Method for preventing dsRNA bacterial liquid from being degraded by ultraviolet, biological preparation for preventing and treating bemisia tabaci and prevention and treatment method

Abstract

The invention discloses a method for preventing dsRNA bacterial liquid from being degraded by ultraviolet, a biological preparation for preventing and controlling bemisia tabaci and a prevention and control method, and belongs to the technical field of pest prevention and control. The method for preventing the dsRNA bacterial liquid from being degraded by ultraviolet comprises the following steps: mixing the dsRNA bacterial liquid with a UV protective agent to prepare a mixed solution of the UV protective agent and the dsRNA bacterial liquid; the UV protective agent is one of potassium humate and sodium humate. The UV protective agent provided by the invention can effectively avoid degradation of dsRNA bacterial liquid, so that the dsGawky can give full play to the fatal effect on bemisia tabaci, and the target of pest control is accurately achieved. The method is efficient, wide in application, easy to formulate, high in insecticidal efficiency and environment-friendly, has the advantages of promoting plant growth and the like, and has a very good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pest control, and specifically relates to a method for avoiding ultraviolet degradation of dsRNA bacterial solution, a biological agent for controlling Bemisia tabaci, and a control method. Background Art

[0002] RNA interference (RNAi) is a mechanism of action triggered by short fragment RNAs (siRNAs) that promotes the degradation of homologous mRNA or inhibits its translation. The RNAi technology can precisely target and silence key genes of pests by specifically inhibiting gene expression, thereby achieving the purpose of efficient pest control.

[0003] The Gawky gene plays a role in gene silencing mediated by micro-RNA (miRNA) and short interfering RNA (siRNA). Inhibiting the expression of this gene will displace other GW body proteins and impair RNAi and microRNA-induced gene silencing.

[0004] When the RNA interference fragment is directly sprayed on the surface of plants, pests can ingest or absorb these fragments. However, the directly sprayed double-stranded dsRNA bacterial solution is easily degraded by nucleases and ultraviolet rays in the air, resulting in the inability to effectively trigger the RNAi reaction, thereby weakening its interference effect on pests. Therefore, exploring strategies to avoid ultraviolet degradation of dsRNA bacterial solution is crucial for promoting the application of RNAi technology in the field of pest biological control. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a method for avoiding ultraviolet degradation of dsRNA bacterial solution, a biological agent for controlling Bemisia tabaci, and a control method.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A method for avoiding ultraviolet degradation of dsRNA bacterial solution, which mixes the dsRNA bacterial solution with a UV protectant to prepare a mixed solution of the UV protectant and the dsRNA bacterial solution; the UV protectant is one of potassium humate and sodium humate.

[0008] The application of the above method for avoiding ultraviolet degradation of dsRNA bacterial solution in RNA interference for controlling agricultural pests.

[0009] A biological agent for controlling Bemisia tabaci, which contains a UV protectant and a dsRNA bacterial solution that interferes with the Gawky gene of Bemisia tabaci; the UV protectant is one of potassium humate and sodium humate.

[0010] Based on the above solution, the dsRNA bacterial solution interfering with the Bemisia tabaci Gawky gene is a bacterial solution transcribed with the Bemisia tabaci Gawky gene dsRNA.

[0011] Based on the above solution, the bacterial solution transcribed with the Bemisia tabaci Gawky gene dsRNA is prepared by the following method:

[0012] Amplify the dsRNA sequence of the Gawky gene using primers, ligate it to an expression vector, construct a recombinant vector, and transform an expression strain; induce the expression strain to transcribe the dsRNA sequence of the Gawky gene, discard the supernatant of the bacterial solution, and dissolve and dilute it with ddH2O to obtain the product.

[0013] Based on the above solution, the method for inducing the expression strain to transcribe the dsRNA sequence of the Gawky gene is as follows:

[0014] Inoculate the recombinant bacterium transformed with the recombinant vector expressing the dsRNA of the Bemisia tabaci Gawky gene into an LB medium containing Amp, culture it with shaking at 37 °C and 20 r / min. When the OD value of the bacterial solution 600 reaches 0.5, add an IPTG solution with a final concentration of 0.5 M for induction for 4 h to obtain the product.

[0015] Based on the above solution, the preparation method of the biological agent for controlling Bemisia tabaci is as follows:

[0016] (1) Disperse the UV protectant in a 0.1% Tween 80 aqueous solution to prepare a UV protectant solution with a concentration of 0.2% - 5%.

[0017] (2) Mix the UV protectant solution and the bacterial solution transcribed with the Bemisia tabaci Gawky gene dsRNA according to a volume ratio of 1:1, and mix them evenly by shaking to obtain the biological agent for controlling Bemisia tabaci.

[0018] Application of the above biological agent for controlling Bemisia tabaci in the biological control of Bemisia tabaci.

[0019] A method for biological control of Bemisia tabaci, spraying the above biological agent for controlling Bemisia tabaci on plants, so that Bemisia tabaci feeds on the sprayed plants, thereby increasing the mortality rate of Bemisia tabaci.

[0020] Based on the above solution, the Bemisia tabaci is Bemisia tabaci Q biotype or Bemisia tabaci B biotype.

[0021] Advantages of the technical solution of the present invention

[0022] The present invention has developed a technology based on the UV protectant KH / SH to avoid the degradation of dsRNA bacterial solution by ultraviolet rays. That is, the humate solution and the dsRNA bacterial solution are shaken and mixed evenly to form a mixed solution of the UV protectant (KH or SH) and the dsRNA bacterial solution. The UV protectant is used to protect the dsRNA bacterial solution from the degradation by ultraviolet rays, so as to achieve the purpose of pest control by making the lethal effect of dsGawky on Bemisia tabaci. This material can effectively protect the dsRNA bacterial solution by absorbing ultraviolet radiation through its specific organic molecules, and reduce the direct damage of ultraviolet rays to the dsRNA bacterial solution. This method is efficient, widely applicable, easy to formulate, has a high insecticidal efficiency, is environmentally friendly, and has many advantages such as promoting plant growth, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixed solution of 1% UV protectant KH and the bacterial solution of transcribed dsGawky on adult Q-type Bemisia tabaci indoors;

[0024] Figure 2 Lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixed solution of 1% UV protectant KH and the bacterial solution of transcribed dsGawky on adult Q-type Bemisia tabaci outdoors;

[0025] Figure 3 Line graph of the lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixed solution of 1% UV protectant KH and the bacterial solution of transcribed dsGawky on adult Q-type Bemisia tabaci outdoors;

[0026] Figure 4 Lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixed solution of 1% UV protectant KH and the bacterial solution of transcribed dsGawky on Q-type Bemisia tabaci eggs outdoors;

[0027] Figure 5 Lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixed solution of 1% UV protectant KH and the bacterial solution of transcribed dsGawky on nymphs of Q-type Bemisia tabaci outdoors

[0028] Figure 6 Lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixed solution of 1% UV protectant KH and the bacterial solution of transcribed dsGawky on adult Q-type Bemisia tabaci under the condition of indoor ultraviolet light irradiation for 2 h;

[0029] Figure 7 Line graph of the lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixed solution of 1% UV protectant KH and the bacterial solution of transcribed dsGawky on adult Q-type Bemisia tabaci under the condition of indoor ultraviolet light irradiation for 2 h;

