Graphene oxide-Beauveria bassiana complex and its application in pest control and improving crop growth

Abstract

The present invention relates to a graphene oxide - Beauveria bassiana complex and its application in pest control and improving crop growth. The invention provides a graphene oxide - Beauveria bassiana complex, and optimizes the ratio of graphene oxide to Beauveria bassiana. Graphene oxide at a concentration of 20 μg / ml - 100 μg / ml has a UV - protecting effect on Beauveria bassiana spores. The experimental results show that the graphene oxide - Beauveria bassiana provided in this application can significantly improve the control effect of Beauveria bassiana on pests, and the seed soaking treatment can also significantly improve the growth indexes of crops such as plant height, leaf length, leaf width and leaf area, having unexpected technical effects and very good agricultural application value.

Description

Technical Field

[0001] The present invention belongs to the field of pest control, and particularly relates to a novel graphene oxide - Beauveria bassiana complex and its application in pest control and improving crop growth. Background Art

[0002] Crops include food crops and cash crops. In particular, food crops are related to the daily necessities of people. As the food crop with the largest planting area in China, the annual planting area of corn is more than 40 million hm², and the annual total output is more than 250 million tons, playing an irreplaceable role in ensuring food security. However, the occurrence of pests and diseases is an important factor threatening the safe production of corn, especially the reduction in production caused by the damage of Lepidoptera pests such as Asian corn borer and fall armyworm. The Asian corn borer (hereinafter referred to as "corn borer") has always been one of the major pests damaging corn in China. In 2019, the fall armyworm, a major migratory pest globally, invaded China and completed the process of invasion, colonization, and damage in China in just one year. Both of these pests can damage corn leaves, stems, cob axes, and grains, seriously affecting the yield and quality of corn. In March 2023, both of these pests were listed in the "List of Class I Crop Pests and Diseases" by the Ministry of Agriculture and Rural Affairs, indicating the severity of their damage. Therefore, doing a good job in the prevention and control of pests such as corn borer and fall armyworm is an important link in ensuring corn yield and food security.

[0003] At present, the prevention and control of corn borer and fall armyworm mainly rely on chemical control. Chemical control is an effective emergency pest control method, but it also inevitably brings problems such as pesticide residues, environmental pollution, and drug resistance. Therefore, it is urgent to explore ecological - friendly, non - resistant, green and safe corn pest control technologies. Entomopathogenic fungi have a wide insecticidal spectrum, strong pathogenicity, and are harmless to the environment, humans, and livestock. They have always been a research hotspot for the control of corn pests such as corn borer and fall armyworm. Summary of the Invention

[0004] The purpose of the present application is to provide a graphene oxide - Beauveria bassiana complex (GO - B094). Through in - depth research, it is found that this complex has both the functions of pest control and improving crop growth, and has very good agricultural promotion and application value.

[0005] The purpose of the present application is to provide a graphene oxide - Beauveria bassiana complex. The main components of the graphene oxide (GO) are natural graphite powder, concentrated sulfuric acid, phosphorus pentoxide, potassium dichromate, and potassium permanganate. The Beauveria bassiana (B094) is adsorbed on the graphene oxide, and the preservation number of the Beauveria bassiana is: CGMCC NO.19034.

[0006] The preparation method of Beauveria bassiana spore suspension includes: picking up the spore powder of Beauveria bassiana strain after activation culture, mixing it with Tween sterilization solution, grinding it evenly with a tissue grinder, measuring the spore content after thoroughly mixing with a vortex oscillator, and finally diluting it with Tween sterilization solution to prepare a spore suspension with the required concentration; preferably, picking up the spore powder of the activated culture strain with a disposable sterile inoculation loop, mixing it with 0.05% Tween-80 sterilization solution, grinding it evenly with a glass tissue grinder, measuring the spore content with a 25-square hemocytometer after thoroughly mixing with a vortex oscillator, counting three times, taking the average value, and finally diluting it with 0.05% Tween-80 sterilization solution to prepare a spore suspension with the required concentration.

[0007] Prepare graphene oxide dispersion; prepare Beauveria bassiana spore suspension; mix the Beauveria bassiana spore suspension and graphene oxide dispersion in a volume ratio of 4:1 and stir at dark room temperature to obtain an aqueous solution of the composite; preferably, the method includes: preparing graphene oxide dispersion (concentration 100 μg / ml); preparing Beauveria bassiana spore suspension (concentration 1×10 8 cells / ml); mix the Beauveria bassiana spore suspension and graphene oxide dispersion in a volume ratio of 4:1 and stir on a magnetic stirrer at 1200 rpm at dark room temperature for 24 h to obtain an aqueous solution of the composite.

[0008] Furthermore, the Beauveria bassiana spore suspension and graphene oxide dispersion are mixed in a volume ratio of 4:1;

[0009] Preferably, the graphene oxide is a 1-100 μg / ml graphene oxide solution; preferably, the graphene oxide is 20-100 μg / ml graphene oxide; the Beauveria bassiana spore suspension is 1×10 8 Beauveria bassiana spores per mL.

[0010] Furthermore, the preparation method of the graphene oxide-Beauveria bassiana composite includes: preparing a spore suspension of Beauveria bassiana conidia with a sterile solution; preparing graphene oxide dispersion, mixing the Beauveria bassiana spore suspension and graphene oxide dispersion to obtain a mixed solution; uniformly stirring the mixed solution with a magnetic stirrer in the dark to generate a graphene oxide-Beauveria bassiana composite solution, and freeze-drying for standby;

[0011] Preferably, the preparation method further includes washing the graphene oxide-Beauveria bassiana, and the specific steps are as follows: centrifuging the generated graphene oxide-Beauveria bassiana composite solution to discard the supernatant, adding glutaraldehyde fixative, fixing at low temperature, rinsing with phosphate buffer solution, performing gradient dehydration with 50%-100% ethanol in sequence, after the dehydration is completed, replacing with ethanol-tert-butanol solution, and finally replacing with 100% tert-butanol twice. After the treatment is completed, the sample is freeze-dried for standby.

