Application of beauveria bassiana spores in prevention and treatment of Asiatic corn borer by reducing application of chlorantraniliprole

Abstract

The invention relates to application of beauveria bassiana spores in prevention and treatment of Asiatic corn borer by reducing application of chlorantraniliprole, and belongs to the technical field of prevention and treatment of Asiatic corn borer. The technical problems that in the prior art, chlorantraniliprole is adopted for preventing and controlling the Asiatic corn borer pests, drug resistance is easily generated, and environmental safety and food safety are affected are solved. The beauveria bassiana bud spore can be applied to prevention and treatment of Asiatic corn borer by reducing the application amount of chlorantraniliprole, and the application amount of chlorantraniliprole is reduced by 10-90%. According to the application, the dosage of chemical pesticides can be reduced, the development of drug resistance can be delayed, the environmental safety can be improved, cross-generation prevention and control can be realized, and bud spores have higher surface adhesiveness and infection activity than conidia and have a more remarkable synergistic interaction effect with chlorantraniliprole.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Ostrinia furnacalis prevention and treatment, and particularly relates to application of Beauveria bassiana blastospores in reducing chlorantraniliprole application for Ostrinia furnacalis prevention and treatment. BACKGROUND

[0002] Ostrinia furnacalis (Guenée) is an important pest of corn in China, which can cause corn yield reduction. In the prior art, the prevention and treatment methods of Ostrinia furnacalis mainly include chemical prevention and treatment and biological prevention and treatment.

[0003] Among them, the chemical prevention and treatment has a quick effect, for example, chlorantraniliprole (CAP) has a good effect on Ostrinia furnacalis prevention and treatment, but the pests are prone to drug resistance, and the environmental safety and food safety are affected. Studies have shown that the resistance multiples of Ostrinia furnacalis field populations to chlorantraniliprole show regional differences: in the Huang-Huai-Hai River Basin, some populations have reached the medium resistance level; and in some local areas of the Yangtze River Basin, the resistance multiple has broken through the 50-fold threshold.

[0004] The biological prevention and treatment with pathogenic microorganisms as the core has better environmental compatibility, but has limitations such as a long prevention and treatment period and insufficient quick-acting property. Beauveria bassiana is a widely used biological insecticide. According to different culture conditions, Beauveria bassiana is divided into three different forms of aerial conidia, blastospores and deep spores. The most commonly used aerial conidia are mainly obtained by surface culture of solid culture medium, and have high environmental tolerance. However, the conidia are completely hydrophobic, and are prone to germination and loss of activity after encountering water, which makes it difficult to prepare liquid formulations; the blastospores are produced through liquid submerged fermentation, and have relatively weak surface hydrophobicity. This characteristic makes the blastospores more suitable for liquid formulation through freeze-drying method to prolong the activity, and the blastospores are more easily attached to the body surface of insects, so compared with other spore forms, the blastospores are more suitable for the preparation of liquid formulations, and the development of new formulations of Beauveria bassiana using blastospores has become one of the key development directions in the field; the deep spores are a special form formed under liquid culture conditions, and have high metabolic activity. SUMMARY

[0005] The application utilizes the characteristics that the blastospores of Beauveria bassiana are easy to culture, the survival and development ability of Ostrinia furnacalis after feeding is reduced, and the sensitivity to chlorantraniliprole is improved, so that the purpose of reducing the amount of chlorantraniliprole is achieved, the generation of drug resistance is delayed, and the environmental safety is improved.

[0006] The technical scheme adopted by the application to achieve the above-mentioned purposes is as follows.

[0007] The application provides application of Beauveria bassiana blastospores in prevention and treatment of Asian corn borer under reduced application of chlorantraniliprole, wherein the reduced application amount of the chlorantraniliprole is 10%-90%.

[0008] Preferably, the Beauveria bassiana is a Beauveria bassiana strain D1-5 with the preservation number ACCC No. 32726.

[0009] Preferably, the reduced application amount of the chlorantraniliprole is 20%-90%, more preferably 30%-90%, more preferably 40%-90%, more preferably 50%-90%, more preferably 60%-90%, and most preferably 70%-90%.

[0010] Preferably, the Beauveria bassiana blastospore suspension and the chlorantraniliprole suspension are fed to Asian corn borer at the egg hatching peak, and the volume ratio of the Beauveria bassiana blastospore suspension to the chlorantraniliprole suspension is 5:1-1:5.

[0011] The concentration of the Beauveria bassiana blastospore suspension is 0.1×10 8 The concentration of the chlorantraniliprole suspension is 0.003mg / kg-0.015mg / kg. 9 The concentration of the chlorantraniliprole suspension is 0.003mg / kg-0.015mg / kg.

[0012] The concentration of the chlorantraniliprole suspension is 0.003mg / kg-0.015mg / kg.

[0013] More preferably, the concentration of the chlorantraniliprole suspension is 0.003mg / kg-0.001mg / kg.

[0014] More preferably, the concentration of the chlorantraniliprole suspension is 0.003mg / kg-0.001mg / kg.

[0015] More preferably, the feeding mode is spraying by using a drone or a sprayer.

[0016] More preferably, the total spraying amount is 50-100mL / acre, and the spraying is repeated once every 7-10 days.

[0017] More preferably, the preparation method of the Beauveria bassiana blastospore suspension is as follows:

[0018] S1. Solid-phase fermentation culture of blastospores: inoculate the Beauveria bassiana on a solid culture medium, perform primary activation culture, transfer to a solid culture medium containing ampicillin for secondary purification culture, and obtain blastospores.

[0019] S2. Liquid-phase fermentation propagation of blastospores: Conidia are taken and an initial suspension is prepared with a 0.01 wt%-0.1 wt% nonionic surfactant solution. The initial suspension is transferred to a liquid culture medium and fermented. The resulting fermentation broth is filtered, centrifuged, and evenly dispersed with sterile saline to obtain a blastospore suspension of Beauveria bassiana.

[0020] More preferably, in S1, Beauveria bassiana is inoculated on a solid culture medium using a four-zone streak method.

[0021] More preferably, in S1, the solid culture medium is SDAY or PDA.

[0022] More preferably, in S1, the temperature of the primary activation culture is 26±1° C., and the culture time is 3 to 6 days, and even more preferably 5 days.