[0030] Figure 8 Lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 1% UV protectant SH and transcription dsGawky bacteria indoors on adult Q-biotype Bemisia tabaci;

[0031] Figure 9 Lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 1% UV protectant SH and transcription dsGawky bacteria outdoors on adult Q-biotype Bemisia tabaci;

[0032] Figure 10 Line graph of the lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 1% UV protectant SH and transcription dsGawky bacteria outdoors on adult Q-biotype Bemisia tabaci;

[0033] Figure 11 Lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 1% UV protectant SH and transcription dsGawky bacteria outdoors on the eggs of Q-biotype Bemisia tabaci;

[0034] Figure 12 Lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 1% UV protectant SH and transcription dsGawky bacteria outdoors on nymphs of Q-biotype Bemisia tabaci

[0035] Figure 13 Lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 1% UV protectant SH and transcription dsGawky bacteria on adult Q-biotype Bemisia tabaci under the condition of indoor ultraviolet irradiation for 2 h;

[0036] Figure 14 Line graph of the lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 1% UV protectant SH and transcription dsGawky bacteria on adult Q-biotype Bemisia tabaci under the condition of indoor ultraviolet irradiation for 2 h;

[0037] Figure 15 Lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 0.2% UV protectant KH and transcription dsGawky bacteria indoors on adult Q-biotype Bemisia tabaci;

[0038] Figure 16 Lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 0.2% UV protectant KH and transcription dsGawky bacteria outdoors on adult Q-biotype Bemisia tabaci;

[0039] Figure 17 Lethal effect of spraying the bacterial liquid of transcription dsGawky bacteria or the mixture of 5% UV protectant KH and transcription dsGawky bacteria indoors on adult Q-biotype Bemisia tabaci;

[0040] Figure 18 Lethal effects of spraying the bacterial solution of dsGawky-expressing bacteria or a mixture of 5% UV protectant KH and dsGawky-expressing bacteria outdoors on adult Q-biotype Bemisia tabaci

[0041] Figure 19 Lethal effects of spraying the bacterial solution of dsGawky-expressing bacteria or a mixture of 0.2% UV protectant SH and dsGawky-expressing bacteria indoors on adult Q-biotype Bemisia tabaci

[0042] Figure 20 Lethal effects of spraying the bacterial solution of dsGawky-expressing bacteria or a mixture of 0.2% UV protectant SH and dsGawky-expressing bacteria outdoors on adult Q-biotype Bemisia tabaci

[0043] Figure 21 Lethal effects of spraying the bacterial solution of dsGawky-expressing bacteria or a mixture of 5% UV protectant SH and dsGawky-expressing bacteria indoors on adult Q-biotype Bemisia tabaci

[0044] Figure 22 Lethal effects of spraying the bacterial solution of dsGawky-expressing bacteria or a mixture of 5% UV protectant SH and dsGawky-expressing bacteria outdoors on adult Q-biotype Bemisia tabaci Detailed implementation manners

[0045] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified. The present invention will be described in further detail below with reference to specific examples and data. The following examples are only for illustrative purposes and do not limit the scope of the present invention in any way.

[0046] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The test materials, reagents, drugs, etc. used in the following examples can be obtained through general channels unless otherwise specified.

[0047] The Bemisia tabaci population used in the following examples was collected from Lingshui City, Hainan Province in January 2017. These insects were reared in an insect-proof cage on the common tobacco variety NC89 for a long time (temperature 27±1°C, relative humidity 60%±5%, light cycle 16L:8D).

[0048] Example 1

[0049] Application of potassium humate in preventing the ultraviolet degradation of dsRNA bacterial solution

[0050] (1) Use a 0.1% (volume fraction) aqueous solution of Tween 80 as a solvent to prepare a 1% (mass fraction) solution of potassium humate as a 1% UV protectant KH solution.

[0051] (2) Mix the UV protectant KH solution and the dsRNA bacterial solution in a volume ratio of 1:1 to avoid the ultraviolet degradation of the dsRNA bacterial solution.

[0052] Example 2

[0053] A method for avoiding the ultraviolet degradation of Bemisia tabaci dsRNA bacterial solution

[0054] (1) Disperse 0.2 mL of Tween 80 in 199.8 mL of water, stir evenly to prepare a 0.1% Tween 80 solution as a solvent;

[0055] (2) Take 1 g of potassium humate and disperse it in 99 mL of the 0.1% Tween 80 solution prepared in step (1), stir well for 30 min until the solution becomes uniform to obtain a 1% UV protectant KH solution;

[0056] (3) Mix the 1% UV protectant KH solution prepared in step (2) with the bacterial solution of Bemisia tabaci dsRNA in a volume ratio of 1:1 to avoid the ultraviolet degradation of the Bemisia tabaci dsRNA bacterial solution.

[0057] Example 3

[0058] A biological control method for Bemisia tabaci based on 1% UV protectant KH is as follows:

[0059] (1) Use a 0.1% (volume fraction) aqueous solution of Tween 80 as a solvent to prepare a 1% (mass fraction) solution of potassium humate as a UV protectant KH solution.

[0060] (2) Mix the 1% UV protectant KH solution with the bacterial solution (concentration: 2 g / L) of the bacteria that transcribe dsGawky (i.e., the bacteria that transcribe the dsRNA of the Bemisia tabaci Gawky gene) in a volume ratio of 1:1, and obtain a mixed solution of 1% UV protectant KH and the bacteria that transcribe dsGawky for Bemisia tabaci RNAi interference after shaking and mixing evenly.

[0061] (3) Spray the above mixed solution of 1% UV protectant KH and the bacteria that transcribe dsGawky on plants, so that Bemisia tabaci feeds on the sprayed plants, thereby increasing the mortality rate of Bemisia tabaci.

[0062] The preparation method of the bacterial solution of the bacteria that transcribe dsGawky is as follows:

[0063] According to the gene sequence of Bemisia tabaci Gawky (SEQ ID NO.1), primers for preparing dsRNA of Gawky gene (SEQ ID NO.2 and SEQ ID NO.3) were designed. Using the cDNA reverse transcribed from the total RNA of Bemisia tabaci as a template, PCR amplification was carried out. The reaction system for PCR was: 10x buffer 2.5 μL; dNTP 2 μL; upstream primer 1 μL; downstream primer 1 μL; rTaq 0.25 μL; H2O 17.25 μL; cDNA 1 μL. The reaction program was: pre-denaturation at 90 °C for 15 min; denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, extension at 72 °C for 10 s, 40 cycles; final extension at 72 °C for 10 min. After PCR was completed, the product was recovered, and the digested amplification product was ligated into the linearized L4440 plasmid using T4 DNA ligase; and then transformed into competent HT115(DE3). Finally, 300 μL was taken and spread on a solid medium containing 100 mg / mL Amp + and cultured overnight at 37 °C in an inverted manner. The next day, single colonies on 6 randomly selected plates were picked and dissolved in the medium to prepare a bacterial solution. The bacteria identified successfully by PCR were the target recombinant bacteria.

