference from the control, and the other 4 dispersants all significantly inhibit the growth of this strain.

[0079] It can be seen from Figure 5 that the wetting agent alkylnaphthalenesulfonate has no significant difference in the growth of this strain compared with the control group.

[0080] Experimental Example 3

[0081] Determination of the quality indicators of the wettable powder of Esteya nematophila prepared in Example 1

[0082] According to the national pesticide standards, the suspension rate, wettability and spore content of the wettable powder were determined. The method for determining the suspension rate refers to "Method for Determining the Suspension Rate of Pesticides (GB / T 14825-2023)", the method for determining the wettability refers to "Method for Determining the Wettability of Wettable Pesticide Powders (GB / T 5451-2001)", and the method for determining the spore content refers to "Entomopathogenic Fungi Beauveria bassiana Part 2: Beauveria bassiana Technical Material (NY / T 2295.1-2012)".

[0083] Experimental results: As shown in Table 1.

[0084] Table 1 Index determination of the wettable powder of Esteya nematophila

[0085] <![CDATA[Sporulation amount (×10 8 cfu / g)]]> Viable spore rate (%) Wetting time (s) Suspension rate (%) Fineness (%) pH 1.45±0.65 92.8±0.86 100±3.42 90±2.16 91±1.45 6.5

[0086] Note: The data in the table are mean ± standard error.

[0087] It can be seen from Table 1 that the spore content of the wettable powder of Esteya nematophila is 1.4×10 8 cfu / g, the viable spore rate is 92.8%; the wetting time is 100 s, meeting the national standard of ≤120 s; the suspension rate is 90%, meeting the national standard requirement of ≥70%; the pH value of the preparation is 6.5, meeting the national standard of 6-8.

[0088] From the above examples and experimental examples, it can be known that the present invention has screened out the types of cryoprotectants and the optimal concentration with better protection effects on the spores of Esteya nematophila, maximizing the viable spore rate of the freeze-dried spore powder. And further screened out the carriers and additives with better biocompatibility for Esteya nematophila, determined the formula of the wettable powder of Esteya nematophila, and the produced wettable powder of Esteya nematophila can not only maintain the activity of the strain, but also be convenient for storage and transportation, and has good application prospects and promotion value for the prevention and control of pine wilt disease.

[0089] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A wettable powder of Esteya nematophila, characterized in that, It comprises the following components in parts by weight: 20 - 30 parts of freeze-dried spores powder of Esteya nematophila, 2 - 6 parts of sodium lignosulfonate, 4 - 8 parts of alkylnaphthalene sulfonate, and 50 - 70 parts of corn starch.

2. The preparation method of the wettable powder of nematode Esteya vermicola according to claim 1, characterized in that, It comprises the following steps: 1) After activating Esteya nematophila, inoculate it into a liquid medium for culture to obtain a seed solution, and ferment the seed solution to obtain a spore suspension of Esteya nematophila; 2) Centrifuge the spore suspension of Esteya nematophila to obtain a spore paste of Esteya nematophila, mix the spore paste of Esteya nematophila with a freeze-drying protectant, and freeze-dry to obtain the freeze-dried spores powder of Esteya nematophila; 3) Mix the freeze-dried spores powder of Esteya nematophila, sodium lignosulfonate, alkylnaphthalene sulfonate, and corn starch to obtain a wettable powder of Esteya nematophila.

3. The preparation method according to claim 2, characterized in that, The freeze-drying protectant in step 2) is a sucrose solution, and the concentration of the sucrose solution is 15 - 20%.

4. The preparation method according to claim 2, characterized in that, The volume ratio of the spore paste of Esteya nematophila to the freeze-drying protectant in step 2) is 1:1 - 3.

5. The preparation method according to claim 2, characterized in that, Pre-freezing is carried out before freeze-drying in step 2), the temperature of the pre-freezing is -40 - -60°C, and the time of the pre-freezing is 1 - 3 h; The temperature of the freeze-drying is -40 - -60°C, and the time of the freeze-drying is 20 - 40 h.

6. The preparation method according to claim 2, wherein The activation medium in step 1) is a potato dextrose agar medium, the activation temperature is 24 - 26°C, and the activation time is 5 - 7 d.

7. The preparation method according to claim 2, characterized in that, The liquid medium in step 1) comprises the following components: 1 - 3% of glucose, 0.3 - 0.7% of yeast extract powder, 0.05 - 0.15% of peptone, 0.3 - 0.8% of calcium chloride, 0.005 - 0.015% of dipotassium hydrogen phosphate, 0.03 - 0.08% of magnesium sulfate, 0.005 - 0.015% of ferrous sulfate, and the balance of water.

8. The preparation method according to claim 2, characterized in that, The culture temperature in step 1) is 24 - 26°C, the culture rotation speed is 180 - 220 rpm, and the culture time is 5 - 7 d.

9. The preparation method according to claim 2, characterized in that, The fermentation temperature in step 1) is 24 - 26°C, and the fermentation time is 3 - 4 d.

10. Use of the wettable powder of Esteya nematophila according to claim 1 or the wettable powder of Esteya nematophila prepared by the preparation method according to any one of claims 2 - 9 in controlling pine wilt disease.

Images