[0023] More preferably, in S1, the final concentration of ampicillin in the solid culture medium is 50 μg·mL -1 -150 μg·mL -1 , and more preferably 100 μg·mL -1 .

[0024] More preferably, in S1, the culture time of the secondary purification culture is 14-21 days.

[0025] More preferably, in S2, the nonionic surfactant is Tween-80, Triton X-100 or NP-40, and the concentration of conidia in the initial suspension is 1×10 4 Spore count / mL or more.

[0026] More preferably, in S2, the liquid culture medium is SDY.

[0027] More preferably, in S2, the water content of the liquid culture medium is 45%-50%.

[0028] More preferably, in S2, the fermentation culture temperature is 25° C., the time is 3-4 days, and the dissolved oxygen concentration of the liquid culture medium in the fermentation culture is ≥30% saturation.

[0029] More preferably, in S2, the filtration is 400 mesh filtration, the centrifugation is gradient centrifugation, the centrifugation temperature is 4°C, the centrifugation speed is 12000×g, the centrifugation time is 15 min, the water content of the precipitate after centrifugation is controlled at 10-15% (w / w), and the shaking is pulse shaking, 30 s×3 times.

[0030] More preferably, in S2, the final concentration of the Beauveria bassiana blastospore suspension is monitored by real-time hemocytometer counting.

[0031] It should be noted that the peak of O. furnacalis egg hatching is the prior art, and the first generation egg hatching peak is usually in late June to early July, and the second generation egg hatching peak is in late August to mid-August.

[0032] The principle of the present application is:

[0033] The present application reduces the amount of chemical pesticides by compounding Beauveria bassiana blastospores and low-dose chlorantraniliprole.

[0034] Growth inhibition: After O. furnacalis larvae feed on Beauveria bassiana blastospores, their body weight and survival rate are significantly reduced, and the pupation rate, adult emergence rate, egg production and hatching rate are also significantly reduced.

[0035] Increased sensitivity to pesticides: Beauveria bassiana blastospores combined with chlorantraniliprole showed significant synergistic effect, and low concentration CAP (LC 10 )+BS+CAP treatment group had significantly lower larval survival rate than CAP treatment group (χ 2 =173.9, p<0.001), and the median lethal time (LT 50 ) was shortened by 30.0%, and the weight of the larvae was reduced by 62.9%; the larval survival rate of the medium concentration CAP (LC 50 )+BS+CAP treatment group was also significantly lower than that of the CAP treatment group (χ 2 =8.73, p<0.05); the synergistic effect was masked at high concentration CAP (LC 100 ); the blastospore synergistic effect is better than that of conidia.

[0036] Intestinal microbial regulation: 16S rRNA sequencing confirmed that Beauveria bassiana blastospores can reduce the abundance of Enterococcaceae in Firmicutes, increase the abundance of Proteobacteria, inhibit immune-related bacteria such as Lactobacillus, cause host nutritional metabolism disorder and immune suppression, and enhance the sensitivity to chlorantraniliprole.

[0037] Transcriptome analysis: A total of 421 significantly differentially expressed genes were identified by differential gene screening, and the differential genes significantly changed in biological processes such as transmembrane transport, carbohydrate metabolism and transcriptional regulation. The specific differential genes were enriched in ribosome pathway (interference of protein synthesis), folic acid biosynthesis pathway (nutritional competition) and peroxisome pathway (weakening of antioxidant defense), revealing that the fungus synergistically enhances the toxicity of the drug through multiple pathways. KEGG pathway analysis showed that the up-regulation of ribosome pathway genes suggests that the fungus may inhibit the immune response function of the host by interfering with the synthesis of host proteins. The enrichment of the folic acid biosynthesis pathway reflects the nutritional competition between the pathogen and the host, and the down-regulation of the peroxisome pathway suggests that Beauveria bassiana may increase the sensitivity of pests to chlorantraniliprole by weakening the antioxidant defense system of the host.

[0038] Compared with the prior art, the beneficial effects of the present application are:

[0039] 1. Reduce the amount of chemical pesticides: The compounding scheme can reduce the amount of chlorantraniliprole by 50%-90% at LC 10 -LC 50 concentration, and the field control effect still reaches more than 90% (compared with the conventional dosage of single agent).

[0040] 2. Delay the development of resistance: By the dual action target of fungus and drug (body wall infection + ryanodine receptor activation), the selection pressure of single agent is reduced, and no significant increase in resistance fold is detected after 5 generations of continuous indoor subculture.

[0041] 3. Improve environmental safety: Beauveria bassiana blastospores have no chemical residues and are harmless to natural enemy insects (such as trichogramma), and the compounding scheme can reduce the decrease of soil microbial diversity index (Shannon) by 23% compared with single agent chemical control.

[0042] 4. Synergistic effect of fungus and drug: Beauveria bassiana blastospores significantly enhance the sensitivity of the host to chlorantraniliprole by inhibiting the growth and development of the pest (such as survival rate reduced to 51.62%, pupation rate reduced to 15.50%) and regulating the intestinal flora, so that the survival rate of larvae at LC 10 dose is reduced by 62.9% compared with single agent, and the LT 50 is shortened by 30%.

[0043] 5. Advantage of blastospores: Blastospores have higher surface adhesion and infection activity than conidospores, and the synergistic effect is more significant (such as the weight of the insect treated with blastospores + CAP is reduced by 42.3% compared with the conidospores + CAP group).

[0044] 6. Cross-generation control effect: Beauveria bassiana blastospores can achieve sustained pest control by affecting the amount of eggs produced by the parent generation (reduced by 24.5%) and the egg hatching rate (reduced by 33.8%), and inhibiting the survival rate of the offspring population (F1 generation survival rate reduced by 7%).

[0045] 7. The preparation method of the Beauveria bassiana blastospore suspension of the present application ensures the purity of the strain through double-stage antibiotic screening, effectively removes metabolic by-products by combining gradient centrifugation technology, and significantly improves the spore dispersity (PDI<0.15) by using pulse oscillation method. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0047] Figure 1 The influence of the blank group (Control) and the Beauveria bassiana blastospores (BS) in the embodiment 1 of the present application on the cumulative survival rate of the Asian corn borer.

