A biocontrol preparation containing Beauveria bassiana and its application in controlling Bemisia tabaci

Abstract

The invention discloses a biocontrol agent, system and application thereof for controlling whitefly, wherein the biocontrol agent comprises Beauveria bassiana and a food attractant, wherein the food attractant comprises plant volatile secondary substances. By using the food attractant in combination with Beauveria bassiana, the amount of attraction to whitefly is increased while the germination of conidia of Beauveria bassiana is not affected, thereby improving the transmission efficiency of whitefly to Beauveria bassiana, and achieving a sustained biological control effect.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and particularly relates to a biological control preparation comprising Beauveria bassiana and application thereof in controlling Bemisia tabaci. Background Art

[0002] Bemisia tabaci (Gennadius) belongs to the family Aleyrodidae and is a worldwide invasive pest. The whitefly has a wide host range. It can not only extract plant sap and secrete honeydew to induce sooty mold, but also spread plant viruses as a vector insect. Because of its small size, short life cycle, overlapping generations, and complex biotype, it is difficult to control. It is recognized by the Food and Agriculture Organization of the United Nations as the second largest pest. At present, the control of whitefly mainly relies on chemical pesticides, but whitefly has developed varying degrees of resistance and cross-resistance to most conventional pesticides such as organophosphates, carbamates, pyrethroids, and neonicotinoid insecticides. Therefore, the development and use of green control methods to reduce the use of chemical pesticides has become a top priority for controlling the whitefly population.

[0003] Beauveria bassiana is one of the most widely used insect pathogenic fungi in pest biological control. It has the characteristics of easy large-scale production, active infection, broad insecticide spectrum, and strong pathogenicity. It has great potential in replacing chemical pesticides to control a variety of pests. However, in practical applications, the pest control effect of insect pathogenic fungi is unstable. Even if the highly toxic strains in indoor bioassays are applied in the field in an inundated manner, the expected control effect is often not achieved. The germination of conidia of Beauveria bassiana on leaves is easily affected by environmental factors such as ultraviolet rays, temperature and humidity. Therefore, how to reduce the inoculation requirements and costs of insect pathogenic fungi and at the same time increase the infection rate of pests is the key to promoting the production and application of biocontrol agents.

[0004] In the process of herbivorous insects identifying and locating host plants, various volatile secondary metabolites produced by plants play an important role. Various food attractants developed by simulating the chemical signals produced by host plants are a green and safe pest control product.

[0005] However, there is no report on how to use food attractants and insect pathogenic fungi together for the control of whiteflies. It is of great significance to develop a new method of combining insect pathogenic fungi and food attractants for effective control of insects. Summary of the invention

[0006] In order to overcome the above defects, the present invention combines a fungal product with infectivity and transmission potential with a food attractant that has an attractant effect on whiteflies to provide a simple, easy and efficient method for controlling whiteflies. Specifically:

[0007] A first aspect of the present invention provides a biocontrol agent for controlling Bemisia tabaci, the biocontrol agent comprising Beauveria bassiana and a food attractant, wherein the food attractant comprises plant volatile secondary metabolites.

[0008] Preferably, the biocontrol agent is a mixture, or the Beauveria bassiana and the food attractant are packaged separately.

[0009] Preferably, the biocontrol agent is a mixture, and the Beauveria bassiana and the food attractant are mixed evenly.

[0010] Preferably, the biocontrol agent is packaged separately, that is, Beauveria bassiana and the food attractant are not mixed, but packaged separately, and can be placed separately or mixed when used.

[0011] Preferably, the Beauveria bassiana includes a Beauveria bassiana strain or spore powder prepared therefrom.

[0012] Preferably, the content of Beauveria bassiana or its spore powder is 1-5 (eg, 1, 2, 3, 4, 5) g.

[0013] More preferably, the Beauveria bassiana includes strain XJWLMQ-1 and / or strain LNTL-1 or spore powder prepared therefrom, wherein the strain XJWLMQ-1 is Beauveria bassiana with a preservation number of CGMCC No.40509, and the strain LNTL-1 is Beauveria bassiana with a preservation number of CGMCC No.40508.