[0064] The above-mentioned recombinant bacteria were inoculated into 15 mL of LB medium (Amp + ), cultured at 37 °C with shaking at 20 r / min. When the OD value of the bacterial solution reached 0.5, an IPTG solution with a final concentration of 0.5 M was added for induction for 4 h to obtain the bacterial solution of dsGawky-transcribing bacteria. The bacterial solution was centrifuged at 4 °C and 5000×g for 10 min to separate the bacteria, and the supernatant was discarded. Then the bacteria were resuspended with a small amount of PBS solution, mixed well, centrifuged at 4 °C and 5000×g for 5 min, and the supernatant was discarded. This was repeated twice. The bacteria were placed in a -20 °C refrigerator and stored for later use.

[0065] When in use, it was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria).

[0066] SEQ ID NO.1 (5’→3’):

[0067]

[0068] The primers for preparing dsRNA of the Gawky gene are as follows:

[0069] Forward primer: dsGawkyF: 5’-taatacgactcactatagggGGAGATCCGTCCTACACGCTACA-3’ (SEQ ID NO.2);

[0070] Reverse primer: dsGawkyR: 5’-taatacgactcactatagggCGAGTTGGTGGAGGTTGCTGAAG-3’ (SEQ ID NO.3).

[0071] Example 4

[0072] Lethal effect of spraying the bacterial solution of transcribed dsGawky bacteria or the mixture of 1% UV protectant KH and transcribed dsGawky bacteria on adult Q-biotype Bemisia tabaci indoors

[0073] The bacterial solution of transcribed dsGawky bacteria prepared in Example 3 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, with 3 replicates for each treatment. It was placed in an incubator without sunlight, at a temperature of 27 ± 1°C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. The adult mortality was counted 7 days after spraying.

[0074] The mixture of 1% UV protectant KH and transcribed dsGawky bacteria prepared in Example 3 was sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, with 3 replicates for each treatment. It was placed in an incubator without sunlight, at a temperature of 27 ± 1°C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. The adult mortality was counted 7 days after spraying.

[0075] The results are as Figure 1 shown. Under indoor conditions, the lethality rate of spraying the bacterial solution of transcribed dsGawky bacteria on adult Q-biotype Bemisia tabaci was 53.9%, and the lethality rate of spraying the mixture of 1% UV protectant KH and transcribed dsGawky bacteria on adult Q-biotype Bemisia tabaci was 68.3%.

[0076] Example 5

[0077] Lethal effect of spraying the bacterial solution of transcribed dsGawky bacteria or the mixture of 1% UV protectant KH and transcribed dsGawky bacteria on adult Q-biotype Bemisia tabaci outdoors

[0078] The transcription dsGawky bacterial solution prepared in Example 3 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Bemisia tabaci Q biotype were released onto the cotton plants. There were 3 replicates for each treatment, and they were placed outdoors with sunlight. The adult mortality rate was counted 7 days after spraying.

[0079] The mixture of 1% UV protectant KH and transcription dsGawky bacteria prepared in Example 3 was sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Bemisia tabaci Q biotype were released onto the cotton plants. There were 3 replicates for each treatment. They were placed outdoors with sunlight. The adult mortality rate was counted 7 days after spraying.

[0080] The results are as Figure 2 and Figure 3 shown. Under outdoor conditions, the lethality rate of the transcription dsGawky bacterial solution sprayed on adult Bemisia tabaci Q biotype was 18%, and the lethality rate of the mixture of 1% UV protectant KH and transcription dsGawky bacteria sprayed on adult Bemisia tabaci Q biotype was 42.8%. There was a significant difference between the two.

[0081] Example 6

[0082] Lethal effect of spraying the bacterial solution of transcription dsGawky bacteria or the mixture of 1% UV protectant KH and transcription dsGawky bacteria on Bemisia tabaci Q biotype eggs outdoors

[0083] 15 pairs of Bemisia tabaci were released onto cotton plants. After the Bemisia tabaci laid eggs for 1 day, the adults were sucked out, and the number of eggs on each cotton plant was recorded under a microscope. The transcription dsGawky bacterial solution prepared in Example 3 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. There were 3 replicates for each treatment, and they were placed outdoors with sunlight. The eggs that did not hatch into nymphs on the 10th day after spraying were considered dead, and the egg mortality rate was counted.

[0084] 15 pairs of Bemisia tabaci were released onto cotton plants. After the Bemisia tabaci laid eggs for 1 day, the adults were sucked out, and the number of eggs on each cotton plant was recorded under a microscope. The mixture of 1% UV protectant KH and transcription dsGawky bacteria prepared in Example 3 was sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. There were 3 replicates for each treatment. They were placed outdoors with sunlight. The eggs that did not hatch into nymphs on the 10th day after spraying were considered dead, and the egg mortality rate was counted.

[0085] The results are as Figure 4As shown, under outdoor conditions, the mortality rate of Q-biotype Bemisia tabaci eggs after spraying the transcription dsGawky bacterial solution was 18%, and the mortality rate of Q-biotype Bemisia tabaci eggs after spraying the mixture of 1% UV protectant KH and transcription dsGawky bacteria was 41.7%. There was a significant difference between the two.

[0086] Example 7

[0087] Lethal effect of spraying the bacterial solution of transcription dsGawky bacteria or the mixture of 1% UV protectant KH and transcription dsGawky bacteria on Q-biotype Bemisia tabaci nymphs outdoors

[0088] Fifteen pairs of Bemisia tabaci were released onto cotton plants. The number of nymphs was observed under a microscope on the 9th day. The transcription dsGawky bacterial solution prepared in Example 3 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto the cotton plants. The front and back of each cotton leaf were sprayed 3 times, with 3 replicates for each treatment, and placed outdoors with sunlight. Since there were slight differences in the emergence time of Bemisia tabaci nymphs, there were newly emerged adults on the 17th day, and it was impossible to accurately determine the death of nymphs under a microscope. On the 17th day, the newly emerged adults were sucked out and counted every day. On the 25th day, there were no newly emerged adults for 3 consecutive days. At this time, the unemerged nymphs were considered dead, and the nymph mortality rate was statistically analyzed.

[0089] Fifteen pairs of Bemisia tabaci were released onto cotton plants. The number of nymphs was observed under a microscope on the 9th day. The mixture of 1% UV protectant KH and transcription dsGawky bacteria prepared in Example 3 was sprayed onto the cotton plants. The front and back of each cotton leaf were sprayed 3 times, with 3 replicates for each treatment. It was placed outdoors with sunlight. Since there were slight differences in the emergence time of Bemisia tabaci nymphs, there were newly emerged adults on the 17th day, and it was impossible to accurately determine the death of nymphs under a microscope. On the 17th day, the newly emerged adults were sucked out and counted every day. On the 25th day, there were no newly emerged adults for 3 consecutive days. At this time, the unemerged nymphs were considered dead, and the nymph mortality rate was statistically analyzed.

[0090] The results are as Figure 5 shown. Under outdoor conditions, the mortality rate of Q-biotype Bemisia tabaci nymphs after spraying the transcription dsGawky bacterial solution was 22%, and the mortality rate of Q-biotype Bemisia tabaci nymphs after spraying the mixture of 1% UV protectant KH and transcription dsGawky bacteria was 50.4%. There was a significant difference between the two.