[0012] Furthermore, the Beauveria bassiana is adsorbed on the graphene oxide sheets, making the surface of the graphene oxide-Beauveria bassiana complex present a rough multi-particle shape.

[0013] The application of the above-mentioned graphene oxide-Beauveria bassiana complex in improving the growth of crops. Preferably, the crops include cereal crops and / or leguminous crops; more preferably, the crops include one or more of the following: wheat, rice, corn, soybean, broad bean, pea, mung bean.

[0014] Furthermore, after disinfecting the surface of the crop seeds, soak them in the suspension of the graphene oxide-Beauveria bassiana complex for 1-2 days, and then air-dry them before sowing.

[0015] The application of the graphene oxide-Beauveria bassiana complex in increasing the plant height, leaf length and / or leaf area of crops.

[0016] A seed treatment method includes: after disinfecting the surface of the crop seeds, soak them in the suspension of the above-mentioned graphene oxide-Beauveria bassiana complex for 1-2 days, and then air-dry them. The crops sown after being treated by the above method grow better and have stronger resistance. Preferably, the crops include one or more of the following: wheat, rice, corn, soybean, broad bean, pea, mung bean.

[0017] The application of the above-mentioned graphene oxide-Beauveria bassiana complex in pest control.

[0018] Furthermore, the pests are corn pests; preferably, the pests include one or more of the following: corn borer, fall armyworm, thrips, black cutworm, yellow cutworm, mole cricket, white grub, wireworm, corn root mealybug, maize leaf beetle, beet webworm, rice leaf roller, cotton leafworm, corn noctuid, armyworm, yellow-bellied tussock moth, red-bordered tussock moth, brown-footed horned leaf beetle, Dolycoris baccarum, red-bearded plant bug, Lygus pratensis, three-spotted leafhopper, big green leafhopper, corn aphid, Tetranychus truncatus, Tetranychus cinnabarinus, Asian corn borer, pink rice borer, peach fruit borer, cotton bollworm, white-spotted flower chafer, small green flower chafer, double-spotted leaf beetle, Nezara viridula.

[0019] Further, the prevention and control method includes: after disinfecting the surface of crop seeds, soaking them in a suspension of graphene oxide - Beauveria bassiana complex for 1 - 2 days, drying them and then sowing. The crops obtained by planting have insect - resistant characteristics.

[0020] A method for improving the growth potential and / or resistance of crops, including: after disinfecting the surface of crop seeds, soaking them in the above - mentioned suspension of graphene oxide - Beauveria bassiana complex for 1 - 2 days, drying them and then sowing.

[0021] The technical advantages of the present invention:

[0022] 1. This application provides a graphene oxide - Beauveria bassiana complex (GO - B094). The Beauveria bassiana in the complex is a strain of Beauveria bassiana screened by the applicant's team, which has good pathogenicity to the larvae of Ostrinia furnacalis and Spodoptera frugiperda. Using graphene oxide (GO) as a carrier can improve the colonization efficiency of Beauveria bassiana in corn and the prevention and control effects on Ostrinia furnacalis and Spodoptera frugiperda.

[0023] 2. Experiments in this application show that graphene oxide at a concentration of 100 μg / ml has no effect on the spore germination rate of Beauveria bassiana. Therefore, it is necessary to control the concentration of graphene oxide. In addition, graphene oxide with a concentration of 20 μg / ml - 100 μg / ml has a UV - protection effect on the spores of Beauveria bassiana. Therefore, it is preferably 20 - 100 μg / ml graphene oxide and 1×10 8 spores / mL of Beauveria bassiana;

[0024] 3. Experiments in this application show that the compounding of graphene oxide - Beauveria bassiana can significantly improve the pathogenicity of Beauveria bassiana to Ostrinia furnacalis and Spodoptera frugiperda, and the effect on Ostrinia furnacalis is more obvious.

[0025] 4. Experiments in this application show that the seed - soaking treatment with graphene oxide - Beauveria bassiana can significantly improve the growth indexes of corn, such as plant height, leaf length, leaf width and leaf area, etc., and has unexpected technical effects. Description of the Drawings

[0026] Figure 1 is the spore germination rate of Beauveria bassiana under different graphene oxide concentrations;

[0027] Figure 2 is the SEM and FT - IR characterization diagrams of the graphene oxide - Beauveria bassiana complex. A: SEM characterization of GO; B: SEM characterization of B094; C: SEM characterization of GO - B094; D: FT - IR characterization of GO - B094;

[0028] Figure 3It is a pathogenicity evaluation diagram of GO-B094 against Ostrinia furnacalis and Spodoptera frugiperda. A: Cumulative infection rate of Ostrinia furnacalis (ACB) and Spodoptera frugiperda (FAW); B: Cumulative corrected mortality rate of Ostrinia furnacalis (ACB) and Spodoptera frugiperda (FAW); C: Larvae of Ostrinia furnacalis after feeding on leaves treated with different treatments.

