Rhodovulum sulfidophilum for Controlling Rice Planthoppers

Abstract

The present patent discloses a method for controlling rice planthoppers by using Rhodovulum sulfidophilum, which is characterized by applying a Rhodovulum sulfidophilum preparation to the rice growth environment. The Rhodovulum sulfidophilum preparation is prepared by inoculating the Rhodovulum sulfidophilum strain IV-1 into a culture medium and culturing it. During the occurrence period of low-instar nymphs of rice planthoppers, the Rhodovulum sulfidophilum preparation is applied to the rice growth environment at a dosage of 100-150 ml per mu, and the application method is to spray evenly after diluting with 30-45 liters of water per mu. Experiments have proved that it has a fast onset and good long-term efficacy against rice planthoppers, and has excellent control effects.

Description

Technical Field

[0001] This patent relates to the biological control of rice planthoppers, and more specifically to a biological product and method for controlling rice planthoppers using bacteria. