A wettable powder of Cordyceps militaris MSC-f1, its preparation method and application

Abstract

This invention relates to the field of biopesticide technology, specifically to a wettable powder of *Cordyceps militaris* MSC-f1, its preparation method, and its application. The wettable powder is composed of the following components by mass percentage: 10.00% *Cordyceps militaris* MSC-f1 conidial powder, 5.00% calcium lignin sulfonate, 5.00% Tween-80, 1.50% humic acid, and 78.50% kaolin. The spore content of this *Cordyceps militaris* wettable powder is 1.77 × 10⁻⁶. 10 Cells / g. Indoor tests showed that the corrected mortality rate against the six tested crop pests reached over 93% after 7 days of treatment; field tests showed a 7-day control efficacy of 76.42% against citrus pseudococcus. This formulation has broad-spectrum insecticidal activity, providing a new approach for the green control of crop pests.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, specifically to a wettable powder of Cordyceps militaris, its preparation method, and its application. Background Technology

[0002] In recent years, the occurrence and damage caused by pests such as the citrus red spider mite, citrus rust mite, citrus psyllid, and rice brown planthopper have become increasingly serious, posing a key obstacle to the development of the citrus and rice industries. For a long time, the control of these pests has relied primarily on chemical pesticides. However, the excessive and irrational use of chemical pesticides has triggered a series of environmental and social problems, most notably excessive pesticide residues, the large-scale killing of natural enemies, and the disruption of the citrus orchard ecosystem balance. Simultaneously, influenced by the ecological adaptation mechanisms of target pests, many pests have developed high levels of resistance to commonly used chemical agents, leading to a year-on-year decline in field control effectiveness and increasing the difficulty of pest control.

[0003] In the field of biological control, the variety of biological insecticides available on the market for the aforementioned pests is currently limited. In particular, there are no commercially available biocontrol agents for the citrus rust mite, making it difficult to meet the current practical needs of green pest control in citrus and rice. Therefore, the development of environmentally friendly biological insecticides has become an important direction for promoting the green control of these crop pests.

[0004] Cordyceps javanica (also known as Javan Cordyceps) is an important entomopathogenic fungus with a wide host range, good environmental compatibility, and low susceptibility to drug resistance, showing promising application prospects in biological pest control. However, currently, there are relatively few formulations developed based on Cordyceps javanica, and research on its application effects on different target pests and its formulation technology is still insufficient, limiting the practical application of this strain. Summary of the Invention

[0005] The purpose of this invention is to provide a wettable powder of Cordyceps javanica MSC-f1, its preparation method, and its application.

[0006] The technical solution adopted in this invention is as follows:

[0007] A wettable powder of Cordyceps militaris, comprising the active ingredient Cordyceps militaris MSC-f1 conidial powder, as well as a carrier, wetting agent, dispersant and ultraviolet protectant;

[0008] Furthermore, the carrier is kaolin;

[0009] Furthermore, the wetting agent is Tween-80;

[0010] Furthermore, the dispersant is calcium lignosulfonate;

[0011] Furthermore, the ultraviolet protectant is humic acid;

[0012] Furthermore, the components, by mass percentage, are as follows: Java Cordyceps MSC-f1 conidial powder 10.00%, dispersant calcium lignosulfonate 5.00%, wetting agent Tween-80 5.00%, UV protectant humic acid 1.50%, and carrier kaolin 78.50%;

[0013] Furthermore, the spore content of the wettable powder is 1.77 × 10⁻⁶. 10 per g.

[0014] A method for preparing the above-mentioned wettable powder of Cordyceps militaris: The conidial powder of Cordyceps militaris MSC-f1 is thoroughly mixed with a carrier, a wetting agent, a dispersant, and a UV protectant in a specific ratio to obtain the wettable powder of Cordyceps militaris.

[0015] The above-mentioned application of Java Cordyceps wettable powder in the control of crop pests;

[0016] Furthermore, the crop pests are selected from one or more of the following: citrus psyllid, citrus rust mite, citrus scale insect, rice brown planthopper, or rice white-backed planthopper.