[0029] Figure 4 It is a spore germination rate diagram of GO-B094 under ultraviolet irradiation;

[0030] Figure 5 It is the colonization situation of B094 in maize after seed soaking treatment with GO-B094 complex;

[0031] Figure 6 It is the colonization situation of B094 in maize leaves observed by fluorescence after seed soaking treatment with GO-B094 complex;

[0032] Figure 7 Diagram of the effect of GO-B094 complex on maize growth. A. Plant height, leaf length, leaf width and leaf area 14 days after emergence; B. Plant height, leaf length, leaf width and leaf area 14 days after emergence; C. Whole-plant fresh weight, root length, chlorophyll a and chlorophyll b 21 days after emergence; D. Plant height 21 days after emergence; E. Root length 21 days after emergence. Data are Means±SE (n = 5); Different letters represent significant differences among treatments (p<0.05);

[0033] Figure 8 It is an indoor resistance diagram of GO-B094 complex against Ostrinia furnacalis after colonization in maize. A. Survival rate of larvae of Ostrinia furnacalis and Spodoptera frugiperda feeding on leaves treated with different seed soaking treatments; B. Larval weight of Spodoptera frugiperda feeding on leaves treated with different seed soaking treatments; C. Larval instars of Ostrinia furnacalis feeding on leaves treated with different seed soaking treatments for 4 days and 7 days;

[0034] Figure 9 It is a field resistance grade distribution diagram of GO-B094 complex against Ostrinia furnacalis after colonization in maize. A. Newly hatched larvae were inoculated 14 days after emergence, and the leaf-eating level was investigated 7 days later; B. Newly hatched larvae were inoculated 14 days after emergence, and the leaf-eating level was investigated 14 days later; C. Newly hatched larvae were inoculated 21 days after emergence, and the leaf-eating level was investigated 7 days later; HR, R, MR, S, HS represent 5 resistance grades of high resistance, insect resistance, medium resistance, susceptible and high susceptible in turn, and the numbers in brackets represent leaf-eating levels from 1 to 9;

[0035] Figure 10 It is a field resistance identification diagram of GO-B094 complex against Spodoptera frugiperda after colonization in maize;

[0036] Figure 11 It is a resistance diagram of GO-B094 complex against natural insect infection after colonization in maize. Detailed implementation methods

[0037] The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0038] Example 1 Test Method

[0039] 1.1 Tested Beauveria bassiana strains, nanomaterials, insects, and corn

[0040] The Beauveria bassiana B094 used in this application was provided by the Grassland Pest Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The preservation number of this strain is: CGMCC NO.19034. It was preserved on December 11, 2019, by the China General Microbiological Culture Collection Center, with the address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Beauveria bassiana was cultured on potato dextrose agar (PDA) medium in an artificial climate chamber at a temperature of (26±1)°C, a relative humidity of (60±5)%, and complete darkness for 10 days. The composition of the PDA medium: 200 g of potato, 20 g of glucose, 20 g of agar, and 1000 mL of distilled water.

[0041] Ostrinia furnacalis and Spodoptera frugiperda were reared in the Corn Pest Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The original populations were collected from the field. After multiple generations of rearing with artificial feed under the conditions of a temperature of 27°C, a humidity of 75±5%, and a photoperiod (L:D) of 16:8, they were used for the tests.

[0042] The graphene oxide (GO) used in this application was synthesized from graphite powder purchased from Qingdao Tianhe Graphite Co., Ltd. by the Hummers method, with a radial size of 70 - 307 nm and a thickness of 1 - 2 nm.

[0043] Use a disposable sterile inoculation loop to pick up the spore powder of the activated Beauveria bassiana strain, mix it with a 0.05% Tween - 80 sterilized solution, grind it evenly with a glass tissue grinder, and then use a 25 - grid hemocytometer to measure the spore content after thoroughly mixing with a vortex oscillator. Count three times and take the average value. Finally, dilute it with a 0.05% Tween - 80 sterilized solution to prepare a Beauveria bassiana spore suspension with the required concentration.

[0044] 1.2 Effects of different concentrations of graphene oxide on the spore germination rate of Beauveria bassiana

[0045] Prepare GO-B094 composite suspensions by mixing B094 spore suspensions with GO solutions at concentrations of 0 μg / mL, 20 μg / mL, 100 μg / mL, and 200 μg / mL. Pour 10 mL of spore germination solution containing 4% glucose and 1% yeast powder into a 50 mL conical flask, and then pour the GO-B094 composite suspension into the conical flask. Place the conical flask in a shaker and shake for 16 h. Set the shaker temperature to 26 ± 1 °C and the rotation speed to 195 rpm / min. Observe the total number of spores and germinated spores under a microscope using a hemocytometer, and generally observe no less than 500 spores.

[0046] Germination rate = (Number of germinated spores / Total number of spores) × 100%

[0047] 1.3 Construction of graphene oxide-Beauveria bassiana complex (GO-B094)

[0048] Based on the results of 1.2, determine the spore concentration that does not affect the spore germination rate. Physically load B094 onto the surface of GO to prepare the GO-B094 complex. Prepare a spore suspension of the test strain B094 at a concentration of 1×10 8 spores / mL using a 0.01% Tween-80 sterile solution. When graphene oxide is solid, add 0.01 g of GO solid and 100 ml of ultrapure water to a beaker, and place it in an ultrasonic cleaner for 30 min to make the GO solid into a 100 μg / mL GO solution. When graphene oxide is liquid, a 100 μg / mL GO solution can be directly prepared. Mix the B094 spore suspension with the GO solution (4:1, V / V) to obtain a mixed solution of GO and B094. Stir the mixed solution evenly with a magnetic stirrer in the dark for 24 h to generate the final GO-B094 composite solution. Centrifuge the GO-B094 composite solution at 8000 r / min for 3 - 5 min, discard the supernatant, add 40 times the sample volume of 2.5% glutaraldehyde fixative, place it in a refrigerator at 4 °C for more than 2 h, rinse it 2 - 3 times with phosphate buffer, and dehydrate it successively with 50%, 70%, 90%, and 100% ethanol, with each dehydration time being 5 - 10 min. After dehydration, displace it with an ethanol-tert-butanol solution (1:1, V / V, the same below) for 20 min, and finally displace it twice with 100% tert-butanol, each time for 20 min. After processing, freeze-dry the sample for standby. The GO-B094 composite solution can also be stored in a refrigerator at 4 °C.