[0091] Example 8

[0092] Lethal effect of spraying the bacterial solution of transcription dsGawky bacteria or the mixture of 1% UV protectant KH and transcription dsGawky bacteria on Q-biotype Bemisia tabaci adults under the condition of indoor ultraviolet light irradiation for 2 h

[0093] The transcription dsGawky bacterial solution prepared in Example 3 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto cotton plants. The front and back of each cotton leaf were sprayed 3 times. After irradiating with an ultraviolet light instrument for 2 h and waiting for the solution to dry, 30 adult Bemisia tabaci Q biotype were released onto the cotton plants, with 3 replicates for each treatment. It was placed in an incubator at a temperature of 27 ± 1 °C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. The adult mortality rate was counted 7 days after spraying.

[0094] The mixture of 1% UV protectant KH and transcription dsGawky bacteria prepared in Example 3 was sprayed onto cotton plants. The front and back of each cotton leaf were sprayed 3 times. After irradiating with an ultraviolet light instrument for 2 h and waiting for the solution to dry, 30 adult Bemisia tabaci Q biotype were released onto the cotton plants, with 3 replicates for each treatment. It was placed in an incubator at a temperature of 27 ± 1 °C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. The adult mortality rate was counted 7 days after spraying.

[0095] The results are as Figure 6 and Figure 7 shown. Under the condition of ultraviolet light irradiation for 2 h, the lethality rate of spraying the transcription dsGawky bacterial solution on adult Bemisia tabaci Q biotype was 30.1%, and the lethality rate of spraying the mixture of UV protectant KH and transcription dsGawky bacteria on adult Bemisia tabaci Q biotype was 43.4%. There was a significant difference between the two.

[0096] Example 9

[0097] Application of sodium humate in avoiding ultraviolet degradation of dsRNA bacterial solution

[0098] (1) Using 0.1% (volume fraction) aqueous Tween 80 solution as a solvent, sodium humate was prepared into a 1% (mass fraction) solution as 1% UV protectant SH solution.

[0099] (2) Mixing the 1% UV protectant SH solution and the dsRNA bacterial solution according to a volume ratio of 1:1 can avoid ultraviolet degradation of the dsRNA bacterial solution.

[0100] Example 10

[0101] A method for avoiding ultraviolet degradation of Bemisia tabaci dsRNA bacterial solution

[0102] (1) Disperse 0.2 mL of Tween 80 in 199.8 mL of water and stir evenly to prepare 0.1% Tween 80 solution as a solvent;

[0103] (2) Weigh 1 g of sodium humate and disperse it in 99 mL of the 0.1% Tween 80 solution prepared in step (1). Stir well for 30 min until the solution becomes uniform, thus obtaining a 1% UV protectant SH solution.

[0104] (3) Mix the 1% UV protectant SH solution prepared in step (2) with the bacterial solution of Bemisia tabaci dsRNA in a volume ratio of 1:1, which can prevent the degradation of the Bemisia tabaci dsRNA bacterial solution by ultraviolet light.

[0105] Example 11

[0106] The Bemisia tabaci biological control method based on 1% UV protectant SH is as follows:

[0107] (1) Using 0.1% (volume fraction) aqueous Tween 80 solution as the solvent, prepare sodium humate into a 1% (mass fraction) solution as the 1% UV protectant SH solution.

[0108] (2) Mix the 1% UV protectant SH solution with the bacterial solution of transcribed dsGawky (i.e., the bacterial solution of transcribed Bemisia tabaci Gawky gene dsRNA) (concentration: 2 g / L) in a volume ratio of 1:1. After shaking and mixing evenly, obtain the mixed solution of 1% UV protectant SH and the bacterial solution of transcribed dsGawky for Bemisia tabaci RNAi interference.

[0109] (3) Spray the above-mentioned mixed solution of UV protectant SH and the bacterial solution of transcribed dsGawky on the plants, enabling Bemisia tabaci to feed on the sprayed plants, thereby increasing the mortality rate of Bemisia tabaci.

[0110] The preparation method of the bacterial solution of transcribed dsGawky is the same as that in Example 3.

[0111] Example 12

[0112] Lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixed solution of 1% UV protectant SH and the bacterial solution of transcribed dsGawky on adult Q-type Bemisia tabaci indoors

[0113] Dissolve and dilute the bacterial solution of transcribed dsGawky prepared in Example 11 with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria). Then spray it onto cotton plants, spraying 3 times on both the front and back of cotton leaves. After the solution dries, release 30 adult Q-type Bemisia tabaci on the cotton plants, with 3 replicates for each treatment. Place them in a constant temperature incubator without sunlight, at a temperature of 27 ± 1°C, relative humidity of 60% ± 5%, and a light cycle of 16L:8D. Count the adult mortality rate after 7 days of spraying.

[0114] The mixture of 1% UV protectant SH prepared in Example 11 and the dsGawky-expressing bacteria was sprayed onto cotton plants, with each side of the cotton leaves sprayed three times. After the solution dried, 30 adult Bemisia tabaci (Q biotype) were released onto each cotton plant, and there were three replicates for each treatment. The plants were placed in an incubator without sunlight, at a temperature of 27 ± 1°C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. The adult mortality was counted 7 days after spraying.

[0115] The results are as Figure 8 shown. Under indoor conditions, the mortality rate of adult Bemisia tabaci (Q biotype) after spraying the dsGawky-expressing bacteria solution was 53.9%, and the mortality rate after spraying the mixture of 1% UV protectant SH and the dsGawky-expressing bacteria was 62.7%.

[0116] Example 13

[0117] Lethal effect of spraying the dsGawky-expressing bacteria solution or the mixture of 1% UV protectant SH and the dsGawky-expressing bacteria on adult Bemisia tabaci (Q biotype) outdoors

[0118] The dsGawky-expressing bacteria solution prepared in Example 11 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto cotton plants, with each side of the cotton leaves sprayed three times. After the solution dried, 30 adult Bemisia tabaci (Q biotype) were released onto each cotton plant, and there were three replicates for each treatment. The plants were placed outdoors with sunlight. The adult mortality was counted 7 days after spraying.

[0119] The mixture of 1% UV protectant SH prepared in Example 11 and the dsGawky-expressing bacteria was sprayed onto cotton plants, with each side of the cotton leaves sprayed three times. After the solution dried, 30 adult Bemisia tabaci (Q biotype) were released onto each cotton plant, and there were three replicates for each treatment. The plants were placed outdoors with sunlight. The adult mortality was counted 7 days after spraying.

[0120] The results are as Figure 9 and Figure 10 shown. Under outdoor conditions, the mortality rate of adult Bemisia tabaci (Q biotype) after spraying the dsGawky-expressing bacteria solution was 18%, and the mortality rate after spraying the mixture of 1% UV protectant SH and the dsGawky-expressing bacteria was 48%, showing a significant difference between the two.

[0121] Example 14

[0122] Lethal effect of spraying the dsGawky-expressing bacteria solution or the mixture of 1% UV protectant SH and the dsGawky-expressing bacteria on Bemisia tabaci (Q biotype) eggs outdoors

[0123] Release 15 pairs of Bemisia tabaci onto cotton plants. After the Bemisia tabaci lay eggs for 1 day, suck out the adults, record the number of eggs on each cotton plant under a microscope. Dissolve and dilute the transcriptional dsGawky bacterial solution prepared in Example 11 with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then spray it onto the cotton plants. Spray three times on both the front and back of the cotton leaves. There are three replicates for each treatment. Place them outdoors with sunlight. Consider the eggs that did not hatch into nymphs on the 10th day after spraying as dead, and count the results of the egg mortality rate.

