Use of tyrophagus putrescentiae as a vector for entomopathogenic fungi

Abstract

The application discloses the application of Tyrophagus putrescentiae ( Tyrophagus putrescentiae ) as a transmission carrier of entomopathogenic fungi, and belongs to the technical field of biological control of forestry pests. The application finds that the Tyrophagus putrescentiae can be used as an efficient transmission carrier of entomopathogenic fungi. The body surface of the mite can carry spores of Metarhizium anisopliae ( Metarhizium anisopliae ), and the carried spores have high germination activity. During the crawling process, the spores can be transmitted to a target environment. After the Tyrophagus putrescentiae is treated by a spore suspension immersion method or a powder spraying method, the amount of the carried spores on the body surface is large, and the number of bacteria produced by the crawling is large. The Tyrophagus putrescentiae carrying the bacteria is released on a pine tree damaged by the pine bark scale Matsucoccus matsumurae ), and the pathogenic fungi can be effectively transmitted to hidden parts such as the pine bark joint gap, so that the fungi can infect the target pests. The application provides a novel biological carrier for field precise application of entomopathogenic fungi, and has the advantages of high transmission efficiency, environmental friendliness and simple operation.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for forest pests, specifically relating to the carrion mite (Typhonium spp.). Tyrophagus putrescentiae Its application as a vector for the transmission of entomopathogenic fungi. Background Technology

[0002] Entomopathogenic fungi are important biological agents in the green control of pests, possessing advantages such as high target specificity, environmental friendliness, and low likelihood of developing resistance. However, in practical applications, the field dispersal efficiency of entomopathogenic fungal spores is often limited: direct spraying of spore suspensions is insufficient to cover all parts of the plant, especially hidden microhabitats such as bark crevices and leaf undersides, leading to inconsistent control efficacy. Therefore, finding efficient spore dispersal vectors has become a key technical issue for improving the field control efficacy of entomopathogenic fungi.

[0003] Mites are common arthropods in the microecological environment. Some mites possess characteristics such as active searching, strong crawling ability, and overlapping habitats with target pests, making them potential vectors for fungal spore dispersal. Previous studies have attempted to utilize predatory mites (such as *Neoseiuys barkii*) Neoseiulus barkeri Neoseui cucumeris Neoseiulus cucumeris They carry pathogenic fungal spores. However, different mites exhibit significant differences in morphological characteristics, body surface structure, and behavioral habits, resulting in varying abilities to carry pathogens and spore dispersal efficiencies. Currently, there are no reports of *Tyrophagus saprophyticus* acting as a vector for entomopathogenic fungi. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for the combined control of Japanese pine scale by combining Tyromitra saprophytica and Metarhizium anisopliae, thereby solving the problem of unstable biological control effects in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of the saprophytic mites as a vector for the transmission of entomopathogenic fungi. The saprophytic mites carry entomopathogenic fungal spores on their body surface and spread the spores to the target environment through crawling activities.

[0006] Furthermore, the entomopathogenic fungus is *Metarhizium anisopliae* (a type of beetle). Metarhizium anisopliae )CQMa421.

[0007] Furthermore, the saprophytic mites are treated with spore suspension immersion or powder spraying to make their body surface carry spores of insect pathogenic fungi.

[0008] Furthermore, the spore suspension immersion method is as follows: the saprophytic mites are immersed in a spore suspension with a concentration of 1×10⁸ spores / mL for 5 seconds, and then removed and air-dried naturally.

[0009] Furthermore, the target environment refers to the branches, trunks, and bark crevices of pine trees where Japanese pine scale insects have caused damage.

[0010] Secondly, the present invention provides a biological carrier for transmitting spores of entomopathogenic fungi, wherein the biological carrier is a carrion mite with spores of entomopathogenic fungi attached to its body surface.

[0011] Secondly, the present invention provides a method for preparing a saprophytic mite carrying spores of an insect pathogenic fungus, comprising the following steps: (1) Prepare a suspension of spores of insect pathogenic fungi with a concentration of 1×10⁻⁶. 8 spores / mL; (2) Immerse adult or nymphal carrion mites in the spore suspension for 5 seconds; (3) Take it out and air dry it naturally to obtain the bacteria-carrying putrefactive mites.