[0049] 1.4 Characterization of graphene oxide-Beauveria bassiana complex

[0050] The wrinkling situation, lamellar structure and other microscopic morphologies of GO, B094, and GO-B094 were characterized and tested using a scanning electron microscope (SEM). Test sample preparation process: Attach a conductive adhesive of appropriate size to the sample stage, then take an appropriate amount of sample powder and evenly smear it on the surface of the conductive adhesive. After sputtering with gold, conduct the test.

[0051] The Fourier transform infrared spectroscopy (FT-IR) analysis of GO, B094, and GO-B094 was carried out using a TENSOR-27 infrared spectrometer. The changes in surface functional groups of GO, B094, and GO-B094 were characterized and tested. Test sample preparation process: Mix the sample to be tested with potassium bromide (1:200) and place it in an agate mortar. Grind it evenly into powder under a drying lamp, place it in a mold and press it into a thin slice for 1 min. Collect the infrared spectrogram in the wavenumber range of 500 - 4000 cm -1 Wavenumber range.

[0052] The STA 409PC type thermal analyzer (TGA) was used to test the thermal stability of GO, B094, and GO-B094. N2 was used as the protective gas, and the heating rate was 10 °C / min. Record the mass change of the sample during the process from 25 - 700 °C. Test sample preparation process: Put 3 mg of the sample to be tested into the instrument, make it in full contact with the crucible, and conduct the test.

[0053] 1.5 Evaluation of the pathogenicity of graphene oxide - Beauveria bassiana complex against Ostrinia furnacalis larvae

[0054] The indoor virulence of the 3rd instar larvae of Ostrinia furnacalis and Spodoptera frugiperda was determined by the leaf dipping method. Collect the conidia of B094 and GO-B094 from the PDA plate, and prepare the conidia suspensions of B094 and GO-B094 with a concentration of 1×10 8 CFU / mL using a 0.01% Tween-80 sterile solution. Take the corn leaves at the 5-leaf stage and cut them into 1 cm × 1 cm squares. Immerse the cut leaves in the conidia suspensions of B094 and GO-B094 for 60 s, take them out and place them on sterile filter paper to dry. After drying, put them into a 24-well tissue bioassay plate. Put 1 3rd instar larva of Ostrinia furnacalis or Spodoptera frugiperda that has been starved for 4 h into each well. After the infected corn leaves are completely eaten, feed them with untreated leaves. Use the leaves soaked in a 0.01% Tween-80 sterile solution and a 20 μg / mL GO solution for 60 s as the control. Each 24 3rd instar larvae are one replicate, and each treatment is replicated 5 times. The larvae are reared in an artificial climate chamber at a temperature of (26 ± 1) °C, a relative humidity of (60 ± 5)%, and a photoperiod of 14L:10D. Observe the death of the larvae every day after inoculation, record the number of deaths, and continuously observe for 7 d. The larvae turning black (mummified insects) or the presence of hyphae on the body surface observed under a microscope is regarded as being infected by Beauveria bassiana.

[0055] Use Excel to organize the experimental data, and calculate the cumulative mortality and cumulative corrected mortality of the 2nd and 3rd instar larvae of Ostrinia furnacalis. The relevant calculation formulas are as follows:

[0056] Cumulative corrected mortality (%) = (Cumulative mortality of treatment - Cumulative mortality of control) / (1 - Cumulative mortality of control) × 100% Cumulative mortality (%) = (Total number of dead insects in treatment / Total number of insects in treatment) × 100%

[0057] Cumulative infection rate (%) = (Total number of infected insects in treatment / Total number of insects in treatment) × 100%

[0058] Use SPSS to test the significance of data differences by the least significant difference method (LSD).

[0059] 1.6 Evaluation of the anti-ultraviolet properties of graphene oxide-Beauveria bassiana complex

[0060] Select GO solutions with different concentrations (0 μg / mL, 20 μg / mL, 100 μg / mL, 200 μg / mL) and 1×10 8 spores / mL of B094 spore suspension to prepare GO-B094 composite suspensions respectively. After the composite suspensions are shaken and germinated, the germinated composite suspensions are placed in an airtight cardboard box under a 20 cm ultraviolet lamp (36 W, 254 nm) for irradiation for multiple time periods (0 h, 3 h, 6 h, 12 h, 24 h). Observe the germination rates of each suspension at different time periods after being irradiated by the ultraviolet lamp under a microscope.

[0061] 1.7 Maize seed soaking treatment

[0062] Select plump, healthy, insect-free and uniform-sized maize seeds for seed soaking treatment. The surface of the seeds needs to be disinfected before soaking. The surface-disinfected maize seeds are soaked in the prepared suspensions of GO, B094, and GO-B094 respectively, and the control maize seeds are soaked in a 0.01% Tween-80 sterile solution. Soak them statically in a beaker at 28°C for 24 h in the laboratory. After soaking, place the seeds in a laminar flow hood, blot the excess water with sterile filter paper, and sow them after fully drying.