[0048] Figure 2 The influence of the blank group (Control) and the Beauveria bassiana blastospores (BS) in the embodiment 1 of the present application on the pupation rate of the Asian corn borer.

[0049] Figure 3 The influence of the blank group (Control) and the Beauveria bassiana blastospores (BS) in the embodiment 1 of the present application on the emergence rate of the Asian corn borer.

[0050] Figure 4 The influence of the blank group (Control) and the Beauveria bassiana blastospores (BS) in the embodiment 1 of the present application on the number of eggs laid by the Asian corn borer.

[0051] Figure 5 The influence of the blank group (Control) and the Beauveria bassiana blastospores (BS) in the embodiment 1 of the present application on the number of newly hatched Asian corn borer larvae.

[0052] Figure 6 The influence of the blank group (Control) and the Beauveria bassiana blastospores (BS) in the embodiment 1 of the present application on the cumulative survival rate of newly hatched Asian corn borer larvae.

[0053] Figure 7Effect of the control (Control), Beauveria bassiana blastospores (BS), chlorantraniliprole (CAP), and the combination of both (BS+CAP) on the survival rate of O. furnacalis in Example 2 of the present application.

[0054] Figure 8 Effect of the combination of B. bassiana blastospores and chlorantraniliprole (BS+CAP), B. bassiana conidia and chlorantraniliprole (CA+CAP), chlorantraniliprole (CAP), B. bassiana blastospores (BS), B. bassiana conidia (AC), and the control (Control) on the survival rate of O. furnacalis in Example 3 of the present application.

[0055] Figure 9 Effect of B. bassiana blastospores (BS), B. bassiana conidia (AC), and the control (Control) on the weight of O. furnacalis at different instars in Example 4 of the present application.

[0056] Figure 10 Effect of the combination of B. bassiana blastospores and chlorantraniliprole (BS+CAP), B. bassiana conidia and chlorantraniliprole (CA+CAP), and chlorantraniliprole (CAP) on the weight of O. furnacalis at different instars in Example 4 of the present application.

[0057] Figure 11 Effect of B. bassiana blastospores (BS), chlorantraniliprole (CAP), and the combination of both (BS+CAP) on the survival rate of O. furnacalis on potted corn in Example 5 of the present application.

[0058] Figure 12 Effect of the combination of B. bassiana blastospores and chlorantraniliprole at different lethal concentrations LC10, LC50, and LC100 on the survival rate of O. furnacalis on potted corn in Example 6 of the present application.

[0059] Figure 13 Bar chart of the species composition of the intestinal bacteria of O. furnacalis (family level) in Example 7 of the present application.

[0060] Figure 14 Bar chart of the species composition of the intestinal bacteria of O. furnacalis (genus level) in Example 7 of the present application. DETAILED DESCRIPTION

[0061] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in conjunction with examples.

[0062] In the following examples, various processes and methods that are not described in detail are conventional methods well known in the art. The materials, reagents, apparatus, instruments, equipment, etc. used in the following examples, unless otherwise specified, are commercially available.

[0063] The method for preparing the Beauveria bassiana blastospore suspension used in Examples 1-7 is as follows:

[0064] S1. Solid-state fermentation culture of conidia: Beauveria bassiana D1-5 was inoculated on SDAY solid medium (Φ90 mm) using a four-zone streaking method for single spore isolation and culture in a 26±1°C constant temperature incubator. After 5 days of primary activation, it was transferred to secondary purification culture on PDA medium containing 100 μg·mL -1 of ampicillin, with a culture period of 14-21 days. The water content of the medium was maintained at 45%-50% during the culture until conidia were formed.

[0065] S2. Liquid fermentation of blastospores: Conidia on the surface of mature colonies were scraped aseptically to prepare an initial suspension (concentration of conidia: 1×10 4 spores / mL) with 0.05% Tween-80 solution. Then, the initial suspension was transferred to a 500 mL conical flask containing 100 mL of SDY liquid medium, and liquid fermentation was performed in a 25°C constant temperature shaking incubator at a rotation speed of 200 rpm for a period of 3.5 days. The dissolved oxygen concentration during the fermentation was ≥30% saturation. Then, the obtained fermentation broth was subjected to primary filtration through 400-mesh sterile gauze (four layers of gauze), and gradient centrifugal purification was performed at 12000×g for 15 min at 4°C. After centrifugation, the supernatant was discarded, and the water content of the precipitate was controlled at 10-15% (w / w). Then, the precipitate was resuspended with sterilized physiological saline, and pulsed shaking was performed using a vortex shaker (Vortex-Genie 2) for 30 s×3 times to ensure uniform dispersion of the spores. Then, the final concentration was calibrated to 1.0×10 8 spores / mL through real-time blood cell counting. Finally, it was divided into sterile cryogenic tubes and stored in the dark at 4°C for standby use (validity period ≤72 h) to obtain the Beauveria bassiana blastospore suspension, with a pH value of 6.8±0.2.

[0066] The feed used in Examples 1-7 was artificial feed composed of cornmeal, soybean meal, yeast powder, agar powder, Vc, sorbic acid, glucose, and H2O at a mass ratio of 60:60:36:8:2:2:30:500.

[0067] Example 1

[0068] Effect of Beauveria bassiana blastospores on the growth and development of Ostrinia furnacalis

[0069] Two treatments were set up:

[0070] Treatment 1: Control group, 50 μL, mixed into 5 g of feed

[0071] Treatment 2: Beauveria bassiana blastospore suspension (BS) group, 50 μL, mixed into 5 g of feed

[0072] Parallel treatment groups were set up, each containing 10 biological replicates. Each replicate was released with 2nd instar larvae in a physiological state (n = 20), with a total of 400 individuals in the cumulative experimental sample. The observation period covered the entire life history stage of O. furnacalis, and life table parameters were collected daily at regular times (09:00 and 16:00): important biological parameter indicators such as the survival rate of O. furnacalis individuals, the number of pupae, the number of adults, the number of eggs, the number of hatchlings, and the survival rate of the new generation of larvae after hatching were recorded. For fecundity parameters, mating combinations were configured in a 1:1 sex ratio during the adult mating period (within 24 hours after emergence), and egg masses were collected using an oviposition cage. The number of eggs laid by a single female was recorded. The F1 generation of larvae was cultured using a standardized feeding regimen, and the survival curve was systematically recorded for subsequent statistical analysis.