[0124] Release 15 pairs of Bemisia tabaci onto cotton plants. After the Bemisia tabaci lay eggs for 1 day, suck out the adults, record the number of eggs on each cotton plant under a microscope. Spray the mixture of 1% UV protectant SH and transcriptional dsGawky bacteria prepared in Example 11 onto the cotton plants. Spray three times on both the front and back of the cotton leaves. There are three replicates for each treatment. Place them outdoors with sunlight. Consider the eggs that did not hatch into nymphs on the 10th day after spraying as dead, and count the results of the egg mortality rate.

[0125] The results are as Figure 11 shown. Under outdoor conditions, the lethality rate of spraying the transcriptional dsGawky bacterial solution on the eggs of Q-type Bemisia tabaci is 18%, and the lethality rate of spraying the mixture of 1% UV protectant SH and transcriptional dsGawky bacteria on the eggs of Q-type Bemisia tabaci is 32%. There is a significant difference between the two.

[0126] Example 15

[0127] Lethal effect of spraying the bacterial solution of transcriptional dsGawky bacteria or the mixture of 1% UV protectant SH and transcriptional dsGawky bacteria outdoors on the nymphs of Q-type Bemisia tabaci

[0128] Release 15 pairs of Bemisia tabaci onto cotton plants. Observe the number of nymphs under a microscope on the 9th day. Dissolve and dilute the transcriptional dsGawky bacterial solution prepared in Example 11 with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then spray it onto the cotton plants. Spray three times on both the front and back of the cotton leaves. There are three replicates for each treatment. Place them outdoors with sunlight. Since there are slight differences in the emergence time of Bemisia tabaci nymphs and there are already newly emerged adults on the 17th day, and it is impossible to accurately determine the death of nymphs under a microscope. On the 17th day, suck out the newly emerged adults every day and record the number. On the 25th day, there have been no newly emerged adults for three consecutive days. At this time, the unemerged nymphs are considered dead, and count the results of the nymph mortality rate.

[0129] Fifteen pairs of Bemisia tabaci were released onto cotton plants. On the 9th day, the number of nymphs was observed under a microscope. The mixture of the UV protectant SH prepared in Example 11 and the bacteria body of transcribed dsGawky was sprayed onto the cotton plants, with 3 sprays on each side of the cotton leaves. Each treatment had 3 replicates. They were placed outdoors under sunlight. Since there were slight differences in the emergence time of Bemisia tabaci nymphs, there were newly emerged adults on the 17th day, and it was impossible to accurately determine the death of nymphs under a microscope. On the 17th day, the newly emerged adults were sucked out and counted every day. On the 25th day, there were no newly emerged adults for 3 consecutive days. At this time, the unemerged nymphs were considered dead, and the nymph mortality rate was statistically analyzed.

[0130] The results are as Figure 12 shown. Under outdoor conditions, the lethal rate of the solution of transcribed dsGawky bacteria to the nymphs of Bemisia tabaci Q biotype was 22%, and the lethal rate of the mixture of 1% UV protectant SH and the bacteria body of transcribed dsGawky to the nymphs of Bemisia tabaci Q biotype was 46.8%. There was a significant difference between the two.

[0131] Example 16

[0132] Under the condition of indoor ultraviolet light irradiation for 2 h, the lethal effect of the bacterial solution of transcribed dsGawky or the mixture of 1% UV protectant SH and the bacteria body of transcribed dsGawky on the adults of Bemisia tabaci Q biotype

[0133] The bacterial solution of transcribed dsGawky prepared in Example 11 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria body), and then sprayed onto the cotton plants, with 3 sprays on each side of the cotton leaves. After irradiating with ultraviolet light for 2 h and waiting for the solution to dry, 30 adults of Bemisia tabaci Q biotype were released onto the cotton plants. Each treatment had 3 replicates. They were placed in an incubator at a temperature of 27 ± 1 °C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. The adult mortality rate was statistically analyzed 7 days after spraying.

[0134] The mixture of 1% UV protectant SH and the bacteria body of transcribed dsGawky prepared in Example 11 was sprayed onto the cotton plants, with 3 sprays on each side of the cotton leaves. After irradiating with ultraviolet light for 2 h and waiting for the solution to dry, 30 adults of Bemisia tabaci Q biotype were released onto the cotton plants. Each treatment had 3 replicates. They were placed in an incubator at a temperature of 27 ± 1 °C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. The adult mortality rate was statistically analyzed 7 days after spraying.

[0135] The results are as Figure 13 and Figure 14As shown, under the condition of ultraviolet light irradiation for 2 h, the lethal rate of spraying the bacterial solution of transcribed dsGawky on the adults of Bemisia tabaci Q type was 30.1%, and the lethal rate of spraying the mixture of 1% UV protectant SH and the bacterial solution of transcribed dsGawky on the adults of Bemisia tabaci Q type was 42.8%, showing a significant difference between the two.

[0136] Example 17

[0137] Application of potassium humate in preventing dsRNA bacterial solution from being degraded by ultraviolet

[0138] (1) Using 0.1% (volume fraction) aqueous solution of Tween 80 as a solvent, potassium humate was prepared into a solution with a concentration of 0.2% (mass fraction) as 0.2% UV protectant KH solution.

[0139] (2) Mixing 0.2% UV protectant KH solution and dsRNA bacterial solution in a volume ratio of 1:1 can prevent the dsRNA bacterial solution from being degraded by ultraviolet.

[0140] Example 18

[0141] A method for preventing the dsRNA bacterial solution of Bemisia tabaci from being degraded by ultraviolet

[0142] (1) 0.2 mL of Tween 80 was dispersed in 199.8 mL of water and stirred evenly to prepare 0.1% Tween 80 solution as a solvent.

[0143] (2) 0.2 g of potassium humate was taken and dispersed in 99.8 mL of the 0.1% Tween 80 solution prepared in step (1), and stirred thoroughly for 30 min until the solution became uniform to obtain 0.2% UV protectant KH solution.

[0144] (3) Mixing the 0.2% UV protectant KH solution prepared in step (2) and the bacterial solution of Bemisia tabaci dsRNA in a volume ratio of 1:1 can prevent the dsRNA bacterial solution of Bemisia tabaci from being degraded by ultraviolet.

[0145] Example 19

[0146] A biological control method of Bemisia tabaci based on 0.2% UV protectant KH is as follows:

[0147] (1) Using 0.1% (volume fraction) aqueous solution of Tween 80 as a solvent, potassium humate was prepared into a solution with a concentration of 0.2% (mass fraction) as 0.2% UV protectant KH solution.

[0148] (2) Mix the 0.2% UV protectant KH solution with the bacterial solution of transcribed dsGawky (i.e., the bacterial solution for transcribing dsRNA of Bemisia tabaci Gawky gene) (concentration: 2 g / L) at a volume ratio of 1:1, and obtain the mixture of 0.2% UV protectant KH and the bacterial body of transcribed dsGawky for RNAi interference of Bemisia tabaci after shaking and mixing evenly.

[0149] (3) Spray the above mixture of 0.2% UV protectant KH and the bacterial body of transcribed dsGawky on plants, so that Bemisia tabaci feeds on the sprayed plants, thereby increasing the mortality rate of Bemisia tabaci.

[0150] The preparation method of the solution of the bacterial body of transcribed dsGawky is the same as that in Example 3.