[0063] 1.8 Field experiment design

[0064] The field experiments were conducted at the Langfang Scientific Research and Middle-scale Base of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Langfang, Hebei, 116.60°E, 39.51°N) from June to August 2023, and at the Dongyang Corn Borer Research Base of the Zhejiang Academy of Agricultural Sciences (Jinhua, Zhejiang, 120.18°E, 29.16°N) from August to October 2023. The colonization rate of Beauveria bassiana in different maize tissues, maize growth indicators, and bioassays of maize leaves treated by seed soaking were carried out at the Langfang Scientific Research and Middle-scale Base of the Chinese Academy of Agricultural Sciences. Field resistance identification was conducted at three locations. However, due to irresistible field factors such as rainfall, the insect inoculation and investigation times at each location were not exactly the same. The specific situation is shown in Table 1.

[0065] All experiments were set with 4 different seed treatments (CK, GO, B094, GO-B094), and each treatment had 4 replicates, adopting a completely randomized block design.

[0066] Table 1 Insect inoculation and investigation schedule

[0067]

[0068]

[0069] 1.9 Evaluation of the colonization rate of the GO-B094 complex in maize roots, stems, and leaves

[0070] At 14 and 21 days after emergence, destructive sampling of the root, stem, and leaf tissues of maize plants was carried out, and the plate method of culture medium was used to evaluate the colonization of Beauveria bassiana in maize. The randomly selected maize plants were repeatedly rinsed with tap water, and then the roots, stems, and leaves of the maize plants were cut. They were rinsed 3 times with sterile distilled water in a laminar flow bench and blotted dry with sterile filter paper to remove excess moisture. The surface-sterilized plant materials were verified. The final rinsing water was plated on PDA medium and cultured at 25°C for 10 days to evaluate the surface sterilization efficiency. If no fungal growth was observed on the PDA medium, it indicated that the surface sterilization efficiency of the maize plants was good, and it was confirmed that the fungi growing from the surface-sterilized plant materials were endophytic fungi originating from the internal plant tissues. Each surface-sterilized plant tissue was cut into 0.5-cm tissue pieces with sterilized scissors, and then the tissue pieces were placed on PDA medium for culture, with 5 - 6 pieces placed on each medium, and repeated 3 times. After culturing at 25 ± 1°C for 3 - 7 days, the number of colony formations was recorded. Colonization rate = number of colony formations / total number of inoculations × 100%.

[0071] 1.10 Effects of the colonization of the GO-B094 complex on maize emergence rate and growth

[0072] The main growth indicators of maize plants were measured 14, 21, and 28 days after maize emergence, including plant height (from the soil surface to the tip of the longest leaf of the maize plant), leaf length (from the base to the tip of the largest true leaf), leaf width (the width at the widest part of the largest true leaf), leaf area, fresh weight of the above-ground part and roots of maize. The total leaf area of each plant was the leaf area of the whole plant. There were 3 replicates, with 20 plants in each replicate, and a total of 60 plants were measured for each treatment. Leaf area = k × leaf length × leaf width, where k = 0.75.

[0073] 1.11 Indoor resistance determination of GO-B094 complex against Ostrinia furnacalis after colonization in maize

[0074] At 21 days after maize emergence, maize leaves colonized with CK, GO, B094, and GO-B094 were collected to feed newly hatched larvae (<24 h) of Spodoptera frugiperda and Ostrinia furnacalis. Whether the larvae were infected with Beauveria bassiana was observed every day, the number of dead insects was recorded, and the total number of dead larvae was counted after 7 days and the surviving insects were weighed. 96 larvae were fed for each treatment.

[0075] 1.12 Field resistance evaluation of GO-B094 complex against Ostrinia furnacalis and Spodoptera frugiperda after colonization in maize

[0076] 1.12.1 Artificial insect inoculation resistance identification

[0077] At the 14th or 21st day after maize emergence, newly hatched larvae of Ostrinia furnacalis and Spodoptera frugiperda were inoculated into the heart leaves of maize plants treated with different seed soaking methods. 40 - 60 larvae were inoculated into each maize plant. After three days of investigation, if obvious damage marks were found, it was regarded as successful insect inoculation. If there were no damage marks, inoculation was carried out again. At the 7th day (21 days after emergence) and the 14th day (28 days after emergence) after insect inoculation, the damage levels of Ostrinia furnacalis and Spodoptera frugiperda to maize leaves were investigated. Insect inoculation was carried out in the evening on sunny days. According to the 9-level classification standard formulated by the International Ostrinia furnacalis Cooperative Group, the leaf-eating level of Ostrinia furnacalis was investigated plant by plant, where levels 1 - 2.9 were highly resistant; levels 3 - 4.9 were resistant; levels 5 - 6.9 were moderately resistant; levels 7 - 8.9 were susceptible; and 9.0 was highly susceptible. According to the Davis Scale, the leaf-eating level of Spodoptera frugiperda was investigated plant by plant, where levels 1 - 2 were highly resistant; levels 2.1 - 4 were resistant; levels 4.1 - 6 were moderately resistant; levels 6.1 - 8 were susceptible; and levels 8.1 - 9 were highly susceptible.

[0078] 1.12.2 Natural insect infection resistance identification

[0079] At 14 days and 21 days after maize emergence, the damage conditions of maize treated with different treatments under natural damage conditions were investigated, including the number of damaged plants and the leaf-eating level of damaged plants. The investigation method was the same as that in 1.12.1.