[0017] The significant advantages of this invention are:

[0018] The wettable powder of this invention is a specialized formulation obtained through screening of the Java Cordyceps MSC-f1 strain, and all components are highly compatible with this strain. Its carrier is made of environmentally friendly inert materials such as kaolin, thus offering greater advantages in terms of greenness and environmental friendliness compared to chemical pesticides. The production process of this formulation is relatively simple, with low production costs and ease of use. Furthermore, this formulation possesses broad-spectrum insecticidal activity, exhibiting significant and stable control effects against a variety of target pests, and due to its complex mechanism of action, it is less likely to induce pesticide resistance in target pests. Attached Figure Description

[0019] Figure 1 : Carrier screening.

[0020] Figure 2 Screening of wetting agents.

[0021] Figure 3 Dispersant screening.

[0022] Figure 4 Screening of UV protectants.

[0023] Figure 5 Infection status of various pests by Java Cordyceps MSC-f1 wettable powder. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments.

[0025] The Cordyceps javanica MSC-f1 used in this invention is also known as Isaria javanica MSC-f1, which has been disclosed in patent CN202111598242.1.

[0026] The liquid culture medium used in this invention is formulated as follows: 15g soybean powder, 45g glucose, 0.50g NaCl, and water is added to a final volume of 1L.

[0027] The formula for the solid fermentation culture medium used in this invention is as follows: take the fermentation substrate carrier (foamed polypropylene (EPP, 3~5mm) and rice husk mixed at a volume ratio of 4:1), add water at a material-to-water ratio of 1:1.2 (mass ratio), and then add corn flour (20% of the mass of the fermentation substrate carrier), soybean flour (16%), yeast powder (1.5%), and NaCl (0.6%), and mix evenly.

[0028] The method for preparing the *Cordyceps militaris* MSC-f1 mycelial cake used in this invention is as follows: *Cordyceps militaris* MSC-f1 stored at 4℃ is inoculated onto PDA medium and thawed and cultured in a constant temperature incubator at 26℃ for 7 days. After the colonies grow pink spores, a mycelial cake with a diameter of 5mm is taken using a punch to obtain the *Cordyceps militaris* MSC-f1 mycelial cake.

[0029] The preparation method of the *Cordyceps militaris* MSC-f1 spore powder used in this invention is as follows: Two *Cordyceps militaris* MSC-f1 mycelial cakes are inoculated into 50 mL of liquid culture medium and cultured on a shaker at 29℃ and 200 r / min for 5 days to obtain seed liquid. Then, the seed liquid is inoculated into solid fermentation medium at an inoculation rate of 16% (v / w, i.e., 16 ml of seed liquid per 100 g of solid fermentation medium) and fermented for 9 days at 28℃, relative humidity of 80%~85%, and aeration rate of 0.8~1.0 vvm. After drying and sieving, *Cordyceps militaris* MSC-f1 spore powder is obtained.

[0030] The Java Cordyceps MSC-f1 spore suspension used in this embodiment of the invention is obtained by diluting Java Cordyceps MSC-f1 spore powder with sterile water.

[0031] Example 1: Screening of the optimal carrier ( Figure 1 )

[0032] Six test carriers—kaolin, talc, diatomaceous earth, calcium carbonate, silica, and bentonite—were mixed with PDA medium to a final concentration of 50 g / L. After autoclaving at 121°C for 15 min, the mixtures were thoroughly shaken and poured into plates. PDA plates without carriers served as a control (CK1), with each treatment replicated three times. A *Cordyceps militaris* MSC-f1 mycelial cake was inoculated into the center of each plate and incubated upside down at 28°C. After 12 days of incubation, the diameter of each colony was measured and recorded as d1. The daily growth rate of colony diameter was calculated using the following formula: Daily growth rate of colony diameter (mm / d) = (d1-5) / 12.

[0033] In the experiment on the effect of carriers on spore germination rate, the preparation methods of plates for each carrier were the same as above. A PDA plate without carrier was used as a control (denoted as CK2). The number of colonies formed by spore germination was determined using the plate dilution method to characterize the spore germination rate. 1.00 × 10⁻⁶ plates were used. 3 A 100 μL suspension of Cordyceps militaris MSC-f1 spores per mL was evenly spread onto a plate, with three replicates per treatment. The plates were incubated upside down at 28°C for 48 hours, and the colony count was observed and recorded.