[0012] It contains at least the following beneficial technical effects: This study discloses for the first time a novel application of *Tetranychus saprophyticus* as a vector for the transmission of entomopathogenic fungi. *Tetranychus saprophyticus* exhibits high bacterial load and well-preserved spore activity, demonstrating significantly higher transmission efficiency than *Neoseiu's parvum*. *Tetranychus saprophyticus* is easily and cost-effective for large-scale indoor rearing, facilitating its widespread application. The bacterial-carrying *Tetranychus saprophyticus* possesses both predatory and fungal transmission functions, achieving a synergistic effect between mites and fungi. This provides a highly efficient and environmentally friendly new biological vector for the field transmission of entomopathogenic fungi. Attached Figure Description

[0013] Figure 1 The cumulative corrected mortality rate (10d) of different developmental stages of Japanese pine scale was calculated using eight entomopathogenic fungi.

[0014] Figure 2 The corrected mortality rate (10 days) of different concentrations of Metarhizium anisopliae CQMa421 on different developmental stages of Japanese pine scale was determined.

[0015] Figure 3 The cumulative mortality rate of adult (left) and nymph (right) Tyromitra saprophytica caused by Metarhizium anisopliae CQMa421.

[0016] Figure 4 The number of spores of different highly virulent strains carried by two types of mites at different time periods was determined.

[0017] Figure 5 The number of colonies produced by two types of mites carrying spores of different highly virulent strains at different time periods.

[0018] Figure 6 The study investigated the combined control effect of indoor saprophytic mites and Metarhizium anisopliae CQMa421 on Japanese pine scale.

[0019] Figure 7To simulate the combined control effect of saprophytic mites and Metarhizium anisopliae CQMa421 on Japanese pine scale in a forest environment. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0026] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0027] Data statistics and analysis Experimental data were processed using WPS Office 2022 and SPSS 27, and regression analysis was performed using probability unit analysis. Predator-prey functional responses were fitted using the Holling II equation. Graphs were generated using Origin 2024, WPS Office, and Adobe Photoshop CC 2019.

[0028] I. Experimental Materials 1. Test insect sources, strains, and mites (1) All tested Japanese pine scale insects were collected from the damaged red pine forest in Culaishan Forest Farm, Tai'an City. The sampling range included eggs, first instar newly hatched nymphs, first instar parasitic nymphs, second instar nymphs, third instar male nymphs, male pupae, female adults, and male adults.

[0029] (2) A total of 8 entomopathogenic fungi were selected in this invention, including two field-collected strains (SDTA-1 and SDTA-2), two commercial strains (Beauveria bassiana ZJU435 and Metarhizium anisopliae CQMa421) and one laboratory-preserved strain (Beauveria bassiana SDTA-3).

[0030] (3) Carrion mites: Carrion mites of multiple generations were raised in the Agricultural Mite Laboratory of Shandong Agricultural University. Carrion mites of uniform size and in good condition were selected for the experiment.

[0031] II. Screening of highly virulent strains of *Pseudomonas japonicus* 1. Virulence determination of five strains against Japanese pine scale The tested entomopathogenic fungi were inoculated onto PDA agar plates and cultured for 7–14 days in an artificial climate chamber at 26±1℃, 75±5% RH, and a photoperiod of L:D=16:8 h. After sufficient sporulation, the spores were washed off with 0.05% Tween-80 sterile water and thoroughly shaken using a vortex mixer to further disperse the fungal spores. A spore suspension of 1×10⁷ spores / mL was prepared by counting the spores using a hemocytometer. Spores of each strain were inoculated onto the surface of healthy Japanese pine scale insects using the immersion method (5 s), with a control treatment using 0.05% Tween-80 solution. Each treatment was replicated three times, with 20 Japanese pine scale insects treated per replicate. After being naturally dried, the processed Japanese pine scale was transferred to a separate humidification chamber and placed in an artificial climate chamber with the same conditions for cultivation. The infection and mortality of each strain of Japanese pine scale were observed and recorded daily for 10 days.

[0032] The pathogenicity of entomopathogenic fungi to different developmental stages of the Japanese pine scale insect varies significantly. Figure 1Two field-collected strains (SDTA-1 and SDTA-2) showed no pathogenicity against eggs, first-instar newly hatched nymphs, first-instar parasitic nymphs, and second-instar nymphs of the Japanese pine scale, but exhibited weak pathogenicity against first-instar newly hatched nymphs, third-instar male nymphs, male adults, and female adults. Beauveria bassiana (ZJU435 and SDTA-3) also showed weak pathogenicity against eggs, first-instar newly hatched nymphs, first-instar parasitic nymphs, and second-instar nymphs of the Japanese pine scale, and its pathogenicity against third-instar male nymphs, male adults, and female adults was only slightly better than SDTA-1 and SDTA-2.