[0073] Data statistics and analysis are as follows:

[0074] The number of dead insects in each replicate group of different treatments was recorded daily until pupation, which was used to calculate the survival rate.

[0075] The number of pupae in each replicate group of different treatments was recorded daily, and the pupation rate was represented by p, with a value of % calculated according to formula (1):

[0076]

[0077] The pupae in each replicate group of different treatments were collected and paired, with 20 pairs selected for each treatment and two replicates. The emergence rate was represented by f, with a value of % calculated according to (2):

[0078]

[0079] In the formula: P-pupation rate, l-number of active insects, n-number of pupae, m-number of pupal shells, F-emergence rate.

[0080] After all the experimental individuals had completed emergence, the egg production of female individuals in each replicate of different treatment groups was quantitatively analyzed. During the experiment, sterile operation techniques were used to collect the eggs of each treatment group and each replicate sample, and accurate counting was performed, while biological indicators such as egg hatching rate were recorded. For successfully hatched larvae, a standardized feeding regimen was used for cultivation. To ensure the reliability of experimental data, fresh feed was replaced daily at regular times.

[0081] To eliminate non-experimental interference, individuals with mechanical damage due to improper operation and individuals that died within the natural mortality range were excluded during data collection. Microsoft Excel 2024 was used to initially organize and calculate the raw experimental data, and biological indicators such as insect cumulative survival rate and insect body weight were calculated. For statistical analysis, all experimental results were processed using IBM SPSS Statistics 25.0 software. Descriptive statistical analysis was first performed on the dataset, and basic statistics such as means and standard deviations were calculated for each indicator. One-way analysis of variance (ANOVA) and log-rank tests were used to compare and assess differences between groups. To visualize the experimental results, data processing was performed using Sigmaplot 12.0 and GraphPad Prism 8.0.2, respectively.

[0082] The feeding experiment of second-instar Asian corn borer lasted for 15 days. The cumulative survival rate of corn borer was Figure 1 As shown in the figure, from the third day of the experiment, the control group was higher than the BS group. From the seventh day, the group treated with Beauveria bassiana blastospores (BS) showed a large number of deaths, which was significantly different from the control group (p < 0.001). On the ninth day, the cumulative survival rate of the control group was (83.90 ± 4.53)%, and the cumulative survival rate of the BS group was (69.12 ± 5.38)%, with a significant difference between the two groups (χ 2 =44.06, p < 0.001). On day 11, the cumulative survival rate of Control was (68.40 ± 8.27)%, and the cumulative survival rate of BS was (51.62 ± 12.17)%. There was a significant difference between the two groups (χ 2 =23.05, p<0.001). This shows that the group treated with Beauveria bassiana blastospores had a significant inhibitory effect on the survival rate of the second-instar larvae of Asian corn borer.

[0083] The pupation period of the Asian corn borer is 6 days. The number of corn borer pupae is as follows: Figure 2 As shown in the figure, Control > BS. On day 1, the pupation rate in the Control group was (12.5±5.89)%, while that in the BS group was 0%, with a significant difference (F=45.05, p<0.05). On day 4, the pupation rate in the Control group was (32.00±10.05)%, while that in the BS group was (15.50±5.50)%, with a significant difference (F=20.75, p<0.001). This indicates that the inoculation of Beauveria bassiana blastospores inhibited both the pupation time and the number of pupae in second-instar larvae of the Asian corn borer.

[0084] The emergence period of the Asian corn borer is 6 days. The number of corn borers emerging is as follows: Figure 3As shown in the figure, the Control group was significantly higher than the BS group from day 2 to day 6 (p < 0.05). On day 2, the eclosion rate of the Control group was (27.50 ± 3.54)%, while that of the BS group was (10.00 ± 7.07), a significant difference between the two groups (F = 48.97, p < 0.001). On day 6, the eclosion rate of the Control group was (67.50 ± 3.53)%, while that of the BS group was (50.00 ± 7.07), a significant difference between the two groups (F = 49.04, p < 0.001). This indicates that the group treated with Beauveria bassiana blastospores inhibited the number of second-instar larvae emerging from the eclosion.

[0085] The Asian corn borer lays eggs for 6 days. Figure 4 As shown in the figure, it can be seen that Control>BS. On day 1, the number of eggs laid by the Control was (51.00±5.65) and that by the BS was 0, with a significant difference between the two (F=84.73, p<0.001). On day 2, the number of eggs laid by the Control was (102.00±1.41) and that by the BS was (77.00±1.41), with a significant difference (F=65.28, p<0.001). On days 3-6, the number of eggs laid by the Control group was significantly higher than that of the BS group (p<0.001). The results showed that the group treated with Beauveria bassiana blastospores had a significant inhibitory effect on the egg-laying time and number of eggs laid by second-instar larvae of the Asian corn borer.

[0086] The Asian corn borer hatching period is 5 days, and the number of corn borer hatching is as follows: Figure 5 As shown in the figure, the control group was significantly higher than the BS group. On day 3, the control group had a population of (91.50±9.19) and the BS group had a population of (57.50±2.12), with a significant difference between the two groups (F=73.03, p<0.01). On day 5, the control group had a population of (154.00±15.56) and the BS group had a population of (102.00±8.48), with a significant difference between the two groups (F=64.15, p<0.001). The results showed that the group treated with Beauveria bassiana blastospores inhibited the hatching time and number of second-instar Asian corn borer larvae.

[0087] The feeding experiment of the new generation of Asian corn borers after hatching lasted for 15 days. The cumulative survival rate of corn borers was as follows: Figure 6 As shown in the figure, BS < Control. On day 11, the cumulative survival rate of Control was (94.00±2.11)%, and the cumulative survival rate of BS was (87.00±3.49)%. There was a significant difference between the two (χ 2= 13.45, p < 0.01). Thus, the inoculation of B. bassiana blastospores inhibited the survival rate of the next generation of the second instar O. furnacalis.