[0034] The results showed that kaolin and talc had no inhibitory effect on the mycelial growth of Cordyceps javanica MSC-f1; kaolin had no adverse effect on spore germination, while the spore germination rate in the talc-treated group was significantly lower than that in the control group, indicating a significant inhibitory effect. Therefore, considering both mycelial growth and spore germination as core biocompatibility indicators, kaolin was ultimately selected as the carrier for the wettable powder of Cordyceps javanica.

[0035] Example 2: Screening of the optimal wetting agent ( Figure 2 )

[0036] Six wetting agents (sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (SDBS), tea seed powder (TSP), sodium butylbenzenesulfonate (SBS), Tween-80, and OP-10) were mixed thoroughly with PDA medium to prepare a series of gradient solutions with final concentrations of 100, 300, 500, 1000, 2000, and 4000 μg / mL. After autoclaving, the solutions were poured into plates. Plates without wetting agents served as a control (CK3). *Cordyceps militaris* MSC-f1 mycelial pellets were inoculated into the center of each plate and incubated upside down at 28°C. After 12 days of incubation, the diameter of each colony was measured, and the daily growth rate of the colony diameter was calculated.

[0037] Effect of wetting agent on spore germination rate: The above gradient solutions were poured into plates, with a plate without wetting agent as the control (denoted as CK4). The method for determining spore germination rate was the same as in Example 1.

[0038] Wetting power determination: The above six wetting agents were mixed with Cordyceps militaris MSC-f1 spore powder and carrier at the following three mass ratios: 1:10:89, 3:10:87, and 5:10:85, respectively, and the mixtures were thoroughly mixed to prepare the test samples. The wetting time of each treatment was determined according to the "Determination of Wetting Power of Pesticide Wettable Powders" (GB / T 5451-2001). The sample without added wetting agent was used as the control (denoted as CK5), and each treatment was repeated in triplicate.

[0039] The results showed that the wetting power of all six tested wetting agents increased with increasing concentration, while the wetting time decreased accordingly. The wetting time of 5% Tween-80 was 16.33 s, significantly lower than other wetting agents. Tween-80 had little effect on mycelial growth and spore germination, and its effect at low concentrations of SDS was also weaker than most other tested wetting agents. Tween-80 had no inhibitory effect on mycelial growth and spore germination at concentrations below 500 μg / mL. At a concentration of 4000 μg / mL, the daily mycelial growth still reached 0.40 cm, with 69.33 colonies. Its effect on the mycelial growth and spore germination of *Cordyceps militaris* MSC-f1 was significantly lower than other adjuvants. Considering both the wetting power of the tested wetting agents and their biocompatibility with *Cordyceps militaris* MSC-f1, 5% Tween-80 was selected as the wetting agent for the wettable powder of *Cordyceps militaris* MSC-f1.

[0040] Example 3: Screening of the optimal dispersant

[0041] Three dispersants—calcium lignosulfonate, sucrose fatty acid ester, and sodium lignosulfonate—were mixed with Cordyceps militaris MSC-f1 spore powder and kaolin clay at three different mass ratios: 1:10:89, 3:10:87, and 5:10:85, respectively, and thoroughly mixed to prepare the test samples. The suspension rate of each treatment was determined according to the "Determination of Suspension Rate of Pesticide Wettable Powders" (GB / T 14825-2006), with the sample without dispersant added serving as the control (CK6). Each treatment was replicated in triplicate.

[0042] The three dispersants were mixed with PDA medium to prepare a series of gradient solutions with final concentrations of 200, 400, 800, 1500, 3000, and 5000 μg / mL. After autoclaving, the solutions were poured into plates. Using plates without dispersants as a control (denoted as CK7), *Cordyceps militaris* MSC-f1 mycelial cakes were inoculated, and the daily growth rate of colony diameter was measured according to the method in Example 1.

[0043] Effect of dispersant on spore germination: Plate preparation was the same as above, with a plate without dispersant used as a control (denoted as CK8). The method for determining spore germination rate was the same as in Example 1.