[0033] Metarhizium anisopliae CQMa421 exhibits significantly higher pathogenicity than other strains, with extremely high mortality rates against first-instar newly hatched nymphs, second-instar nymphs, third-instar male nymphs, male adults, and female adults. Furthermore, its overall virulence is significantly superior to other tested strains.

[0034] 2. The effect of highly virulent strains on eggs and newly hatched nymphs of the Japanese pine scale. The selected highly virulent strains were diluted with 0.05% Tween-80 sterile water to create five experimental concentration gradients (1×10⁻⁶). 4 1×10 5 1×10 6 1×10 7 1×10 8 (Spores / mL) Eggs and newly hatched nymphs of the Japanese pine scale were dipped in the spore suspension for 5 seconds using a fine brush, then removed, air-dried, and placed in a small chamber. Sterile water containing 0.05 mL / L Tween-80 was used as a control. Each treatment group consisted of 60 scales, and each treatment was replicated three times. The scales were incubated in an incubator at 26±1℃, 75±5%RH, and a photoperiod of 16:8 (L:D). The infection and mortality of each strain of Japanese pine scale were observed and recorded daily.

[0035] 3. Virulence determination of highly virulent strains against first-instar and second-instar nymphs of the Japanese pine scale. The spore suspension was prepared as above. Pine branches infested with first-instar and second-instar nymphs of the Japanese pine scale were peeled back under a stereomicroscope with tweezers to expose the nymphs. The branches were then immersed in the spore suspension for 5 seconds, removed, and air-dried before being placed in the experimental apparatus. Sterile water containing 0.05 mL / L Tween-80 was used as a control. Each treatment group contained 60 nymphs, and each treatment was repeated three times. The apparatus was incubated at 26±1℃, 75±5%RH, and a photoperiod of 16:8 (L:D). The infection and mortality of each strain of Japanese pine scale were observed and recorded daily, and photographs were taken.

[0036] 4. Virulence determination of highly virulent strains against third instar male nymphs, male adults, and female adults of the Japanese pine scale. The spore suspension was prepared as above. Third-instar male nymphs, male adults, and female adults were immersed in spore suspensions of different concentrations for 5 seconds, then removed and placed in petri dishes. After air drying, they were placed in the experimental chamber. Sterile water containing 0.05 mL / L Tween-80 was used as a control. Each treatment group consisted of 20 insects, and each treatment was repeated 3 times. The insects were placed in an incubator at 26±1℃, 75±5%RH, and a photoperiod of 16:8 (L:D). The infection and mortality of each strain on the Japanese pine scale were observed and recorded and photographed daily.

[0037] from Figure 2 The mortality rates of *Metarhizium anisopliae* CQMa421 against different life stages of *Pseudomonas japonicus* showed that the corrected mortality rates for each life stage increased significantly with increasing spore concentration. At a spore concentration of 1×10⁻⁶, the corrected mortality rate for each life stage was significantly higher. 7 At a spore concentration of 10^6 spores / mL, the corrected mortality rate for first-instar newly hatched nymphs, male adults, third-instar male nymphs, and female adults can reach over 90%. At high concentrations (1×10^6 spores / mL), the mortality rate can reach over 90%. 8 At a concentration of spores / mL, the corrected mortality rate of *Metarhizium anisopliae* CQMa421 against first-instar newly hatched nymphs, adult males, third-instar male nymphs, and adult females reached 100%. Simultaneously, *Metarhizium anisopliae* CQMa421 exhibited strong toxicity against second-instar nymphs, with high concentrations (1×10⁻⁶) showing significant activity. 8 At a concentration of spores / mL, the corrected mortality rate reached 69.4%. First-instar nymphs, third-instar male nymphs, male adults, and female adults of the Japanese pine scale were all highly susceptible to both strains. This may be related to the lack of a waxy layer on their body surface and the presence of numerous folds, which greatly increases the attachment sites for fungal spores, facilitating germination and infection. In contrast, first-instar parasitic nymphs, second-instar nymphs, and male pupae were less susceptible to fungal infection, especially at low concentrations, with extremely low mortality rates. This may be due to their more robust protective structure on their body surface.