[0080] Results and analysis of Example 2

[0081] 2.1 Effects of Graphene Oxide at Different Concentrations on the Spore Germination Rate of Beauveria bassiana B094

[0082] After mixing the GO at 0 μg / ml, 20 μg / ml, 100 μg / ml, and 200 μg / ml with the 1×10 8 spore suspension of B094 at a concentration of 6 spores / mL for 18 h, the number of germinated spores was observed and counted under a microscope. As Figure 1 shown, the spore germination rates of B094 at 20 μg / ml and 100 μg / ml were both above 90%, showing no significant difference compared with the control (p>0.05), while the spore germination rate of B094 was only 4.9% when the concentration was 200 μg / ml.

[0083] 2.2 Construction and Characterization

[0084] SEM morphology characterization was carried out on GO, B094, and GO-B094. The morphology of GO is as Figure 2 shown in A, presenting a typical wrinkled morphology with a multi-layered rough sheet structure. The SEM image of B094 is as Figure 2 shown in B. The spore powder of Beauveria bassiana B094 presents a typical round shape with a relatively smooth surface. The loading of B094 results in the appearance of a large number of round particles on the surface of GO Figure 2 as shown in C. Due to the adsorption of B094 on the GO sheet, the surface of GO-B094 presents a rough multi-particle shape.

[0085] Fourier transform infrared spectroscopy is a common characterization method for determining the chemical bonds and surface functional groups of compound molecules. The FT-IR spectra of GO, B094, and GO-B094 are as Figure 2 shown in D. In the spectrum of GO, the O-H stretching vibration appears at 3,363 cm -1 , the peak of the graphite skeleton is at 1,649 cm -1 , and the COOH stretching vibration and C-O stretching vibration appear at 1,649 cm -1 . In the spectrum of B094, the PO4 3- vibration appears at 1,080 cm -1 , and the stretching vibration of the amide bond appears at 1,649 cm -1 . The spectrum of GO-B094 contains all the characteristic peaks of GO and B094, indicating that B094 and GO are physically combined and do not change each other's chemical properties.

[0086] 2.3 Pathogenicity of Graphene Oxide-Beauveria bassiana Complex against the 3rd Instar Larvae of Ostrinia furnacalis and Spodoptera frugiperda

[0087] The results of indoor virulence determination showed that: at a concentration of 1×10 8The cumulative infection rates of the complex (GO-B094) formed by Beauveria bassiana B094 at 1×10 Figure 3 individuals / mL and graphene oxide (GO) against the 3rd instar larvae of Ostrinia furnacalis and Spodoptera frugiperda were (61.23±3.75)% and (56.83±3.66)% respectively, which were significantly higher than the infection rates of B094 alone, (40.37±1.44)% and (40.5±1.5)%( Figure 3 A). The cumulative corrected mortality rates of GO-B094 against the 3rd instar larvae of Ostrinia furnacalis and Spodoptera frugiperda were (74.14±5.83)% and (62.83±3.22)% respectively, both of which were significantly higher than the cumulative corrected mortality rates of B094 alone, (54.65±1.74)% and (49.84±1.67)%( Figure 3 B).

[0088] 2.4 Evaluation of the anti-ultraviolet effect of the graphene oxide-Beauveria bassiana complex

[0089] Figure 4 The spore germination rates of Beauveria bassiana B094 at 1×10 8 individuals / mL after being compounded with different concentrations of GO under ultraviolet light irradiation were measured. Among them, CK was the germination rate of B094 spores without ultraviolet light irradiation, which was 91.25%. The spore germination rates of Beauveria bassiana spores with a GO concentration of 0 decreased to 49.75%, 37.84% and 24.83% after being irradiated with ultraviolet light for 12 h, 24 h and 48 h respectively. It can be seen that ultraviolet light has a strong killing effect on Beauveria bassiana spores. However, 20 μg / ml and 100 μg / ml of GO have a certain ultraviolet protection efficiency for the spores. When irradiated with ultraviolet light for 12 h, the spore germination rates of GO-B094 at 20 μg / ml and 100 μg / ml were 76.21% and 77.09% respectively. When irradiated for 24 h, the spore germination rates of GO-B094 at 20 μg / ml and 100 μg / ml concentrations were 69.51 and 68.85% respectively, both of which were significantly higher than the spore germination rates without GO. When irradiated with ultraviolet light for 48 h, 20 μg / ml and 100 μg / ml of GO still had a certain protection efficiency for the spores, but the germination rates were only 38.47 and 40.48%. It shows that there is an obvious protection efficiency for the spores within 24 h of ultraviolet light irradiation, and there is no significant difference in the protection rates of spores between 20 μg / ml and 100 μg / ml of GO.

[0090] 2.5 Evaluation of the Colonization of the GO-B094 Complex in Maize

[0091] For the maize tissue parts after soaking seeds with B094 and GO-B094, when inoculated on the culture medium plates for about 3 days, white hyphae began to grow from the roots and stems, and the leaves began to grow at 4 - 5 days( Figure 5 ). The colonization results of B094 in maize after soaking seeds with B094 and GO-B094 are shown in Table 2. The colonization rate varied with different maize tissue parts and measured seedling ages. The colonization rate of B094 after soaking seeds with the GO-B094 complex was the highest in maize leaves, followed by stems, and the lowest in roots. At 14 days and 21 days after emergence, the colonization rates of GO-B094 in different tissue parts were significantly higher than those of B094. Among them, the colonization rates of GO-B094 in leaves at 14 days and 21 days after emergence could reach 100% and 83.33% respectively, as shown in Table 2.