[0044] The results showed that the addition of all three dispersants significantly improved the conidial suspension rate of *Cordyceps javanica* MSC-f1. Furthermore, the dispersing efficacy of each dispersant increased with increasing concentration; the conidial suspension rate with 5% dispersant was superior to that with 1% and 3% concentrations. With the addition of 5% calcium lignin sulfonate, the suspension rate reached 84.73%. Among the three dispersants, calcium lignin sulfonate showed significantly better dispersing efficacy than sodium lignin sulfonate and sucrose fatty acid ester. The addition of low concentrations of calcium lignin sulfonate, sodium lignin sulfonate, and sucrose fatty acid ester had virtually no effect on mycelial growth and spore germination of *Cordyceps javanica* MSC-f1, while calcium lignin sulfonate concentrations of 200 and 400 μg / mL promoted mycelial growth and spore germination. Taking into account the dispersing efficiency of different concentrations of various dispersants, their biocompatibility with Cordyceps javanica MSC-f1, as well as cost and economic benefits, 5% calcium lignosulfonate was selected as the dispersant for the wettable powder of Cordyceps javanica MSC-f1.

[0045] The results of the dispersant screening are shown below. Figure 3 .

[0046] Example 4: Screening of the optimal UV protectant

[0047] Three UV protectants—humic acid, sodium fluorescein, and sodium alginate—were mixed with PDA medium to prepare a series of gradient media with final concentrations of 5.00, 10.00, and 15.00 g / L. After autoclaving, the media were plated, with a plate without added UV protectant serving as the control (denoted as CK9). Compatibility tests (daily colony diameter growth and spore germination rate) were performed as in Example 1.

[0048] Two UV protectants, humic acid and sodium fluorescein, were thoroughly mixed with Cordyceps javanica MSC-f1 spore powder, calcium lignin sulfonate (CLS), Tween-80, and kaolin in three mass ratios: 0.5:10:5:5:79.5, 1:10:5:5:79, and 1.5:10:5:5:78.5. 1 g of each mixed sample was weighed, evenly spread on sterile filter paper, and placed 30 cm directly below a 30W UV lamp for 10, 20, and 30 minutes of irradiation. After irradiation, the spore germination rate of each treatment was determined using the plate dilution method. Samples without UV protectants and without UV irradiation served as negative controls (CK10), and samples without UV protectants but irradiated for 10, 20, and 30 minutes served as controls (CK11). Each treatment was replicated in triplicate.

[0049] Compatibility tests showed that humic acid and sodium fluorescein had no significant inhibitory effect on the germination of *Cordyceps militaris* conidia at the tested concentrations. The germination rate of conidia in the sodium alginate treatment group was significantly lower than that in the humic acid and sodium fluorescein treatment groups. UV protection efficacy tests showed that all UV protectants provided varying degrees of protection to *Cordyceps militaris*, and the germination rate of conidia decreased with increasing UV irradiation time. Among the adjuvants, 1.50% humic acid showed the best protective efficacy; after 30 minutes of UV irradiation, the germination rate of conidia still reached 77.00%, significantly higher than other tested adjuvants. In conclusion, 1.50% humic acid was selected as the UV protectant for *Cordyceps militaris* wettable powder.

[0050] The results of the UV protectant screening are shown below. Figure 4 .

[0051] Example 5: Formulation and quality indicators of wettable powder

[0052] Based on the screening results of Examples 1-4, the final formula of the wettable powder of *Cordyceps militaris* was determined as follows (by mass percentage): the active ingredient is *Cordyceps militaris* MSC-f1 conidia powder, with a content of 10.00%; the dispersant is calcium lignosulfonate (CLS), with a content of 5.00%; the wetting agent is Tween-80, with a content of 5.00%; the ultraviolet protectant is humic acid, with a content of 1.50%; and the carrier is kaolin, with a content of 78.50%. The *Cordyceps militaris* MSC-f1 conidia powder, carrier, wetting agent, dispersant, and ultraviolet protectant are thoroughly mixed in the specified proportions to obtain the wettable powder of *Cordyceps militaris*.