[0088] Example 2

[0089] The effect of the synergistic action of B. bassiana blastospores and chlorantraniliprole on the survival rate of O. furnacalis was studied by setting up four treatment groups (the concentration of chlorantraniliprole suspension used was 0.003 mg / kg):

[0090] Treatment 1: Control group, 50 μL of water

[0091] Treatment 2: B. bassiana blastospore suspension (BS) treatment group, 50 μL

[0092] Treatment 3: B. bassiana blastospore suspension + chlorantraniliprole suspension (BS + CAP) treatment group, a total of 60 μL, with a volume ratio of 5:1

[0093] Treatment 4: Chlorantraniliprole suspension (CAP) treatment group, 50 μL

[0094] Each group contained 10 biological replicates, and 20 healthy, uniform 3rd instar larvae were introduced into each replicate. The total sample size was 800 larvae to ensure the reliability and statistical power of the data.

[0095] Experimental method: The samples in each treatment group were evenly mixed with 5 g of feed, and the larvae after 24 h of starvation were introduced. The larvae were reared at 25 ± 1°C and 75% ± 5% RH, and the mortality rate was observed and recorded regularly.

[0096] The experiment lasted for 11 days, and the cumulative survival rate of O. furnacalis was as shown in Table 2. Figure 7 As can be seen from the figure, the cumulative survival rates of the various treatment groups were significantly different, with the Control group > the BS group > the CAP group > the BS + CAP group. The survival rate of the BS + CAP group was significantly lower than that of the CAP group (p < 0.001), indicating that the synergistic action of B. bassiana blastospores and chemical pesticides significantly enhanced the insecticidal effect of CAP. Specifically, the survival rate of the BS + CAP group decreased to 0% on the 7th day, while that of the CAP group was 35.83%, and the difference between the two was significant (χ2= 173.9, p < 0.001). The results showed that the infection of B. bassiana blastospores significantly improved the sensitivity of O. furnacalis larvae to CAP. 2

[0097] Example 3

[0098] ​Effects of Beauveria bassiana blastospores and conidia on survival rate and weight of O. furnacalis larvae treated with chlorantraniliprole

[0099] Six treatment groups were set up (the concentration of chlorantraniliprole suspension used was 0.003 mg / kg, and the concentration of B. bassiana conidia suspension was 1.0 x 10 8

[0100] Treatment 1: Control group, 50 μL of water

[0101] Treatment 2: B. bassiana blastospore suspension (BS) treatment group, 50 μL

[0102] Treatment 3: B. bassiana blastospore suspension + chlorantraniliprole suspension (BS + CAP) treatment group, a total of 60 μL, with a volume ratio of 5:1

[0103] Treatment 4: B. bassiana conidia suspension (AC) treatment group

[0104] Treatment 5: B. bassiana conidia suspension + chlorantraniliprole suspension (AC + CAP) treatment group, a total of 60 μL, with a volume ratio of 5:1

[0105] Treatment 6: Chlorantraniliprole suspension (CAP) treatment group, 50 μL

[0106] Each group contained 10 biological replicates, and 20 healthy, uniform 3rd instar larvae were introduced into each replicate. The total sample size was 1200 larvae to ensure the reliability and statistical power of the data.

[0107] Experimental method: The samples in each treatment group were evenly mixed into 5 g of feed, and the larvae were introduced after 24 h of starvation. The larvae were reared at 25 ± 1°C and 75% ± 5% RH, and the mortality rate was observed and recorded regularly.

[0108] The experiment lasted for 13 days, and the cumulative survival rate of O. furnacalis is shown in Table 1. As can be seen from the table, the cumulative survival rates of the various treatment groups showed a significant gradient difference, in the order of: Control group > AC group > BS group > CAP group > AC + CAP group > BS + CAP group. Specifically, the survival rate of the BS + CAP group was 41.41% on the 5th day, while that of the AC + CAP group was 63.07%, and the two groups showed a significant difference (χ2= 6.87, P < 0.05). Figure 8 2 ​​= 23.41, p < 0.001). The survival rate of BS+CAP treatment group had dropped to 0 at day 7, while the survival rate of AC+CAP treatment group and CAP treatment group had dropped to 0 at day 9 and day 11, respectively. This phenomenon revealed that there was a significant difference in the enhancing effect of the two spore forms on the sensitivity of O. furnacalis to CAP. The inoculation of BS treatment group and AC treatment group both enhanced the sensitivity of O. furnacalis to CAP, and the enhancing effect of BS treatment group was significantly higher than that of AC treatment group.

[0109] Example 4

[0110] Effects of B. bassiana blastospores and conidia on the weight of O. furnacalis larvae at different ages and the effects of B. bassiana blastospores and conidia combined with chlorantraniliprole on the weight of O. furnacalis larvae at different ages

[0111] Six treatment groups were set (same as in Example 3):

[0112] Each group contained 30 biological replicates, 10 replicates of 20 healthy, uniform 3rd instar larvae, 10 replicates of 20 healthy, uniform 2nd instar larvae, and 10 replicates of 20 healthy, uniform 4th instar larvae were inoculated;

[0113] Experimental method: The samples in each treatment group were evenly mixed with 5 g of feed, and the larvae after 24 h of starvation treatment were inoculated. The larvae were reared in an environment with a temperature of 25 ± 1 °C and a relative humidity of 75% ± 5%. The growth and development of the larvae were observed and recorded regularly. The weight of the larvae was measured using an electronic analytical balance (precision 0.0001 g). Before weighing, the larvae were starved for 24 h to empty their intestinal contents, ensuring the accuracy of the data.

[0114] Effects of B. bassiana blastospores and conidia on the weight of O. furnacalis larvae at different ages and the effects of B. bassiana blastospores and conidia combined with chlorantraniliprole on the weight of O. furnacalis larvae at different ages Figure 9 As shown in the table, there was a significant difference in the effect of different spore forms of B. bassiana on the weight of 4th instar O. furnacalis larvae. There was a significant difference in the weight of larvae between the BS treatment group and the Control treatment group, and the difference between the AC treatment group and the Control treatment group also reached a significant level. The weight of larvae in the BS treatment group (0.37 ± 0.03 g) was significantly lower than that in the AC treatment group (0.53 ± 0.04 g) (F = 4.73, p < 0.05), indicating that the inhibitory effect of blastospores on the growth and development of larvae was more significant.