[0092] The results of fluorescence microscopy observation are as Figure 6 shown. No obvious fluorescence signals were produced in the leaves of maize seedlings at 14 and 21 days after emergence treated with CK and GO by soaking seeds, while obvious fluorescence signals could be observed in the leaves of maize seedlings at 14 and 21 days after emergence treated with B094 and GO-B094 by soaking seeds. Among them, the fluorescence signal in the leaves treated with GO-B094 by soaking seeds was stronger. It shows that GO can promote the distribution of Beauveria bassiana in maize leaves.

[0093] Table 2 Colonization Rates of B094 in Maize after Soaking Seeds with the GO-B094 Complex

[0094] Note: Different capital letters in the same column represent the differences in the colonization rates of B094 and GO-B094 in the same tissue (P<0.05),

[0095]

[0096] and different lowercase letters in the same row represent the differences in the colonization rates of different parts (P<0.05)

[0097] 2.6 Effects of the GO-B094 Complex on Maize Growth

[0098] The biomass and plant height of plants reflect the growth status of plants. The physiological basis and material prerequisite for plant growth and development are photosynthesis and the organic matter produced by it. Chlorophyll is the material basis of photosynthesis, and the chlorophyll content can indirectly reflect the intensity of photosynthesis. There were significant differences among the growth indexes of maize after soaking seeds with different treatments( Figure 7 ). At 14 days after emergence, compared with CK, soaking seeds with GO, B094, and GO-B094 could significantly increase the growth indexes of maize such as plant height, leaf length, leaf width, and leaf area. Among them, the growth-promoting effect of GO-B094 was significantly higher than that of GO and B094(Figure 7 A). At 21 days after emergence, except that B094 had little effect on leaf width, seed soaking treatments with GO, B094, and GO-B094 could all significantly increase the growth indexes of maize, such as plant height, leaf length, leaf width, and leaf area, and the growth-promoting effect of GO-B094 was the most obvious. Figure 7 B). At 21 days after emergence, the fresh weight of the whole maize plants treated with CK, GO, B094, and GO-B094 was in the order of GO-B094 > GO > B094 > CK. Figure 7 C, D), but only GO-B094 significantly increased the root length of maize. Figure 7 C, E).

[0099] 2.7 Control effects of GO-B094 complex on Ostrinia furnacalis and Spodoptera frugiperda

[0100] To clarify the insecticidal effects of maize leaves after different seed soaking treatments on the larvae of Ostrinia furnacalis and Spodoptera frugiperda, we fed the newly hatched larvae of Ostrinia furnacalis and Spodoptera frugiperda with maize leaves at 21 days after emergence, investigated the larval mortality after continuous feeding for 7 days, and weighed the surviving larvae. The results showed that the leaves treated with GO seed soaking had no significant effect on the larval mortality of Ostrinia furnacalis and Spodoptera frugiperda, while B094 and GO-B094 both significantly increased the larval mortality of Ostrinia furnacalis and Spodoptera frugiperda. Among them, the lethal rate of the leaves treated with GO-B094 was more obvious, reaching 89% for Ostrinia furnacalis and 52% for Spodoptera frugiperda, indicating that GO-B094 had a more obvious control effect on Ostrinia furnacalis. Due to the too high mortality of Ostrinia furnacalis larvae feeding on the leaves treated with GO-B094, we only measured the body weight of Spodoptera frugiperda larvae fed for 8 days. The results of insect weight measurement showed that the Spodoptera frugiperda larvae feeding on the leaves treated with GO seed soaking were the heaviest, followed by the larvae feeding on the control and B094-treated leaves, while the Spodoptera frugiperda larvae feeding on the leaves treated with GO-B094 were the lightest. Figure 8 B). Figure 8 C showed that no infection was found in the dead Ostrinia furnacalis larvae colonized by CK and GO, while the dead larvae colonized by B094 and GO-B094 were all infected. The larval mortality and infection rate of the larvae fed with the leaves colonized by GO-B094 were higher than those of B094, and the amount of fungi growing on the body surface of the larvae feeding on the leaves treated with GO-B094 was more than that of the larvae feeding on the leaves treated with B094.

[0101] The results of field artificial inoculation resistance identification showed that Figure 9), 14 days after seedlings, corn borer larvae were inoculated. When investigated on the 21st day, the leaves of the four soaking treatments were all lightly damaged, among which the resistance level of the leaves treated with GO and GO-B094 soaking was high resistance, and the resistance level of the leaves treated with CK and B094 soaking was resistance. However, from the proportion of plants with different resistance, it can be seen that the proportion of high resistance in the plants treated with GO-B094 soaking reached 90%, which was much higher than the proportion of high resistance in the plants treated with CK and GO. When investigated on the 28th day, the degree of damage to the leaves of different treatments was significantly aggravated. Among them, the average leaf-eating level of the plants treated with GO-B094 soaking was 4.02±0.09, showing insect resistance, the average leaf-eating level of the plants treated with B094 and GO soaking was 5.84±0.11 and 5.88±0.12, respectively, both showing medium resistance, and the average leaf-eating level of the control plants was 7.06±0.11, which had reached the insect-susceptible level. From the proportion of plants with different resistance, it can be seen that 78% of the plants in the GO-B094 soaking treatment were resistant to insects, 88% and 72% of the plants in the B094 and GO soaking treatments were moderately resistant, and 78% of the plants in the CK were susceptible to insects. The results of the 28-day survey of newly hatched corn borer larvae after 21 days after seedlings were similar to those of the 14-day survey of 21 days after inoculation: the leaves of the four soaking treatments were less damaged, among which the resistance level of the leaves treated with GO-B094 was high resistance, and the leaves of the other three treatments were resistant to insects. The proportion of plants treated with GO-B094 soaking that showed high resistance was also the highest.