[0053] The quality indicators were tested according to the following methods: wettability was determined according to GB / T 5451-2001 "Determination of wettability of pesticide wettable powders"; suspension rate was determined according to GB / T 14825-2023 "Determination of suspension rate of pesticides"; fineness was determined according to GB / T 16150-1995 "Determination of fineness of pesticide powders and wettable powders"; pH value was determined according to GB / T 1601-1993 "Determination of pH value of pesticides"; and viable spore rate was determined by the plate dilution coating method (1g of wettable powder was serially diluted and coated onto a PDA plate, incubated at 28℃ for 48h, the number of colonies was counted, and the viable spore rate was calculated).

[0054] The results showed that the spore content of the wettable powder of Cordyceps militaris was 1.77 × 10⁻⁶. 10 The product has the following characteristics: spores / g, viable spore rate of 90.21%, drying weight loss of 5.72%, wetting time of 39s, spore suspension rate of 82.19%, pH value of 6.93, and fineness (passing through a 175μm test sieve) of 92.44%. All indicators meet the requirements for fungal microbial pesticide formulations.

[0055] Example 6: Indoor pathogenicity of wettable powder against various crop pests

[0056] Select citrus leaves of uniform size, clean them thoroughly, and disinfect their surface with 75% alcohol. Place the leaves, back side up, on sterile filter paper in a sterile petri dish, and add sterile water to the filter paper to maintain moisture. Randomly select adult citrus psyllids, citrus rust mites, and citrus psyllids of the same instar and transfer them to the leaves, 30-40 per leaf. Cut branches with scale insects of the same instar, retaining 30-40 insects on each branch. Separately, collect adult brown planthoppers and white-backed planthoppers of the same instar and inoculate them onto rice seedlings in seedling cups, 20-30 per cup.

[0057] Java Cordyceps wettable powder (1.77×10) 10 Prepare a 50-fold dilution (using sterile water containing 0.1% Tween-80) by diluting spores / g at a mass ratio. Apply the solution evenly to the surface of the tested pests and their habitat using a small handheld sprayer (2 mL capacity). Use sterile water containing 0.1% Tween-80 as a control. Each treatment was replicated 5 times.

[0058] After spraying, the petri dishes were transferred to a light incubator for cultivation under the following conditions: photoperiod 16L:8D, temperature (26±2)℃, and relative humidity (70±5)%. The mortality of pests was observed and recorded daily, with the appearance of *Cordyceps militaris* mycelium or spores on the insect surface considered an effective cause of death. Results were investigated 3 and 7 days after treatment. The corrected mortality rate was calculated using the following formula: Corrected mortality rate (%) = (Treatment mortality rate - Control mortality rate) / (1 - Control mortality rate) × 100%.

[0059] The results showed that the Java Cordyceps wettable powder had good control effects on all six tested crop pests. Three days after treatment, the corrected mortality rates for citrus psyllids, citrus rust mites, and citrus rust mites all exceeded 50%, with the highest pathogenicity rate of citrus psyllids at 62.03%. Seven days after treatment, the corrected mortality rates for all six crop pests, including citrus psyllids and rice brown planthoppers, exceeded 93%. This indicates that the Java Cordyceps MSC-f1 wettable powder has broad-spectrum insecticidal activity and can be used for the control of various crop pests.

[0060] Infection status see Figure 5 .

[0061] Table 1. Indoor pathogenicity of MSC-f1 wettable powder against various pests.

[0062]

[0063] Example 7: Field control efficacy of wettable powder against citrus pseudomitr

[0064] The experimental site was selected at the Zhejiang Provincial Citrus Research Institute's main experimental station, using a 3-year-old Red Beauty mandarin orange tree as the test variety. On the morning of October 5, 2022, the insect population was assessed and pesticide was sprayed. Within 14 days after spraying, the highest daily average temperature was 26℃ and the lowest was 18℃, with an average relative humidity of 71.12%.

[0065] The experiment consisted of 5 treatments: Treatment A was the wettable powder of Cordyceps militaris MSC-f1 (1.77 × 10⁻⁶). 10 Treatment B consisted of a suspension of Cordyceps militaris MSC-f1 spores (1.77 × 10⁻⁶ spores / g). 10 Treatment C was Paecilomyces lilacinus ZJPL08 wettable powder (1.98 × 10⁻⁶ spores / mL). 10 Spores / g (Patent No.: ZL 202010683602.7). Treatments A, B, and C were prepared with water at a mass ratio of 1:50. Treatment D was prepared with 30% etoxazole suspension (Baozhuo) and water at a mass ratio of 1:3000. Treatment E was a water control.