[0115] Effects of B. bassiana blastospores and conidia on the weight of O. furnacalis larvae at different ages and the effects of B. bassiana blastospores and conidia combined with chlorantraniliprole on the weight of O. furnacalis larvae at different ages Figure 10The results showed that the combination of B. bassiana conidia and chlorantraniliprole had a significant synergistic inhibitory effect on the weight of 4th instar larvae of O. furnacalis. One-way ANOVA showed that the weight of the BS+CAP treatment group (0.13±0.03 g) was 62.9% lower than that of the CAP treatment group (0.35±0.04 g) (F=33.34, p<0.001), and was significantly lower than that of the AC+CAP treatment group (0.26±0.03 g) (F=4.64, p<0.05). This indicates that the combination of conidia and chemical pesticides can produce a significant synergistic effect, and the inhibitory intensity is significantly higher than that of the AC+CAP treatment group.

[0116] Example 5

[0117] Effect of the combination of B. bassiana conidia and chlorantraniliprole on the survival rate of O. furnacalis on potted corn

[0118] Three treatment groups were set up (the concentration of chlorantraniliprole suspension used was 0.003 mg / kg):

[0119] Treatment 1: B. bassiana conidia suspension (BS) treatment group, 50 μL

[0120] Treatment 2: B. bassiana conidia suspension + chlorantraniliprole suspension (BS+CAP) treatment group, a total of 60 μL, the volume ratio of the two was 5:1

[0121] Treatment 3: chlorantraniliprole suspension (CAP) treatment group, 50 μL

[0122] Experimental method: The effect of different treatments on the survival rate of 3rd instar larvae of O. furnacalis was evaluated by potting simulation experiments. Three treatment groups were set up, each containing 3 replicates, a total of 9 experimental units. Each potting unit was planted with 10 corn plants at the jointing stage. The test larvae were healthy 3rd instar larvae bred in the laboratory, which were introduced at a density of 5 per corn plant, with a total of 50 introduced per potting unit, and a cumulative test insect amount of 450. During the experiment, the greenhouse environmental parameters were strictly controlled at a temperature of 25±1°C, a relative humidity of 70±5%, and a light cycle of 14L:10D. The survival state of the larvae was observed twice a day at 08:00 and 20:00. The method of administration was spraying, with a total spraying amount of 50-100 mL / acre, and repeated once every 7-10 days.

[0123] The effect of the combination of B. bassiana conidia and chlorantraniliprole on the survival rate of O. furnacalis was systematically evaluated through a 11-day potting simulation experiment. As Figure 11As shown, the cumulative survival rates of each treatment group showed significant differences, and the order was: BS treatment group > BS+CAP treatment group > CAP treatment group. The survival rate of the BS+CAP treatment group was significantly lower than that of the CAP treatment group, specifically, the survival rate of the BS+CAP treatment group decreased to 32.5±4.2% at the 7th day, while the survival rate of the CAP treatment group was 43.7±3.2% at the 7th day (χ2=59.34, p<0.001), in addition, the median lethal time (LT50) of the BS+CAP treatment group (4.5 days) was significantly shorter than that of the CAP treatment group (6.2 days) (p<0.01), further confirming the synergistic effect, indicating that the sensitivity of the conidial spores to the chemical agent was significantly enhanced. 2 50 =4.5 days) was significantly shorter than that of the CAP treatment group (6.2 days) (p<0.01), further confirming the synergistic effect, indicating that the sensitivity of the conidial spores to the chemical agent was significantly enhanced.

[0124] Example 6

[0125] Synergistic effect of Beauveria bassiana conidial spores and different lethal concentrations of chlorantraniliprole on the survival rate of Ostrinia furnacalis on potted corn 10 (0.003 mg / kg), LC 50 (0.015 mg / kg), and LC 100 (0.03 mg / kg) on the survival rate of Ostrinia furnacalis on potted corn

[0126] Eight treatment groups were set up:

[0127] Treatment 1: Control group treated with water

[0128] Treatment 2: Beauveria bassiana conidial spore suspension (BS) treatment group

[0129] Treatment 3: Beauveria bassiana conidial spore suspension + lethal concentration LC 10 (0.003 mg / kg) chlorantraniliprole suspension (BS+LC 10 ) treatment group

[0130] Treatment 4: chlorantraniliprole suspension with lethal concentration LC 10 (LC 10 ) treatment group

[0131] Treatment 5: Beauveria bassiana conidial spore suspension + lethal concentration LC 50 chlorantraniliprole suspension (CAP+LC 50 ) treatment group

[0132] Treatment 6: chlorantraniliprole suspension with lethal concentration LC 50 (LC 50 ) treatment group

[0133] Treatment 7: Beauveria bassiana conidial spore suspension + lethal concentration LC​100 Chlorantraniliprole suspension concentrate (CAP+LC 100 ) treatment group

[0134] Treatment 8: Inoculation lethal concentration is LC 100 Chlorantraniliprole (LC 100 ) treatment group

[0135] Ten corn seedlings at the jointing stage were planted in each pot. The experiment set up 8 treatment groups, each containing 3 biological replicates, for a total of 24 experimental units. The test larvae were healthy third-instar larvae raised in the laboratory, and were inoculated at a density of 5 larvae per corn plant. The total inoculation density for a single pot unit was 50, and the cumulative number of test insects reached 1,200. The larvae were starved for 24 hours before inoculation to standardize their initial physiological state, and individuals with deformities or abnormal activity were screened and eliminated using a stereomicroscope. Survival status was observed twice a day at 08:00 and 16:00. The drug administration method was spraying, with a total spraying volume of 50-100 mL / mu, repeated once every 7-10 days.

[0136] The blastospores of Beauveria bassiana and chlorantraniliprole at different lethal concentrations (LC) were systematically evaluated through an 11-day potted plant simulation experiment. 10 , LC 50 , LC 100 ) on the survival rate of Asian corn borer. Figure 12 As shown, the cumulative survival rate of each treatment group showed significant gradient differences, and the order was: Control treatment group > BS treatment group > LC 10 Treatment group>BS+LC 10 Treatment group>LC 50 Treatment group>BS+LC 50 Treatment group>LC 100 Treatment group>BS+LC 100 Treatment group. The log-rank test showed that BS+LC 10 The survival rate of the treated group was significantly lower than that of the LC 10 Treatment group (χ 2 =42.15, p<0.001), BS+LC 50 The treated group was compared with LC 50 The treatment groups also showed significant differences (χ 2 =8.73, p<0.05), while BS+LC 100 Treatment group and LC 100 There was no statistical difference among the treatment groups (χ 2 =1.26, p>0.05). This result indicates that the synergistic effect of Beauveria bassiana blastospores and chlorantraniliprole is concentration-dependent. 10), BS pretreatment can significantly enhance the insecticidal effect of the pesticide, while in the high concentration range (LC 100 ), BS pretreatment has no obvious enhancement of the insecticidal effect of the pesticide, indicating that the synergistic effect of fungi decreases with the increase of pesticide concentration.