[0038] III. Safety of Highly Virulent Strains at Different Concentrations to Mites The spore suspension was prepared as above. Adult and nymphal mites of Tyrophagus saprophyticus were immersed in the spore suspension for 5 seconds, then removed and air-dried before being placed in a small chamber. Sterile water containing 0.05 mL / L Tween-80 was used as a control. Each treatment group consisted of 20 mites, and each treatment was replicated three times. The mites were incubated in an incubator at 26±1℃, 75±5%RH, and a photoperiod of 16:8 (L:D). The infection and mortality of each strain of Tyrophagus saprophyticus were observed and recorded daily.

[0039] Metarhizium anisopliae CQMa421 showed certain toxicity against both nymphs and adults of Tyrofoetida saprophyticus, and the toxicity was closely related to the strain concentration, treatment time, and stage of the tested mites. Figure 3 ).

[0040] In the toxicity test of Metarhizium anisopliae CQMa421 against Tyromitra saprophytica, the mortality rates of both nymphs and adults of Tyromitra saprophytica increased with the increase of spore concentration and treatment days. The mortality rate of adults was slightly higher than that of nymphs, but the overall mortality rate was low, indicating that it was relatively friendly to Tyromitra saprophytica.

[0041] IV. Quantity and activity of fungal spores carried by the tested mites 1. Number of spores carried by the test mites at different time points after spore suspension treatment. The preparation concentration is 1×10 8 The test mites were immersed in a spore suspension of spores / mL for 5 seconds and then removed. After 2 h, 12 h, and 24 h, the test mites were removed and placed in centrifuge tubes containing 200µL of 0.05% Tween-80 sterile water. The mixture was centrifuged and shaken to wash away the spores on the test mites. The spore content in the solution was determined by a hemocytometer, and the number of spores carried on the surface of each mite was calculated.

[0042] To avoid the influence of prey on the number of spores carried on the mite's body surface, the test mites were not fed during the experiment. Any test mites that died due to starvation or other reasons were removed with a brush and not included in the experimental results. Each treatment was repeated 5 times, with 5 mites tested each time.

[0043] 2. Number of spores carried by the test mites at different time points after powder spraying treatment. Using the spray method, the test mites were placed in the device and removed after 5 seconds, then placed in a small chamber. After 2 hours, 12 hours, and 24 hours, the test mites were removed and placed in centrifuge tubes containing 200 µL of 0.05% Tween-80 sterile water. The mixture was centrifuged and shaken to wash away the spores on the test mites. The spore content in the solution was determined by a hemocytometer, and the number of spores carried on the surface of each mite was calculated.

[0044] To avoid the influence of prey on the number of spores carried on the mite's body surface, the mites were not fed during the experiment. Mites that died due to starvation or other reasons had their bodies removed with a brush and were not included in the experimental results. Each treatment was repeated 5 times, with 5 mites tested each time.

[0045] 3. Number of colonies produced by the crawling of test mites after spore suspension treatment. The preparation concentration is 1×10 8Spores / mL of *Metarhizium anisopliae* CQMa421 spore suspension were used to immerse test mites in the spore suspension for 5 seconds. After air-drying, the mites were placed in a small chamber. At 2 h, 12 h, and 24 h, the mites were removed and inoculated into 3 cm diameter petri dishes containing PDA medium, 5 mites per dish. The petri dishes were sealed with plastic wrap with pinholes to maintain ventilation. The petri dishes were placed in an artificial climate chamber at 26±1℃, 75±5% relative humidity, and a light-dark ratio of 16:8. After 24 h, the test mites were removed, and after 72 h, the colony units formed on the culture medium were recorded. Each treatment was repeated 10 times.

[0046] 4. Number of colonies produced by test mites crawling after powder spraying treatment Using the powder spraying method, test mites were placed in the apparatus, sprayed with powder, and then placed in a small chamber. After 2 h, 12 h, and 24 h, the mites were removed and inoculated into 3 cm diameter petri dishes containing PDA medium, 5 mites per dish. The petri dishes were sealed with plastic wrap. Ventilation holes were made with an insect pin, and the petri dishes were placed in an artificial climate chamber at 26±1°C, 75±5% relative humidity, and a light-dark ratio of 16:8 (L:D). After 24 h, the test mites were removed, and colony units formed on the culture medium were recorded after 72 h. Each treatment was repeated 10 times.