[0151] Example 20

[0152] Lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixture of 0.2% UV protectant KH and the bacterial body of transcribed dsGawky on adult Q-type Bemisia tabaci indoors

[0153] Dissolve and dilute the bacterial solution of transcribed dsGawky prepared in Example 19 with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacterial body), then spray it on cotton plants, spray 3 times on both the front and back of cotton leaves. After the solution dries, release 30 adult Q-type Bemisia tabaci on the cotton plants, with 3 replicates for each treatment. Place them in an incubator without sunlight, at a temperature of 27 ± 1°C, relative humidity of 60% ± 5%, and a light cycle of 16L:8D. Statistically analyze the adult mortality rate 7 days after spraying.

[0154] Spray the mixture of 0.2% UV protectant KH and the bacterial body of transcribed dsGawky prepared in Example 19 on cotton plants, spray 3 times on both the front and back of cotton leaves. After the solution dries, release 30 adult Q-type Bemisia tabaci on the cotton plants, with 3 replicates for each treatment. Place them in an incubator without sunlight, at a temperature of 27 ± 1°C, relative humidity of 60% ± 5%, and a light cycle of 16L:8D. Statistically analyze the adult mortality rate 7 days after spraying.

[0155] The results are as Figure 15 shown. Under indoor conditions, the lethality rate of spraying the bacterial solution of transcribed dsGawky on adult Q-type Bemisia tabaci is 53.9%, and the lethality rate of spraying the mixture of 0.2% UV protectant KH and the bacterial body of transcribed dsGawky on adult Q-type Bemisia tabaci is 51.1%.

[0156] Example 21

[0157] The lethal effect of spraying the bacterial solution of transcription dsGawky bacteria or the mixture of 0.2% UV protectant KH and transcription dsGawky bacteria on the adults of Bemisia tabaci Q biotype outdoors;

[0158] Dissolve and dilute the transcription dsGawky bacterial solution prepared in Example 19 with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then spray it onto cotton plants. Spray 3 times on both the front and back of the cotton leaves. After the solution dries, release 30 adult Bemisia tabaci Q biotype onto the cotton plants. There are 3 replicates for each treatment and place them outdoors with sunlight. After spraying for 7 days, count the results of adult mortality.

[0159] Spray the mixture of 0.2% UV protectant KH and transcription dsGawky bacteria prepared in Example 19 onto cotton plants. Spray 3 times on both the front and back of the cotton leaves. After the solution dries, release 30 adult Bemisia tabaci Q biotype onto the cotton plants. There are 3 replicates for each treatment. Place them outdoors with sunlight. After spraying for 7 days, count the results of adult mortality.

[0160] The results are as Figure 16 shown. Under outdoor conditions, the lethal rate of spraying the transcription dsGawky bacterial solution on the adults of Bemisia tabaci Q biotype is 18%, and the lethal rate of spraying the mixture of 0.2% UV protectant KH and transcription dsGawky bacteria on the adults of Bemisia tabaci Q biotype is 32.4%. There are significant differences between the two.

[0161] Example 22

[0162] Application of potassium humate in avoiding the ultraviolet degradation of dsRNA bacterial solution

[0163] (1) Using 0.1% (volume fraction) aqueous solution of Tween 80 as a solvent, prepare potassium humate into a solution with a concentration of 5% (mass fraction) as a 5% UV protectant KH solution.

[0164] (2) Mix the 5% UV protectant KH solution and the dsRNA bacterial solution in a volume ratio of 1:1, which can avoid the ultraviolet degradation of the dsRNA bacterial solution.

[0165] Example 23

[0166] A method for avoiding the ultraviolet degradation of Bemisia tabaci dsRNA bacterial solution

[0167] (1) Disperse 0.2 mL of Tween 80 in 199.8 mL of water, stir evenly to obtain a 0.1% Tween 80 solution as a solvent;

[0168] (2) Take 5 g of potassium humate and disperse it in 95 mL of the 0.1% Tween 80 solution prepared in step (1), and stir well for 30 min until the solution becomes homogeneous, thus obtaining a 5% UV protectant KH solution.

[0169] (3) Mix the 5% UV protectant KH solution prepared in step (2) with the bacterial solution of Bemisia tabaci dsRNA in a volume ratio of 1:1, which can prevent the Bemisia tabaci dsRNA bacterial solution from being degraded by ultraviolet light.

[0170] Example 24

[0171] A biological control method for Bemisia tabaci based on 5% UV protectant KH, the steps are as follows:

[0172] (1) Use 0.1% (volume fraction) aqueous Tween 80 solution as a solvent to prepare a potassium humate solution with a concentration of 5% (mass fraction) as a 5% UV protectant KH solution.

[0173] (2) Mix the 5% UV protectant KH solution with the bacterial solution of transcribed dsGawky (i.e., the bacterial solution of transcribed Bemisia tabaci Gawky gene dsRNA) (concentration: 2 g / L) in a volume ratio of 1:1, and after shaking and mixing evenly, obtain a mixture of 5% UV protectant KH and the bacterial solution of transcribed dsGawky for Bemisia tabaci RNAi interference.

[0174] (3) Spray the above mixture of 5% UV protectant KH and the bacterial solution of transcribed dsGawky on plants, so that Bemisia tabaci feeds on the sprayed plants, thereby increasing the mortality rate of Bemisia tabaci.

[0175] The preparation method of the bacterial solution of the transcribed dsGawky is the same as that in Example 3.

[0176] Example 25

[0177] The lethal effect of spraying the bacterial solution of transcribed dsGawky or the mixture of 5% UV protectant KH and the bacterial solution of transcribed dsGawky on adult Q-type Bemisia tabaci indoors;

[0178] Dissolve and dilute the bacterial solution of transcribed dsGawky prepared in Example 24 with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), then spray it onto cotton plants, spray 3 times on both the front and back of cotton leaves, and after the solution dries, release 30 adult Q-type Bemisia tabaci on the cotton plants, with 3 replicates for each treatment. Place them in an incubator without sunlight, at a temperature of 27 ± 1°C, relative humidity of 60% ± 5%, and a light cycle of 16L:8D. After spraying for 7 days, count the results of adult mortality.

[0179] The mixture of 5% UV protectant KH prepared in Example 24 and the transcribed dsGawky bacterial cells was sprayed onto cotton plants, with each side of the cotton leaves sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, and there were 3 replicates for each treatment. They were placed in an incubator without sunlight, at a temperature of 27 ± 1 °C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. The adult mortality rate was counted 7 days after spraying.

[0180] The results are as Figure 17 shown. Under indoor conditions, the lethality rate of the transcribed dsGawky bacterial cell solution against adult Q-biotype Bemisia tabaci was 53.9%, and the lethality rate of the mixture of 5% UV protectant KH and the transcribed dsGawky bacterial cells against adult Q-biotype Bemisia tabaci was 54.9%.

[0181] Example 26

[0182] Lethality effect of spraying the bacterial solution of transcribed dsGawky or the mixture of 5% UV protectant KH and the transcribed dsGawky on adult Q-biotype Bemisia tabaci outdoors;

[0183] The bacterial solution of transcribed dsGawky prepared in Example 24 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacterial cells), and then it was sprayed onto cotton plants, with each side of the cotton leaves sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, and there were 3 replicates for each treatment. They were placed outdoors with sunlight. The adult mortality rate was counted 7 days after spraying.