[0137] Example 6

[0138] Effect of Beauveria bassiana blastospores on the intestinal flora of Ostrinia furnacalis

[0139] Each petri dish was weighed 5g of feed, and 50μL of Beauveria bassiana blastospore suspension (set up water treatment as control group) was added to the surface of the feed using a pipette, and then a sterile glass rod was used to stir thoroughly, so that the Beauveria bassiana blastospore suspension and the feed were completely mixed. Healthy and consistent 4-5 instar Ostrinia furnacalis larvae were selected, and they were starved for 24h before the experiment to remove the intestinal contents and improve the feeding rate of the larvae on the treated feed. The starved larvae were randomly transferred to the prepared petri dishes and placed in an artificial climate chamber for 72h of feeding.

[0140] The Ostrinia furnacalis treated with Beauveria bassiana blastospores were dissected, and two treatment groups were set up. Thirty healthy 4-5 instar larvae and 30 Ostrinia furnacalis infected with Beauveria bassiana were selected and dissected after 24h of starvation in a clean bench. First, rinse with sterile water for 1-2min until there is no obvious impurity on the surface of the larvae, then soak the larvae in 75% alcohol for 3min, and finally rinse with sterile water for 3-5 times. The larvae were dissected under sterile conditions, and the insect intestines were dissected with sterilized tweezers, and then placed in a 1.5mL centrifuge tube, frozen in liquid nitrogen, and stored at -80℃ for subsequent experiments.

[0141] DNA was extracted from each sample using the Omega Bio-tek Soil DNA Isolation Kit. DNA purity and concentration were determined using a NanoDrop 2000. PCR amplification: PCR primers corresponding to the following regions: 16SV4 region primers (515F and 806R): Identify bacterial diversity. 18SV4 region primers (528F and 706R): Identify eukaryotic microbial diversity. ITS1 region primers (ITS5-1737F and ITS2-2043R): Identify fungal diversity. In addition, amplified regions include 16SV3-V4, 16SV4-V5, and 16SV5-V7; Archaeal 16SV4-V5 and Archaeal 16SV8; and 18SV9 and ITS2 regions. All PCR mixtures were added with 15 μL Phusion High-Fidelity PCR Master Mix, 0.2 μM primers, and 10 ng genomic DNA template. The first denaturation was performed at 98°C for 1 minute, followed by denaturation at 98°C for 10 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds, for 30 cycles. Finally, the final extension was maintained at 72°C for 5 minutes. The PCR products were mixed and detected by gel electrophoresis. The PCR products were purified using the AxyPrep DNA Gel Extraction Kit. The PCR products were purified using the Quantus TM Fluorometer was used for detection and quantification. The library was constructed using the DNAPCR-Free Sample Preparation Kit. The constructed library was quantified by Qubit and qPCR, and after the library was qualified, it was sequenced on the NovaSeq6000. Finally, data analysis was performed. For example, OTUs were analyzed by abundance, alpha diversity calculation, and petal plot to obtain information on species richness and uniformity within the sample, as well as information on common and unique OTUs between different samples or groups. Principal Coordinates Analysis (PCoA) and Non-Metric Multi-Dimensional Scaling (NMDS) were used to analyze the differences in community structure between different groups. Statistical analysis methods such as Metagenomeseq were used to test the significance of differences in species composition and community structure of grouped samples.

[0142] Figure 13The column chart of the species composition of the gut bacteria of O. furnacalis at the genus level is shown in FIG. 6. As can be seen from the chart, the gut microbiota of different sample groups at the genus level is significantly different. The relative abundance of each group of microorganisms is composed of a variety of genera, including Lactiplantibacillus, Enterococcus, Aquabacterium, Serratia, etc., and the proportion of dominant genera in different samples presents dynamic changes. The vertical axis relative abundance shows that the microbial community structure of the control group and the treatment group has significant distinction in composition ratio. Lactiplantibacillus occupies an absolute dominant position in the control group Control, especially in Control 1, its relative abundance is close to 0.75, becoming the core genus in the community. However, in the BS1, BS2, BS3 treatment groups, the relative abundance of Lactiplantibacillus decreased significantly, among which the BS3 group decreased most obviously, and the relative abundance decreased to about 0.25. This change shows that the Beauveria bassiana conidium may inhibit the abundance of Lactiplantibacillus, leading to the weakening of the dominant position of this genus in the treatment group. Enterococcus slightly increased in the control group and the treatment group, Aquabacterium and Serratia and other genera had low abundance in the control group and showed a slight upward trend in some treatment groups, suggesting that Beauveria bassiana conidium treatment may provide more suitable survival conditions for these genera. The microbial community structure of the control group is relatively similar, and the community composition is relatively single; while the microbial community structure of the treatment group is more diverse. This difference shows that Beauveria bassiana conidium treatment breaks the community structure in the control group, promoting the diversification of the microbial community.

[0143] Figure 14The column chart of the species composition of the intestinal bacteria of the Asian corn borer at the genus level is shown in the figure. It can be seen from the figure that the intestinal flora of different sample groups at the genus level is obviously different. The relative abundance of each group of microorganisms is composed of a variety of genera, including Lactiplantibacillus, Enterococcus, Aquabacterium, Serratia, etc., and the proportion of the dominant genus in different samples presents dynamic changes. The relative abundance of the vertical axis shows that the microbial community structure of the control group and the treatment group has significant distinction in composition ratio. Lactiplantibacillus occupies an absolute dominant position in the control group Control, especially in Control 1, its relative abundance is close to 0.75, becoming the core genus in the community. However, in the BS1, BS2, BS3 treatment groups, the relative abundance of Lactiplantibacillus decreased significantly, among which the BS3 group decreased most obviously, and the relative abundance decreased to about 0.25. This change shows that the Beauveria bassiana blastospores may inhibit the abundance of Lactiplantibacillus, leading to the weakening of the dominant position of this genus in the treatment group. Enterococcus slightly increased in the control group and the treatment group, Aquabacterium and Serratia and other genera had low abundance in the control group and showed a slight upward trend in some treatment groups, suggesting that Beauveria bassiana blastospore treatment may provide more suitable survival conditions for these genera. The microbial community structure of the control group is similar, and the community composition is relatively single; while the microbial community structure of the treatment group is more diverse. This difference shows that the Beauveria bassiana blastospore treatment breaks the community structure in the control group and promotes the diversification of the microbial community.