[0014] Preferably, the method for preparing the spore powder comprises:

[0015] 1) Preparation of activated spore powder: Inoculate Beauveria bassiana XJWLMQ-1 and / or LNTL-1 on Sabouraud medium SDAY, wherein the Sabouraud medium SDAY contains 1% peptone, 1% yeast extract, 4% glucose and 2% agar. More preferably, the culture conditions are to maintain the temperature at 25-27° C. and culture for 7-10 days to produce activated spore powder of XJWLMQ-1 and / or LNTL-1.

[0016] More preferably, the spore powder culture method further comprises:

[0017] The activated spore powder is prepared by the steps of 2) primary seed liquid culture, 3) secondary seed liquid culture, 4) solid phase fermentation and 5) drying and harvesting the spore powder.

[0018] More preferably,

[0019] 2) Cultivation of primary seed solution

[0020] The activated spore powder of XJWLMQ-1 and / or LNTL-1 was taken and inoculated into Sabouraud culture medium SDY to obtain seed solution. The Sabouraud culture medium SDY contained 1% peptone, 1% yeast extract, and 4% glucose. The initial spore concentration of the seed solution was 1.0×10 6 pc.ml -1 , at 25℃, 120r·min -1 The mixture was shaken and cultured for 48 hours to obtain a primary seed solution.

[0021] 3) Cultivation of secondary seed solution

[0022] The first-level seed solution was inoculated with Sabouraud culture medium SDY culture medium at a 5% inoculation rate and cultured in a 7-liter liquid fermentation tank. 0.5% soybean oil was added to the volume of the fermentation liquid. The culture temperature was 25-27°C, the rotation speed was 100-200rpm, and the initial dissolved oxygen content was above 50%. After culturing for 40 hours, the second-level seed solution was obtained.

[0023] 4) Solid phase fermentation

[0024] Soak the rice in boiling water for 30 minutes, remove and drain excess water, sterilize at 121℃ for 30 minutes, inoculate the rice with 15ml of secondary seed solution per 100g of rice, and use a 20×30cm plastic bag as the fermentation container. After inoculation, lay the rice at 25-26℃ and culture for 7-10 days.

[0025] 5) Drying and harvesting of spore powder

[0026] The solid phase fermented material is dried at 30°C for 3 days, and then spore powder is harvested using a spore harvester, such as a harvester from Mycoharvester Company, UK.

[0027] Preferably, the plant volatile secondary substances include cis-3-hexenal, linalool and eugenol.

[0028] Preferably, the mass ratio of the cis-3-hexenal, linalool and eugenol is (0.1-0.5):(0.01-0.1):(0.5-1), for example, (0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.5):(0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1):(0.5, 0.6, 0.7, 0.8, 0.9, 1).

[0029] Preferably, the food attractant may also include a slow-release synergist, which includes one or more of vegetable oil, butter, honey, paraffin oil, glycerol, vaseline, n-hexane and / or animal oil. Further preferably, the slow-release synergist is paraffin oil.

[0030] More preferably, the volume ratio of the above-mentioned plant volatile secondary substances to the sustained-release enhancer is 1:(1-10), for example 1:(1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0031] Preferably, the food attractant may also include nutrients.

[0032] Preferably, the nutrients include honey, glucose and / or corn syrup.

[0033] Preferably, the food attractant inhibits the germination of Beauveria bassiana or the food attractant does not affect the germination of Beauveria bassiana.

[0034] More preferably, the food attractant inhibits the germination of Beauveria bassiana, and the food attractant and Beauveria bassiana are separately packaged.

[0035] More preferably, the food attractant does not affect the germination of Beauveria bassiana, and the food attractant and Beauveria bassiana are mixed or packaged separately.

[0036] The second aspect of the present invention provides a device for controlling Bemisia tabaci, wherein the device can contain any of the above-mentioned biological control agents.

[0037] Preferably, the device comprises a container having a certain internal space, and the biocontrol agent is contained in the internal space of the container.

[0038] More preferably, the container is in the shape of a triangular prism.

[0039] Preferably, holes are provided on the side of the container to facilitate entry and exit of whiteflies.

[0040] Preferably, the device also includes components for fixing the container, such as a hook, a bracket, etc.

[0041] Preferably, the container is made of any material, and further preferably, the material is a waterproof material.

[0042] Preferably, the size of the device is suitable for placing the Beauveria bassiana and the food attractant at a certain distance. Further preferably, the distance between the food attractant and the Beauveria bassiana is 0-20 (e.g., 0, 5, 10, 15, 20) cm.