[0047] like Figure 4 As shown, the number of Metarhizium anisopliae CQMa421 spores carried on the surface of Tyrofomys carrionus all showed a decreasing trend over time.

[0048] The spore load of *Metarhizium anisopliae* CQMa421 on mites was generally higher under dusting treatment than under spore suspension treatment, indicating that dusting is more likely to cause a large number of spores to adhere to the surface of mites. Figure 5 As shown, the number of colonies produced by *Metarhizium anisopliae* CQMa421 carried by *Typhonium saprophyticum* var. *scarioides* showed a decreasing trend over time. Under dusting, more spores carried by *Typhonium saprophyticum* var. *scarioides* were scattered, and their activity was higher.

[0049] V. The predatory ability of saprophytic mites on different life stages of Japanese pine scale insects 1. The predatory ability of healthy saprophytic mites on different life stages of the Japanese pine scale. Predation of first-instar nymphs by Tyrophagos saprophyticus: 1, 3, 5, and 7 first-instar nymphs were selected and placed in a small chamber. Then, a Tyrophagos saprophyticus mite that had been starved for 24 hours was introduced into the chamber. The predation ability of the Tyrophagos saprophyticus mite on the first-instar nymphs was observed within 24 hours.

[0050] Predation of third-instar male nymphs by Tyrophagos saprophyticus: 1, 2, and 3 third-instar male nymphs were selected and placed in a small chamber. Then, 10 Tyrophagos saprophyticus mites that had been starved for 24 hours were introduced. The predation ability of Tyrophagos saprophyticus mites on third-instar male nymphs was observed over 24 hours.

[0051] Predation of female adults by Tyrophagocytic mites: 1, 2, or 3 female adults were selected and placed in a small chamber, followed by 10 Tyrophagocytic mites that had been starved for 24 hours. The predation ability of Tyrophagocytic mites on female adults was observed after 3 days.

[0052] 2. The predatory ability of saprophytic mites treated with spore suspension against different life stages of *Typhonium japonicum*. Using the immersion method, one adult Tyromitra saprophytica mite was selected and immersed in a solution with a concentration of 1×10⁻⁶. 7 Soak the insects in a spore suspension of 1 spore / mL for 5 seconds, then air dry them naturally before transferring them into a small chamber containing Japanese pine scale insects at different developmental stages. The remaining procedures are the same as above.

[0053] 3. The predatory ability of saprophytic mites on *Pseudomonas japonicus* scale treated with spore suspension. Using the immersion method, different stages of the Japanese pine scale insect were selected and immersed in a solution with a concentration of 1×10⁻⁶. 7 Soak the spores in a spore suspension of 1 spore / mL for 5 seconds, allow them to air dry, then transfer them to a small chamber and place the carrion mites inside. Repeat the above steps.

[0054] According to the results in Table 1, the predation function of *Typhonium spp.* var. *spp.* 'Japonicum'' under different treatments conformed to the Hollings type II model, and the R² values ​​of each treatment group were all above 0.9, indicating a good model fit. At a prey density of 10 individuals, uninfected *Typhonium spp.* var. * ...

[0055] Table 1. Predation ability of *Tyrophagus saprophyticus* on first-instar nymphs of *Pinus japonicus* under different treatments. According to the results in Table 2, when 10 carrion mites prey together, the predation amount of third-instar male nymphs increases with the increase of prey density. When the prey density is 4 mites, the maximum predation amount per day when 10 carrion mites prey together is 1.46 mites.

[0056] Table 2. Predation ability of *Tyrophagus saprophyticus* on 3rd instar male nymphs of *Pinus japonicus* under different treatments. According to the results in Table 3, the predation rate of carrion mites on female adult Japanese pine scale insects also increased with the increase of prey density. When the prey density was 3 insects, the maximum daily predation rate when 10 carrion mites were preying on each insect was 1.26 insects.

[0057] Table 3. Predation ability of *Tyrophagus saprophyticus* on female adult *Pinus japonicus* under different treatments. VI. Combined control efficacy of Tyrofoetida saprophyticus and Metarhizium anisopliae CQMa421 against Pine Scale Insecta japonicus 1. Combined control efficacy of Tyrofoetida saprophyticus and Metarhizium anisopliae CQMa421 against Pine Scale in a small laboratory. All the pine scale insects tested (eggs, first instar newly hatched nymphs, second instar nymphs, third instar male nymphs, and adult females) were collected from healthy individuals in the same pine forest in Culaishan National Forest Park for the experiment.