[0184] The mixture of 5% UV protectant KH prepared in Example 24 and the transcribed dsGawky bacterial cells was sprayed onto cotton plants, with each side of the cotton leaves sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, and there were 3 replicates for each treatment. They were placed outdoors with sunlight. The adult mortality rate was counted 7 days after spraying.

[0185] The results are as Figure 18 shown. Under outdoor conditions, the lethality rate of the transcribed dsGawky bacterial cell solution against adult Q-biotype Bemisia tabaci was 18%, and the lethality rate of the mixture of 5% sprayed UV protectant KH and the transcribed dsGawky bacterial cells against adult Q-biotype Bemisia tabaci was 59.6%, showing a significant difference between the two.

[0186] Example 27

[0187] Application of sodium humate in preventing the ultraviolet degradation of dsRNA bacterial solution

[0188] (1) Using 0.1% (volume fraction) aqueous Tween80 solution as a solvent, prepare sodium humate into a solution with a concentration of 0.2% (mass fraction) as 0.2% UV protectant SH solution.

[0189] (2) Mix the 0.2% UV protectant SH solution and the dsRNA bacterial solution in a volume ratio of 1:1, then the dsRNA bacterial solution can be prevented from being degraded by ultraviolet light.

[0190] Example 28

[0191] A method for preventing the dsRNA bacterial solution of Bemisia tabaci from being degraded by ultraviolet light

[0192] (1) Disperse 0.2 mL of Tween80 in 199.8 mL of water, stir evenly to prepare 0.1% Tween80 solution as a solvent;

[0193] (2) Take 0.2 g of sodium humate and disperse it in 99.8 mL of the 0.1% Tween80 solution prepared in step (1), stir well for 30 min until the solution becomes uniform, then 0.2% UV protectant SH solution is obtained;

[0194] (3) Mix the 0.2% UV protectant SH solution prepared in step (2) and the bacterial solution of Bemisia tabaci dsRNA in a volume ratio of 1:1, then the dsRNA bacterial solution of Bemisia tabaci can be prevented from being degraded by ultraviolet light.

[0195] Example 29

[0196] The Bemisia tabaci biological control method based on 0.2% UV protectant SH is as follows:

[0197] (1) Using 0.1% (volume fraction) aqueous Tween80 solution as a solvent, prepare sodium humate into a solution with a concentration of 0.2% (mass fraction) as 1% UV protectant SH solution.

[0198] (2) Mix the 0.2% UV protectant SH solution and the bacterial solution of transcribed dsGawky (i.e., the bacterial solution of transcribed dsRNA of Bemisia tabaci Gawky gene) with a concentration of 2 g / L in a volume ratio of 1:1, and after shaking and mixing evenly, the mixture of 0.2% UV protectant SH and the bacterial solution of transcribed dsGawky for RNA interference of Bemisia tabaci is obtained.

[0199] (3) Spray the above mixture of 0.2% UV protectant SH and the bacterial solution of transcribed dsGawky on plants, so that Bemisia tabaci feeds on the sprayed plants, thereby increasing the mortality rate of Bemisia tabaci.

[0200] The preparation method of the bacterial solution of the transcribed dsGawky is the same as that in Example 3.

[0201] Example 30

[0202] Lethal effect of spraying the bacterial solution of transcription dsGawky bacteria or the mixture of 0.2% UV protectant SH and transcription dsGawky bacteria indoors on adult Q-biotype Bemisia tabaci;

[0203] The transcription dsGawky bacteria solution prepared in Example 29 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, with 3 replicates for each treatment. They were placed in an incubator without sunlight, at a temperature of 27±1°C, a relative humidity of 60%±5%, and a light cycle of 16L:8D. The adult mortality rate was counted 7 days after spraying.

[0204] The mixture of 0.2% UV protectant SH and transcription dsGawky bacteria prepared in Example 29 was sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, with 3 replicates for each treatment. They were placed in an incubator without sunlight, at a temperature of 27±1°C, a relative humidity of 60%±5%, and a light cycle of 16L:8D. The adult mortality rate was counted 7 days after spraying.

[0205] The results are as Figure 19 shown. Under indoor conditions, the lethal rate of spraying the transcription dsGawky bacteria solution on adult Q-biotype Bemisia tabaci was 53.9%, and the lethal rate of spraying the mixture of 0.2% UV protectant SH and transcription dsGawky bacteria on adult Q-biotype Bemisia tabaci was 49.5%.

[0206] Example 31

[0207] Lethal effect of spraying the bacterial solution of transcription dsGawky bacteria or the mixture of 0.2% UV protectant SH and transcription dsGawky bacteria outdoors on adult Q-biotype Bemisia tabaci;

[0208] The transcription dsGawky bacteria solution prepared in Example 29 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, with 3 replicates for each treatment, and placed outdoors with sunlight. The adult mortality rate was counted 7 days after spraying.

[0209] The mixture of 0.2% UV protectant SH prepared in Example 29 and the bacteria solution of transcribed dsGawky was sprayed onto cotton plants, with each side of the cotton leaves sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants, and there were 3 replicates for each treatment. They were placed outdoors with sunlight. The adult mortality was counted 7 days after spraying.

[0210] The results are as Figure 20 shown. Under outdoor conditions, the lethality rate of the bacteria solution of transcribed dsGawky to adult Q-biotype Bemisia tabaci was 18%, and the lethality rate of the mixture of 0.2% UV protectant SH and the bacteria solution of transcribed dsGawky to adult Q-biotype Bemisia tabaci was 33%. There was a significant difference between the two.

[0211] Example 32

[0212] Application of sodium humate in preventing ultraviolet degradation of dsRNA bacteria solution

[0213] (1) Using an aqueous solution of 0.1% (volume fraction) Tween 80 as a solvent, sodium humate was prepared into a solution with a concentration of 5% (mass fraction) as a 5% UV protectant SH solution.

[0214] (2) Mixing the 5% UV protectant SH solution and the dsRNA bacteria solution in a volume ratio of 1:1 can prevent the dsRNA bacteria solution from being degraded by ultraviolet light.

[0215] Example 33

[0216] A method for preventing ultraviolet degradation of Bemisia tabaci dsRNA bacteria solution

[0217] (1) Disperse 0.2 mL of Tween 80 in 199.8 mL of water and stir evenly to obtain a 0.1% Tween 80 solution as a solvent;

[0218] (2) Take 5 g of sodium humate and disperse it in 95 mL of the 0.1% Tween 80 solution prepared in step (1), and stir well for 30 min until the solution becomes uniform to obtain a 5% UV protectant SH solution;

[0219] (3) Mix the 5% UV protectant SH solution prepared in step (2) with the bacteria solution of Bemisia tabaci dsRNA in a volume ratio of 1:1 to prevent the Bemisia tabaci dsRNA bacteria solution from being degraded by ultraviolet light.

[0220] Example 34

[0221] The Bemisia tabaci biological control method based on 5% UV protectant SH is as follows:

[0222] (1) Using an aqueous solution of 0.1% (volume fraction) Tween 80 as a solvent, sodium humate was prepared into a solution with a concentration of 5% (mass fraction) as a 1% UV protectant SH solution.