[0043] A third aspect of the present invention provides a system for controlling Bemisia tabaci.

[0044] Preferably, the system comprises the above-mentioned biocontrol agent and the above-mentioned device.

[0045] A fourth aspect of the present invention provides a use of the above-mentioned biological control agent, the above-mentioned device or the above-mentioned system in controlling Bemisia tabaci.

[0046] Preferably, the replacement time of Beauveria bassiana is more than 3 days, more preferably, more than 6 days, for example, more than 7, 8, 9, 10, 11, 12, 13, 14, 15 days, etc.

[0047] Preferably, the device is placed for no less than 1 day, and the device is placed for no less than 1, 2, 3, 4, 5, 7, 9, 10, 13, or 14 days.

[0048] A fifth aspect of the present invention provides a method for controlling Bemisia tabaci, wherein the method comprises using the above-mentioned biological control agent, the above-mentioned device or the above-mentioned system for control.

[0049] Preferably, when the biocontrol agents are used separately, the distance between the food attractant and Beauveria bassiana is 0-20 (eg, 0, 5, 10, 15, 20) cm.

[0050] Preferably, the replacement time of Beauveria bassiana is more than 3 days, more preferably, more than 6 days, for example, more than 7, 8, 9, 10, 11, 12, 13, 14, 15 days, etc.

[0051] Preferably, the device or system is placed for no less than 1 day, and the device or system is placed for no less than 1, 2, 3, 4, 5, 7, 9, 10, 13, or 14 days.

[0052] Preferably, the distance between the device or system and the target plant is 0-20 (e.g., 0, 5, 10, 15, 20) cm.

[0053] The term “include” or “comprising” described in the present invention is an open description, which includes the specified components or steps described and other specified components or steps that will not be substantially affected.

[0054] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0055] The advantages of the present invention include but are not limited to:

[0056] 1) The biocontrol agent of the present invention includes both fungal biological control and chemical attractant, and combines the advantages of the two. By establishing an "attract-infect-disseminate" system, the attractant is used to attract the whiteflies to a device where fungal propagules (e.g., spores) are placed, and the whiteflies infected by the fungi spread the fungi. Less Beauveria bassiana can be used to control more whiteflies, thereby improving the efficiency of fungal use and reducing waste, so as to achieve environmentally friendly green control of whiteflies.

[0057] 2) The biocontrol agent of the present invention increases the amount of attraction to whiteflies while not affecting the germination of conidia of Beauveria bassiana, thereby improving the transmission efficiency of whiteflies to Beauveria bassiana and achieving a sustained biological control effect.

[0058] 3) The device of the present invention is configured to accommodate the biocontrol agent and facilitate the entry and exit of whiteflies, thereby protecting the biocontrol agent, especially the fungus Beauveria bassiana, and avoiding the adverse effects of the external environment (such as ultraviolet rays, rain, etc.) on the fungal agent. At the same time, the size of the device is suitable for placing the Beauveria bassiana and the food attractant at a certain distance, thereby avoiding the inhibitory effect of the food attractant on the germination rate of Beauveria bassiana, and also extending the effective use period of the Beauveria bassiana.

[0059] 4) The present invention uses a combination of spore powder and food attractant to attract and infect whiteflies, avoiding the waste caused by flooding spraying of fungal liquid in the prior art. At the same time, the spore powder used in the present invention has a longer survival period and a smaller usage amount than the fungal liquid in the prior art.

[0060] 5) The method for controlling whitefly of the present invention is low in cost, simple to operate, easy to implement and highly effective.

[0061] Strain deposit information:

[0062] 1) Strain XJWLMQ-1

[0063] Depository: China General Microbiology Center (CGMCC);

[0064] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0065] Accession number: CGMCC No.40509;

[0066] Deposit date: February 27, 2023;

[0067] Classification name: Beauveria bassiana.

[0068] 2) Strain LNTL-1

[0069] Depository: China General Microbiology Center (CGMCC);

[0070] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0071] Accession number: CGMCC No.40508;

[0072] Deposit date: February 27, 2023;

[0073] Classification name: Beauveria bassiana. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 : Toxicity of highly virulent strains to adult Bemisia tabaci, where a, b, c, and d represent significant differences between the treatment group and the control group, and between the treatment groups when P < 0.001, and CK is the control group.