[0102] The above results show that when corn borers infest for 7 days, the damage is generally mild, but the plants treated with GO-B094 can also show the effect of reducing damage at 14-21 days and 21-28 days. As the time and degree of damage increase, the effect of GO-B094 in reducing corn borer damage becomes more obvious. When the control shows insect-susceptible conditions, the plants treated with GO-B094 can still maintain the insect-resistant level. That is, 28 days after seedlings, compared with the use of Beauveria bassiana alone, GO-B094 has a better control effect on corn borers.

[0103] The results of the resistance identification of artificially inoculated fall armyworm in the field showed that ( Figure 10) At 14 and 21 days after emergence, newly hatched Spodoptera frugiperda larvae were inoculated respectively. After 7 days, that is, at 21 and 28 days for investigation, the leaf damage of the 4 seed soaking treatments was significantly more serious than that of Ostrinia furnacalis. When investigated at 21 and 28 days, the plants treated with GO-B094 showed moderate resistance. Although the average damage levels of the plants treated with GO and B094 were lower than those of the CK plants, they were all susceptible to insects like the CK plants. When the insects were inoculated at 14 days after emergence and investigated at 28 days, the leaf damage of different treatments was significantly aggravated. Among them, the average leaf consumption level of the plants treated with GO-B094 was 6.51±0.22, showing susceptibility to insects, while the average leaf consumption levels of the plants treated with the other three seed soaking treatments were all close to 9, showing high susceptibility. It can also be seen from the proportion of plants with different resistance levels that all the plants treated with CK, GO and B094 showed susceptibility or high susceptibility to insects, and more than 80% of them reached the high susceptibility level. However, only 24% of the plants treated with GO-B094 reached the high susceptibility level, and 32% of the plants reached the susceptible level, indicating that GO-B094 seed soaking can significantly reduce the damage of Spodoptera frugiperda.

[0104] The results of the investigation on the field resistance to natural insect infestation showed ( Figure 11 ) that when investigated at 14 days after emergence, due to the suitable temperature and humidity at this stage, the maize seedlings grew well and had reached the stage of 5 leaves and 1 heart. All the seedlings treated with the 4 seed soaking treatments were damaged, mainly by Ostrinia furnacalis, and also by Spodoptera exigua. However, there were obvious differences in the damage degree. About 80% of the plants treated with GO-B094 had only pinhole-like damage about 1 mm on individual leaves. All the plants treated with CK, GO and B094 showed susceptibility to insects. Among them, 60% of the plants treated with B094 showed susceptibility or high susceptibility to insects, while the proportion of plants with susceptibility or high susceptibility in the plants treated with CK and GO was higher, accounting for more than 90% ( Figure 11 A and 11B). When investigated at 21 days after emergence, although the resistance levels of the seedlings treated with the 4 seed soaking treatments all reached susceptibility to insects, the resistance levels of CK, GO and B094 were close to high susceptibility, while the resistance level of the plants treated with GO-B094 was close to moderate resistance. It can also be seen from the proportion of plants with different resistance levels that 100% of the plants treated with CK, GO and B094 showed susceptibility or high susceptibility to insects, and 78-90% of the plants reached the high susceptibility level. However, the proportion of plants with susceptibility and high susceptibility in the plants treated with GO-B094 was only about 60%. This indicates that GO-B094 seed soaking has a good insect-resistant effect.

Claims

1. A graphene oxide-Beauveria bassiana composite, characterized in that: The Beauveria bassiana is adsorbed on graphene oxide, the preservation number of the Beauveria bassiana is: CGMCC NO.19034, the graphene oxide is 1-100ug / ml graphene oxide solution; the Beauveria bassiana is 1×10 8 spores / mL of Beauveria bassiana. The preparation method of the graphene oxide-Beauveria bassiana composite comprises: preparing a spore suspension of conidia of Beauveria bassiana with a sterile solution; preparing a graphene oxide solution, mixing the Beauveria bassiana spore suspension with the graphene oxide solution to obtain a mixed solution; uniformly stirring the mixed solution in the dark with a magnetic stirrer to generate a graphene oxide-Beauveria bassiana composite solution; centrifuging the generated graphene oxide-Beauveria bassiana composite solution to discard the supernatant, adding glutaraldehyde fixative, fixing at low temperature, washing with phosphate buffer, and sequentially dehydrating with 50%-100% ethanol for gradient dehydration, replacing with ethanol-tert-butanol solution after dehydration, and finally replacing twice with 100% tert-butanol, and freeze-drying the sample for standby use after the treatment is completed; the Beauveria bassiana is adsorbed on the graphene oxide sheet, so that the surface of the graphene oxide-Beauveria bassiana composite presents a rough multi-granular shape.

2. The graphene oxide-Beauveria bassiana composite according to claim 1, characterized in that: The graphene oxide is a 20-100 ug / ml graphene oxide solution.

3. Use of the graphene oxide-Beauveria bassiana complex according to any one of claims 1 to 2 in increasing plant height, leaf length and / or leaf area of ​​corn.

4. Use of the graphene oxide-Beauveria bassiana complex according to any one of claims 1 to 2 in controlling pests, wherein the pests include Ostrinia nubilalis and Spodoptera frugiperda.

5. A method for improving the growth and / or resistance of corn, characterized in that: The method comprises: after disinfecting the surface of corn seeds, soaking them in the graphene oxide-Beauveria bassiana composite suspension according to any one of claims 1 to 2 for 1 to 2 days, and sowing them after drying.

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