[0223] (2) The 5% UV protectant SH solution was mixed with the solution of the bacteria body for transcribing dsGawky (i.e., the bacteria body for transcribing dsRNA of the Bemisia tabaci Gawky gene) (concentration: 2 g / L) at a volume ratio of 1:1, and after shaking and mixing evenly, a mixed solution of 0.2% UV protectant SH and the bacteria body for transcribing dsGawky for RNAi interference of Bemisia tabaci was obtained.

[0224] (3) The above-mentioned mixed solution of 5% UV protectant SH and the bacteria body for transcribing dsGawky was sprayed on plants, enabling Bemisia tabaci to feed on the sprayed plants, thereby increasing the mortality rate of Bemisia tabaci.

[0225] The preparation method of the solution of the bacteria body for transcribing dsGawky is the same as that in Example 3.

[0226] Example 35

[0227] Lethal effects of spraying the bacterial solution of the bacteria body for transcribing dsGawky or the mixed solution of 5% UV protectant SH and the bacteria body for transcribing dsGawky on adult Q-type Bemisia tabaci indoors;

[0228] The bacterial solution of the bacteria body for transcribing dsGawky prepared in Example 34 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria body), and then sprayed onto cotton plants. Spray 3 times on both the front and back of the cotton leaves. After the solution dries, release 30 adult Q-type Bemisia tabaci on the cotton plants, with 3 replicates for each treatment. Place them in a constant temperature incubator without sunlight, at a temperature of 27 ± 1°C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. After spraying for 7 days, count the results of adult mortality.

[0229] The mixed solution of 5% UV protectant SH and the bacteria body for transcribing dsGawky prepared in Example 34 was sprayed onto cotton plants. Spray 3 times on both the front and back of the cotton leaves. After the solution dries, release 30 adult Q-type Bemisia tabaci on the cotton plants, with 3 replicates for each treatment. Place them in a constant temperature incubator without sunlight, at a temperature of 27 ± 1°C, a relative humidity of 60% ± 5%, and a light cycle of 16L:8D. After spraying for 7 days, count the results of adult mortality.

[0230] The results are as Figure 21 shown. Under indoor conditions, the lethal rate of spraying the bacterial solution of the bacteria body for transcribing dsGawky on adult Q-type Bemisia tabaci was 53.9%, and the lethal rate of spraying the mixed solution of 5% UV protectant SH and the bacteria body for transcribing dsGawky on adult Q-type Bemisia tabaci was 52.6%.

[0231] Example 36

[0232] Lethal effect of spraying the bacterial solution of transcribed dsGawky bacteria or the mixture of 5% UV protectant SH and transcribed dsGawky bacteria outdoors on adult Q-biotype Bemisia tabaci;

[0233] The transcribed dsGawky bacterial solution prepared in Example 34 was dissolved and diluted with ddH2O to a concentration of 2 g / L of the bacterial solution (wet bacteria), and then sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants. There were 3 replicates for each treatment and they were placed outdoors with sunlight. The adult mortality rate was counted 7 days after spraying.

[0234] The mixture of 5% UV protectant SH and transcribed dsGawky bacteria prepared in Example 34 was sprayed onto cotton plants. Each side of the cotton leaves was sprayed 3 times. After the solution dried, 30 adult Q-biotype Bemisia tabaci were released onto the cotton plants. There were 3 replicates for each treatment. They were placed outdoors with sunlight. The adult mortality rate was counted 7 days after spraying.

[0235] The results are as Figure 22 shown. Under outdoor conditions, the lethal rate of spraying the transcribed dsGawky bacterial solution on adult Q-biotype Bemisia tabaci was 18%, and the lethal rate of spraying the mixture of 5% UV protectant SH and transcribed dsGawky bacteria on adult Q-biotype Bemisia tabaci was 64.8%. There was a significant difference between the two.

[0236] Spraying different mixtures of protectants and transcribed dsGawky bacteria on cotton under outdoor conditions, the lethal effect on adult Q-biotype Bemisia tabaci 7 days later: The control effects of 0.2% UV protectant KH and 0.2% UV protectant SH did not meet expectations. Although the control effects of 5% UV protectant KH and 5% UV protectant SH were better than those of 1% UV protectant KH and 1% UV protectant SH, the material costs were higher. Considering factors such as cost, 1% UV protectant KH and 1% UV protectant SH were selected.

[0237] The above are only the preferred embodiments of the present invention, and are not limitations to the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for avoiding the degradation of dsRNA bacterial solution by ultraviolet light, characterized in that, Mix the dsRNA bacterial solution with a UV protectant to prepare a mixed solution of the UV protectant and the dsRNA bacterial solution; the UV protectant is one of potassium humate and sodium humate.

2. Application of the method for preventing the dsRNA bacterial solution from being degraded by ultraviolet light as described in claim 1 in the control of agricultural pests by RNA interference.

3. A biological agent for controlling Bemisia tabaci, characterized in that, A biological agent comprising a UV protectant and a dsRNA bacterial solution that interferes with the Gawky gene of Bemisia tabaci; the UV protectant is one of potassium humate and sodium humate.

4. The biological agent for preventing and controlling Bemisia tabaci according to claim 3, wherein, The dsRNA bacterial solution that interferes with the Gawky gene of Bemisia tabaci is a bacterial solution transcribed with the dsRNA of the Gawky gene of Bemisia tabaci.

5. The biological agent for controlling Bemisia tabaci according to claim 4, wherein The bacterial solution transcribed with the dsRNA of the Gawky gene of Bemisia tabaci is prepared by the following method: Amplify the dsRNA sequence of the Gawky gene using primers, ligate it to an expression vector to construct a recombinant vector, and transform the expression strain; induce the expression strain to transcribe the dsRNA sequence of the Gawky gene, discard the supernatant of the bacterial solution, and dissolve and dilute it with ddH2O to obtain the product.

6. The biological agent for controlling Bemisia tabaci according to claim 5, characterized in that, The method for inducing the expression strain to transcribe the dsRNA sequence of the Gawky gene is as follows: The recombinant bacteria transformed with the recombinant vector expressing dsRNA of Bemisia tabaci Gawky gene were inoculated into LB medium containing Amp, and cultured with shaking at 37 °C and 20 r / min. When the OD 600 value reached 0.5, an IPTG solution with a final concentration of 0.5 M was added for induction for 4 h to obtain the product.

7. The biological agent for controlling Bemisia tabaci according to any one of claims 3 to 6, characterized in that, The preparation method is as follows: (1) Disperse the UV protectant in a 0.1% aqueous solution of Tween 80 to prepare a UV protectant solution with a concentration of 0.2% - 5%. (2) Mix the UV protectant solution and the bacterial solution transcribed with the dsRNA of the Gawky gene of Bemisia tabaci according to a volume ratio of 1:1, and mix well by shaking to obtain a biological agent for controlling Bemisia tabaci.

8. Application of the biological agent for controlling Bemisia tabaci as described in claim 7 in the biological control of Bemisia tabaci.

9. A biological control method for Bemisia tabaci, characterized in that, Spray the plant with the biological agent for controlling Bemisia tabaci as described in claim 7, so that Bemisia tabaci feeds on the sprayed plant, thereby increasing the mortality rate of Bemisia tabaci.

10. The Bemisia tabaci biological control method according to claim 9, characterized in that, The Bemisia tabaci is Q-type Bemisia tabaci or B-type Bemisia tabaci.

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