[0075] Figure 2 :Determination of spore production of highly virulent strains.

[0076] Figure 3 : Schematic diagram of the automatic inoculation device for food attractants and Beauveria bassiana. DETAILED DESCRIPTION

[0077] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0078] Data processing in the embodiment was performed using Excel 2016 to process the raw data of each experiment, infection rate, lethal time LT 50 SAS 9.2 was used to analyze the significant differences of LT 50 Calculated by POLO software.

[0079] Based on bioassay data

[0080] Infection rate = number of infected insects / total number of treated insects × 100%

[0081] Example 1: Preparation of Beauveria bassiana conidia and food attractants

[0082] 1 Test insects

[0083] Bemisia tabaci were initially collected from tobacco in a greenhouse in Langfang. Bemisia tabaci were reared in the laboratory for multiple generations using tomato seedlings, Solanum lycopersicum L. The rearing conditions were 26±2℃, with a photoperiod of 12:12 (L:D), and 4th instar nymphs and 5-day-old adults after eclosion were selected for use.

[0084] 2 Test strains

[0085] The 20 strains of Beauveria bassiana for testing were collected from insect bodies in different regions of the country. After being isolated and purified indoors, they were inoculated on a slant and stored at 4°C. During the test, the strains of Beauveria bassiana were inoculated on Goettel spore-forming medium for activation culture. The formula is 2% corn flour, 1% wheat bran, 0.5% peptone, 0.3% KH2PO4, 0.1% MgSO4·7H2O, 0.1% NH4NO3, 2% agar, and 0.5% cicada shells. Strains with good biological properties were selected for further bioassays. The spore powder of the test strains obtained by culture on Goettel medium was dissolved in 0.05% Tween-80 solution and prepared to a final concentration of 1×10 8 spores / ml of spore suspension; 0.05% Tween-80 solution was used as blank control.

[0086] 3 Food attractant recipes

[0087] According to the food attractant screened in the early stage and the ratio improvement: the mass ratio of cis-3-hexenal: linalool: eugenol is 0.25:0.05:0.7 (each component is purchased from Beijing Yikeman Biotechnology Co., Ltd.). The food attractant is mixed evenly with liquid paraffin (Shanghai Aladdin Biochemical Technology Co., Ltd.) (v:v=1:9).

[0088] 4 Determination of infection rate of the tested strains on Bemisia tabaci

[0089] 4.1 Whitefly nymphs

[0090] Select a single tomato leaf containing 30 4th instar nymphs of whitefly and immediately immerse it in the prepared 1×10 8 spore / ml suspension for 10s, then take out the leaves and place them on sterile filter paper for 10-20min to dry naturally. The control group was treated in the same way with 0.05% Tween-80 solution. The dried leaves were immediately placed in a culture dish (diameter: 90mm) containing 2% agar, the culture dish was sealed with a sealing film, and cultured in a light incubator at 26±1℃ and 12:12 (L:D). The number of infected whiteflies was recorded every day, each test was repeated 4 times, and the records were kept for 8 consecutive days to calculate the lethal median time of each strain to the 4th instar nymphs of whiteflies.

[0091] 4.2 Adult Bemisia tabaci

[0092] Five strains with high toxicity to whitefly nymphs were used for bioassay on adults. 0.1 g of dry conidia of each strain was evenly distributed on the cotton velvet cloth at the bottom of a petri dish (90 mm diameter). 30 whitefly adults were moved to the cotton velvet cloth at the bottom of a petri dish (90 mm diameter) and walked on the velvet cloth for 1 minute. Then, 30 adults were moved from the velvet cloth into a clean and ventilated glass cage (150×150×200 mm) and tomato seedlings were placed in the cage as a food source. Adults in the control group were exposed to the velvet cloth without dry conidia and then transferred to the same cage. The treatment was completely randomized and repeated 4 times. The whitefly rearing conditions were maintained at a temperature of 25±1°C, a relative humidity of 60%-80%, and a photoperiod of 14L:10D.

[0093] Record the number of dead insects for 11 consecutive days. The dead insects were surface disinfected with 70% alcohol, rinsed three times in distilled water, and transferred to a petri dish with moist sterile filter paper. Mycelium grew on the surface of the dead insects.