[0066] Each treatment was repeated three times in a randomized block design. Three trees were surveyed in each plot. Conventional canopy spraying was used, with a standard single-tube sprayer. The sprayer was applied evenly to both the upper and lower surfaces of the leaves, ensuring no dripping. Surveys were conducted 3 and 7 days post-treatment. The number of live mites on both the upper and lower surfaces of the leaves was counted by direct visual observation (4 leaves were randomly selected from the east, south, west, north, and center of each tree, for a total of 20 leaves).

[0067] Efficacy calculation method: Mite population reduction rate (%) = (Number of live mites before application - Number of live mites after application) / Number of live mites before application × 100; Control effect (%) = (Mite population reduction rate in the treatment area - Mite population reduction rate in the control area) / (100 - Mite population reduction rate in the control area) × 100.

[0068] The results showed that the wettable powder of Cordyceps javanica MSC-f1 had a certain control effect on citrus pseudococcus var. citrus under greenhouse cultivation conditions. Seven days after application, a 50-fold dilution of the wettable powder of Cordyceps javanica MSC-f1 showed a control efficacy of 76.42% against citrus pseudococcus var. citrus. This was slightly lower than the 82.59% efficacy of the chemical pesticide 30% etoxazole suspension, but significantly higher than the 42.41% efficacy of unprocessed Cordyceps javanica MSC-f1 spore suspension and the 60.14% efficacy of a 50-fold dilution of Paecilomyces lilacinus ZJPL08 wettable powder.

[0069] Table 2 Field control efficacy of MSC-f1 wettable powder against citrus pseudococcus mite

[0070]

[0071] The above results indicate that processing Cordyceps militaris into a wettable powder can effectively enhance its control efficacy against citrus psyllid mites. However, compared to the chemical agent etoxazole, this fungicide still lags behind in terms of rapid action. Therefore, in practical applications, it can be considered for application alone or in combination with acaricides to reduce the amount of chemical pesticides used while ensuring control efficacy, thus providing a feasible approach for the green control of citrus psyllid mites.

Claims

1. A wettable powder of Cordyceps militaris, characterized in that: It contains the active ingredient Cordyceps javanica MSC-f1 conidia powder, as well as a carrier, wetting agent, dispersant and UV protectant.

2. The Java Cordyceps wettable powder according to claim 1, characterized in that: The carrier is kaolin.

3. The Java Cordyceps wettable powder according to claim 1, characterized in that: The wetting agent is Tween-80.

4. The Java Cordyceps wettable powder according to claim 1, characterized in that: The dispersant is calcium lignosulfonate.

5. The Java Cordyceps wettable powder according to claim 1, characterized in that: The ultraviolet protectant is humic acid.

6. The Java Cordyceps wettable powder according to claim 1, characterized in that: The components, by mass percentage, are: Java Cordyceps MSC-f1 conidial powder 10.00%, dispersant calcium lignosulfonate 5.00%, wetting agent Tween-80 5.00%, UV protectant humic acid 1.50%, and carrier kaolin 78.50%.

7. The Java Cordyceps wettable powder according to any one of claims 1 to 6, characterized in that: The spore content of the wettable powder is 1.77 × 10⁻⁶. 10 per g.

8. A method for preparing the wettable powder of Cordyceps militaris according to any one of claims 1 to 7, characterized in that: The Java Cordyceps MSC-f1 conidial powder is thoroughly mixed with a carrier, wetting agent, dispersant and UV protectant in a certain proportion to obtain Java Cordyceps wettable powder.

9. The application of the wettable powder of Cordyceps militaris according to any one of claims 1 to 7 in the control of crop pests.

10. The application according to claim 9, characterized in that: The crop pests are selected from one or more of the following: citrus psyllid, citrus rust mite, citrus scale insect, rice brown planthopper, or rice white-backed planthopper.

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