[0058] Before the experiment, the *Metarhizium anisopliae* CQMa421 was rinsed with 0.05% Tween-80 sterile water, filtered through a 300-mesh screen to remove hyphae and impurities, counted using a hemocytometer, and the spore suspension concentration was adjusted to 1×10⁻⁶. 8 Spores / mL were prepared for use, while the control group used 0.05% Tween-80 solution. Experiments with eggs, first-instar newly hatched nymphs, third-instar male nymphs, and adult females were conducted in 6 cm diameter petri dishes lined with moist, sterile filter paper. For the egg and first-instar newly hatched nymph groups, 50 healthy eggs / 1st-instar newly hatched nymphs were placed in each petri dish; for the third-instar male nymphs and adult females, 5 individuals were placed in each compartment. The second-instar nymph experiment was conducted in 6 cm diameter, 10 cm high containers, with 20 second-instar nymphs per container. Ten *Tyrophagus saprophyticus* / *Neoseiu's pasteurellium* mites were added to the corresponding treatment groups. Each treatment was repeated three times and incubated in an incubator at 26±1℃, 75±5%RH, and a photoperiod of 16:8 (L:D). Infection and mortality rates were observed and recorded daily for 5 consecutive days. The mortality criteria were shriveling and blackening of the insect body, and the growth of mycelium on the body surface. A small amount of sterile water was added daily to keep the filter paper moist. This experiment included seven treatment groups: Treatment 1) Spraying only with spore suspension; Treatment 2) Spraying with spore powder using a powder sprayer; Treatment 3) Releasing *Tetranychus saprophyticus* carrying spore powder; Treatment 4) Releasing *Neoseiu's pasteurellium* carrying spore powder; Treatment 5) Releasing only *Tetranychus saprophyticus*; Treatment 6) Spraying with mycelium 1 day after releasing *Tetranychus saprophyticus*; Treatment 7) Releasing *Tetranychus saprophyticus* 1 day after spraying with mycelium. according to Figure 6It can be seen that there are significant differences in the corrected mortality rates of different treatment groups for each insect developmental stage. In treatment groups 1, 3, 5, 6, and 7, the corrected mortality rate of first-instar newly hatched nymphs reached 100%, making them the easiest to control. Third-instar male nymphs and female adults also showed high corrected mortality rates in most treatment groups, especially in treatment groups 3, 4, 6, and 7, where the mortality rate approached 100%, indicating that these two developmental stages can be controlled using entomopathogenic fungi and predatory mites. In contrast, the control efficacy against eggs and second-instar nymphs was lower in all treatments, with mortality occurring only in some treatment groups (1, 6, and 7), but the mortality rate was low, with the overall mortality rate of eggs being lower than that of other developmental stages.

[0059] From the perspective of treatment methods, the control effect of using only spore powder (treatment 2) on Japanese pine scale was limited. In the treatment groups with bacteria-carrying *Neoseiuthis basilaria* and *Tyrophagus saprophyticus* (treatments 3 and 4), only *Tyrophagus saprophyticus* carrying bacteria caused a high mortality rate in first-instar newly hatched nymphs, third-instar male nymphs, and adult females. However, there was no significant difference between treatment groups 3 and 5. The high mortality rate was mainly due to the predatory action of *Tyrophagus saprophyticus*. *Neoseiuthis basilaria* carrying bacteria did not have a significant control effect on Japanese pine scale. Spraying bacteria alone (treatment 1) or "*Tyrophagus saprophyticus* + bacterial solution" (treatments 6 and 7) showed the best control efficacy against first-instar newly hatched nymphs, third-instar male nymphs, and adult females. Overall, the control effect of releasing only *Tyrophagus saprophyticus* or releasing bacteria alone was limited, while the combined treatment of "mites + bacteria" significantly improved the corrected mortality rate of all life stages of Japanese pine scale.

[0060] 2. Combined control efficacy of saprophytic mites and Metarhizium anisopliae CQMa421 against Japanese pine scale in simulated forest environment. The first-instar newly hatched nymphs, third-instar male nymphs and female adults of Japanese pine scale were collected from healthy individuals in the same pine forest in Culaishan National Forest Park for the experiment.