[0094] 5. Determination of spore production of test strains

[0095] To determine the spore production of each strain, the spores of the highly virulent strains selected above were prepared with 0.05% Tween-80 solution to a concentration of 1×10 6 Spores / ml suspension. Take 0.1ml of the suspension on 90mm Goettle spore medium (corn flour 2%, bran 1%, peptone 0.5%, KH2PO4 0.3%, MgSO4·7H2O 0.1%, NH4NO3 0.1%, agar 2%), and spread it evenly with a sterile triangular glass rod. Repeat 4 times for each strain. The culture medium was cultured in the dark at 26±1℃ for 15d, and 5 points were randomly selected from the culture medium with a sterile hole puncher with a diameter of 4mm and placed in 5ml of 0.05% Tween-80 solution. The suspension was sonicated in an ultrasonic cleaner for 15min to destroy the spore block structure, and then vortexed with a vortex oscillator for 10min to obtain a uniformly dispersed spore suspension. After the suspension was diluted 10 times, the number of spores was determined using a hemocytometer, and each was repeated 3 times, and then the number of spores per square centimeter was calculated.

[0096] Results of the pathogenicity test of 6 test strains to Bemisia tabaci nymphs

[0097] As shown in Table 1, the tested Beauveria bassiana strains have certain toxicity to whitefly nymphs, and the nymphs in most treatments reached the peak of death in 4-6 days. Among them, the infection rate of 5 strains was above 75%, namely LNTL-1, LNTL-5, JXNC-5, HNLY-46, and XJWLMQ-1. The dead nymphs were moisturized and cultured, and white hyphae grew out of them. It was identified that they were caused by infection with Beauveria bassiana.

[0098] Table 1: Numbers, collection locations, and median lethal time (LT) of 20 strains of Beauveria bassiana 50 ±SE

[0099]

[0100]

[0101] Results of the pathogenicity test of the 7 tested strains to Bemisia tabaci adults

[0102] Five strains with infection rates of more than 75% on whitefly nymphs were selected, namely LNTL-1, LNTL-5, JXNC-5, HNLY-46, and XJWLMQ-1, and their toxicity to whitefly adults was compared. Figure 1 As shown, 5 days after inoculation, the average mortality rate of the control group was 5.21±1.1%, and the mortality rate of the experimental group was 80%-95.9%. The mortality rate of adults inoculated with strains XJWLMQ-1 and LNTL-1 was the highest, at 95.9% and 94.5%, respectively.

[0103] Determination results of spore production of 8 strains

[0104] The results of spore production of 20 strains of Beauveria bassiana are as follows Figure 2 The conidia production of the 20 strains of Beauveria bassiana ranged from 2.4×10 8 ~3.9×10 8 Pieces / cm 2 The average conidia production was 3.01×10 8 Pieces / cm 2 Among them, the strain with the highest spore production was LNTL-1, with a spore production of 3.85×10 8 Pieces / cm 2 The second was XJWLMQ-1, with a spore production of 3.74×10 8 Pieces / cm 2In the screening of toxic strains against several pests, LNTL-1 and XJWLMQ-1 were more toxic than other strains, and the lethal time of infection against several pests was shorter. In addition, these two strains had a higher spore production rate, and the high spore production rate strains played an important role in the vigorous production in the application. In summary, it was shown that Beauveria bassiana XJWLMQ-1, LNTL-1, etc. were entomogenous fungi with great application prospects in biological control of Bemisia tabaci.

[0105] Example 2: Indoor experiment to test the control effect of the automatic inoculation device on Bemisia tabaci

[0106] 1. Combined effects of indoor food attractants and Beauveria bassiana on Bemisia tabaci

[0107] The food attractants and Beauveria bassiana inoculation device used in this study were Figure 3 A triangular prism-shaped paper box (triangle side length 24 cm, prism length 30 cm), a food attractant is hung in the center of the paper box (20 cm from the bottom), a sticky flannel (24 cm × 24 cm) is laid flat on the bottom, and about 5 g of conidia of Beauveria bassiana XJWLMQ-1 are evenly distributed on the flannel.