[0144] Based on the detection results of Example 6, it can be shown that Beauveria bassiana blastospore treatment can reduce the diversity of the intestinal microorganisms of the Asian corn borer and reshape the community structure. Specifically, under the Alpha diversity index, the abundance of the dominant bacterial family Lactobacillaceae decreases, the genus-specific enrichment of Serratia, Aquabacterium, etc. is enriched, and the community composition in PCoA, NMDS analysis shows obvious grouping characteristics.

[0145] Example 7

[0146] Transcriptome research on the interaction between Beauveria bassiana blastospores and the Asian corn borer

[0147] The samples stored at -80℃ were sent to Beijing Nuoweziyuan Technology Co., Ltd. for transcriptomic sequencing and library construction analysis, and the process is briefly described as follows: total RNA of all samples was extracted, and raw data was obtained after library construction and sequencing. The remaining high-quality data was mapped to the sample reference genome, and DESeq2 software (1.42.0) was used for differential expression analysis between sample groups. According to the gene expression level, it can be divided into up-regulated genes and down-regulated genes. Based on the difference in gene expression between the two biological conditions, Padj≤0.05 and |log2(fold change)|≥1 were used as the threshold for significant differential expression. The screened differential genes were subjected to enrichment analysis using Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. By comparing the distribution of transcription factors of all genes and differential genes, transcription factors with obvious proportion difference were screened out. In the transcriptomic study, three biological replicates were set for each parallel group.

[0148] It was found through Example 7 that the GO function enrichment results showed that the differential genes were mainly involved in transmembrane transport, carbohydrate metabolism and transcriptional regulation in biological process BP; the cell component CC related gene was enriched in cell membrane, ribosome and other structures; and the molecular function MF was mainly transport protein and catalytic activity. KEGG pathway analysis further showed that the differential genes were significantly enriched in ribosome, folate biosynthesis and cancer-related proteoglycan pathway, among which the number of up-regulated genes in the ribosome pathway was the most, suggesting that Beauveria bassiana infection may achieve pathogenesis by interfering with host protein synthesis and immune response.

[0149] Obviously, the above examples are only examples for the purpose of clarity, and are not limitations of the examples. For ordinary skilled persons in the art, other different forms of changes or variations can be made on the basis of the above description. It is not necessary and impossible to exhaust all examples. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Use of blastospores of Beauveria bassiana in controlling Asian corn borer by reducing the application of chlorantraniliprole, wherein the application amount of chlorantraniliprole is reduced by 10%-90%.

2. The use according to claim 1, characterized in that The Beauveria bassiana is the Beauveria bassiana strain D1-5, with the preservation number ACCC No. 32726.

3. The use according to claim 1, characterized in that The application amount of chlorantraniliprole is reduced by 50%-90%.

4. The use according to claim 1, characterized in that During the peak hatching period of Asian corn borer eggs, the fish were fed with a suspension of blastospores of Beauveria bassiana and a suspension concentrate of chlorantraniliprole, with the volume ratio of the suspension of blastospores of Beauveria bassiana to the suspension concentrate of chlorantraniliprole being 5:1-1:

5. The concentration of the Beauveria bassiana blastospore suspension is 0.1×10 8 Spore count / mL-1×10 9 spore count / mL, pH 6.8 ± 0.2; The concentration of the chlorantraniliprole suspension concentrate is 0.003 mg / kg-0.015 mg / kg.

5. The use according to claim 4, characterized in that The feeding method is to use a drone or a sprayer for spraying, with a total spraying amount of 50-100 mL / mu, and repeat once every 7-10 days.

6. The use according to claim 4, characterized in that The preparation method of the Beauveria bassiana blastospore suspension is as follows: S1. Beauveria bassiana is inoculated on a solid culture medium for primary activation culture, and then transferred to a solid culture medium containing ampicillin for secondary purification culture until conidia are obtained; S2. Take conidia and prepare an initial suspension with a 0.01wt%-0.1wt% nonionic surfactant solution. Transfer the initial suspension to a liquid culture medium and ferment it. The resulting fermentation broth is filtered, centrifuged, and evenly dispersed with sterile saline to obtain a spore suspension of Beauveria bassiana.

7. The use according to claim 6, characterized in that In S1: Beauveria bassiana was inoculated on solid culture medium using the four-zone streak method. The solid culture medium is SDAY or PDA; The temperature of the primary activation culture is 26±1°C, and the culture time is 3-6 days; The final concentration of ampicillin in the solid culture medium is 50 μg·mL -1 -150 μg·mL -1 ; The culture time of the secondary purification culture is 14-21 days.

8. The use according to claim 6, characterized in that In S2: The nonionic surfactant is Tween-80, Triton X-100 or NP-40; The concentration of conidia in the initial suspension was 1×10 4 Spore count / mL or more; The liquid culture medium is SDY; The water content of the liquid culture medium is 45%-50%; The fermentation culture temperature is 25° C., the fermentation culture time is 3-4 days, and the dissolved oxygen concentration of the liquid culture medium in the fermentation culture is ≥30% saturation.

9. The use according to claim 6, characterized in that In S2: The filtration is 400 mesh filtration; The centrifugation is gradient centrifugation, the centrifugation temperature is 4°C, the centrifugation speed is 12000×g, the centrifugation time is 15 min, and the water content of the precipitate after centrifugation is controlled at 10%-15% (w / w); The oscillation is a pulsed oscillation, 30s×3 times; The final concentration of the Beauveria bassiana blastospore suspension was monitored by real-time hemocytometer counting.

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