[0061] Before the experiment, the *Metarhizium anisopliae* CQMa421 was rinsed with 0.05% Tween-80 sterile water, filtered through a 300-mesh screen to remove hyphae and impurities, counted using a hemocytometer, and the spore suspension concentration was adjusted to 1×10⁻⁶. 8 Spores / mL were prepared for use, while the control group used 0.05% Tween-80 solution. To evaluate the combined control effect under different application strategies, this experiment set up 5 treatment groups. The corresponding number of insects in each treatment group was 50 carrion mites, 50 newly hatched nymphs, and 10 third-instar male nymphs / female adults. Each treatment was replicated 3 times.

[0062] The experiment consisted of five treatment groups: Treatment 1) released bacteria-laden Tyromitra saprophytica; Treatment 2) only sprayed with 1×10 8Spore suspension at spores / mL; Treatment 3) only introduced carrion mites; Treatment 4) introduced carrion mites first, then sprayed with spore suspension after 1 day; Treatment 5) sprayed with spore suspension first, then introduced carrion mites 1 day later. The entire experiment was observed for 7 days. The criteria for judging death were that the insect body was shriveled and blackened and mycelium grew on the body surface.

[0063] From the overall pest control effect ( Figure 7 Treatment 5 (spraying fungal spores first, followed by the introduction of fungal-carrying saprophytic mites 1 day later) showed the best synergistic control effect. After 7 days of treatment, the mortality rate of newly hatched nymphs was 48%, third-instar male nymphs 76.6%, and the highest among adult females at 83.3%, significantly higher than other treatment groups. This result indicates that spraying fungal spores first, followed by the introduction of saprophytic mites as predators, can quickly suppress the population of *Pinus koraiensis*. Treatment 4 (introducing fungal-carrying saprophytic mites first, followed by fungal spore spraying 1 day later) showed the second best control effect, but the results were not significantly different from treatment 5; both treatments effectively reduced the number of *Pinus koraiensis*. In contrast, treatment 2 (spraying only 1×10⁶ fungal spores) showed the best control effect. 8 The spore suspension (spores / mL) showed the second-highest control efficacy against the Japanese pine scale, second only to treatments 4 and 5, with corrected mortality rates of 73.3% for adult females and 70% for third-instar male nymphs, but lower mortality rates for newly hatched nymphs. Treatments 1 and 3 showed the lowest control efficacy against adult females and third-instar male nymphs, but higher mortality rates for first-instar newly hatched nymphs than the treatments that only sprayed the bacterial suspension. Overall, the single treatment groups (treatments 1, 2, and 3) showed lower control efficacy against all stages of the Japanese pine scale than the combined treatment groups. In the combined treatment groups, the application strategy of first spraying Metarhizium anisopliae CQMa421 followed by the release of bacteria-carrying carrion mites could fully utilize the synergistic effect of fungal infection and mite predation, significantly improving the control efficacy against different stages of the Japanese pine scale.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications 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. Tyrophagus putrificans ( Tyrophagus putrescentiae Its application as a vector for the transmission of entomopathogenic fungi is characterized by, The saprophytic mites carry spores of insect pathogenic fungi on their body surface and spread the spores to the target environment through crawling activities.

2. The application according to claim 1, characterized in that, The entomopathogenic fungus is *Metarhizium anisopliae* (a type of beetle). Metarhizium anisopliae )CQMa421.

3. The application according to claim 1, characterized in that, The saprophytic mites are treated by spore suspension impregnation or powder spraying to make their body surface carry spores of insect pathogenic fungi.

4. The application according to claim 3, characterized in that, The spore suspension impregnation method is as follows: *Tyromitra saprophytica* is immersed in a solution with a concentration of 1×10⁻⁶. 8 Place in a spore suspension of spores / mL for 5 seconds, then remove and air dry naturally.

5. The application according to claim 1, characterized in that, The target environment refers to the branches, trunks, and bark crevices of pine trees where Japanese pine scale insects have caused damage.

6. A biological vector for transmitting spores of entomopathogenic fungi, characterized in that, The biological carrier is the saprophytic mites, whose body surface is covered with spores of insect pathogenic fungi.

7. A method for preparing a saprophytic mite carrying spores of an entomopathogenic fungus, characterized in that, Includes the following steps: (1) Prepare a suspension of spores of insect pathogenic fungi with a concentration of 1×10⁻⁶. 8 spores / mL; (2) Immerse adult or nymphal carrion mites in the spore suspension for 5 seconds; (3) Take it out and air dry it naturally to obtain the bacteria-carrying putrefactive mites.

Images