[0108] 2. The effect of the measuring device on the attraction of whiteflies

[0109] Thirty 5-day-old whiteflies were placed in an insect cage (80×80×80cm), and 14-day-old tomato seedlings (about 20cm high) were planted as food sources. The flannel cloth with conidia powder of Beauveria bassiana at the bottom of the automatic inoculation device was replaced with a sticky board, which was hung with a rope at a level of 20cm from the top bud of the tomato seedling. The number of whiteflies captured by the sticky board was recorded 1d, 3d, 5d, 7d, and 14d after release. The experiment was repeated four times.

[0110] 3. Determination of the amount of fungi captured and the transmission rate of whiteflies

[0111] 30 5-day-old whiteflies were placed in an insect cage (80×80×80cm), and 14-day-old tomato seedlings (about 20cm high) were planted as food sources. The automatic inoculation device was suspended with a rope at a level of 20cm from the top buds of the tomato seedlings. Ten whiteflies were randomly captured at 1d, 3d, 5d, 7d, and 14d after release, and repeated four times. The whiteflies were transferred to a 2ml centrifuge tube and 1ml of sterile 0.05% Triton X-100 solution was added. Vortex for 2-3min to shake out conidia from the insect body, and the concentration of conidia was determined using a hemocytometer.

[0112] 4. The effect of indoor automatic inoculation device on the control of whitefly

[0113] As shown in Table 2, the attraction rate of the automatic inoculation device to the adult whiteflies showed an increasing trend with the sampling time. The attraction rate of the automatic inoculation device to the adult whiteflies was the highest 14 days after treatment. The number of conidia obtained from the inoculation device for 5 whiteflies was 7.49×10 4 ~1.17×10 5 The average number of spores carried by a single whitefly exposed to the automatic inoculation device continued to increase with time.

[0114] Table 2: The attraction rate of the automatic inoculation device and the amount of fungi captured by whiteflies

[0115] After treatment (days) The attraction rate of whitefly adults The amount of fungi obtained from 5 adult whiteflies (spores / ml) 1d 74.16％±0.31c <![CDATA[7.49×10 4 ±0.15c]]> 3d 80.00％±0.27b <![CDATA[7.84×10 4 ±0.15bc]]> 5d 85.83％±0.31a <![CDATA[8.11×10 4 ±0.15b]]> 7d 86.66％±0.27a <![CDATA[9.98×10 4 ±0.04a]]> 14d 89.99％±0.27a <![CDATA[1.17×10 5 ±0.06a]]>

[0116] The control effect of strain LNTL-1 is basically the same as that of XJWLMQ-1.

[0117] Example 3: Field experiment to test the control effect of automatic inoculation device on Bemisia tabaci

[0118] 1. Effect of field combined inoculation device on infection rate and amount of fungi obtained from Bemisia tabaci

[0119] The experiment was conducted in August 2023 in a tomato greenhouse in Shoubonong Ecological Park, Fangshan District, Beijing. Two greenhouses with the same growth status and damage by whiteflies were selected, with an area of ​​80m 2 No chemical pesticides were applied during the experiment, and other management was normal. The greenhouse was divided into 6 small areas, and the small areas were separated by more than 5m to reduce interference between different treatments.

[0120] In the experimental group, one automatic inoculation device was placed in each plot in the greenhouse, and XJWLMQ-1 spores were evenly spread on the flannel of each device, and the device was hung 20 cm above the plant canopy. In the control group, one automatic inoculation device was placed in each plot in the greenhouse, and the flannel of each device was not inoculated with spores, and the device was hung 20 cm above the plant canopy.

[0121] On the 1st, 3rd, 5th, 7th and 14th day after the device was placed, 10 plants were randomly selected from each plot and 20 whiteflies were captured in 2ml centrifuge tubes. They were brought back to the laboratory and recorded for 11 consecutive days. The number of dead insects was recorded at regular intervals every day. The dead insects were surface disinfected with 70% alcohol, rinsed three times in distilled water, and transferred to a culture dish with moist sterile filter paper. Mycelium grew on the surface of the dead insects.

[0122] Of the 20 whiteflies initially captured, 5 were used to determine the number of conidia obtained per whitefly in each treatment. The insects were transferred to a 2 ml centrifuge tube and 1 ml of sterile 0.05% Triton X-100 solution was added. The conidia were shaken out of the insects by vortexing for 2 to 3 minutes, and the conidia concentration was determined using a hemocytometer.

[0123] 2 Results of the control of Bemisia tabaci by the automatic field inoculation device

[0124] As shown in Table 3, the lethal median time of whiteflies around the experimental group was significantly lower than that of the control group. The lethal median time of whiteflies shortened with the increase of sampling time. The dead insects were kept moist and cultured, and white hyphae grew, which was identified as infection by Beauveria bassiana. In the experimental group, the amount of fungi obtained by whiteflies on the first day was 8.06×10 4 spores / ml, and the bacterial count on the 14th day reached 1.40×10 4 spores / ml. The average amount of fungi increased with the increase of sampling time, which was 1st day < 3rd day < 5th day < 7th day < 14th day. No spores of Beauveria bassiana were detected in the control group of whiteflies.

[0125] Table 3 The median lethal time and the amount of fungi obtained in field determination of Bemisia tabaci

[0126]

[0127] The field control effect of strain LNTL-1 is basically the same as that of XJWLMQ-1.

[0128] The above experimental results showed that all 20 strains were pathogenic to Bemisia tabaci. Among them, the virulence and lethality of XJWLMQ-1 and LNTL-1 were superior to those of other strains. The lethality of LNTL-1 to nymphs was 3.95 days, the mortality rate of adults was 94.5%, and the spore production was 3.85×10 8 Pieces / cm 2 The median lethal time of XJWLMQ-1 to nymphs was 3.78 days, the mortality rate of adults was 95.9%, and the conidia production was 3.74×10 8 Pieces / cm 2 . Subsequently, the combined inoculation device was evaluated indoors and in the field, using the highly virulent Beauveria bassiana strains XJWLMQ-1 or LNTL-1. The device was loaded with 5 g of dried conidia of Beauveria bassiana. In the field experiment, the mortality rate of adult whiteflies captured from the greenhouse with Beauveria bassiana conidia was significantly lower than that from the greenhouse without Beauveria bassiana conidia. The automatic inoculation device of Beauveria bassiana and food attractants can establish an "attraction-infection-transmission" system, which provides the possibility of using fungal self-inoculation devices to control whiteflies.

[0129] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0130] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0131] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A biological control agent for controlling Bemisia tabaci, characterized in that: The biocontrol preparation comprises Beauveria bassiana and a food attractant, the food attractant comprises plant volatile secondary metabolites, the plant volatile secondary metabolites are cis-3-hexenal, linalool and eugenol, the Beauveria bassiana is strain XJWLMQ-1 and / or strain LNTL-1, the strain XJWLMQ-1 is Beauveria bassiana with a preservation number of CGMCC No.40509, the strain LNTL-1 is Beauveria bassiana with a preservation number of CGMCC No.40508, and the mass ratio of cis-3-hexenal, linalool and eugenol is (0.1-0.5):(0.01-0.1):(0.5-1).

2. The biocontrol agent according to claim 1, characterized in that The food attractant also includes a slow-release synergist.

3. The biocontrol agent according to claim 2, characterized in that The sustained-release enhancer includes one or more of vegetable oil, butter, honey, paraffin oil, glycerin, vaseline, n-hexane and / or animal oil.

4. The biocontrol agent according to claim 3, characterized in that The volume ratio of the plant volatile secondary substances to the slow-release synergist is 1:(1-10).

5. The biocontrol agent according to any one of claims 1 to 4, characterized in that The Beauveria bassiana includes a Beauveria bassiana strain or spore powder prepared therefrom.

6. A system for controlling whiteflies, characterized in that: The system comprises the biocontrol agent according to any one of claims 1 to 5 and a device for controlling whiteflies, wherein the device can contain the biocontrol agent according to any one of claims 1 to 5, and the device comprises a container having a certain internal space, and the biocontrol agent is contained in the internal space of the container.

7. The system according to claim 6, characterized in that The side of the container is provided with holes to facilitate the entry and exit of whiteflies.

8. Use of the biological control agent according to any one of claims 1 to 5 or the system according to any one of claims 6 to 7 in controlling Bemisia tabaci.

9. A method for controlling Bemisia tabaci, characterized in that: The method comprises using the biocontrol agent according to any one of claims 1 to 5 or the system according to any one of claims 6 to 7 for control.

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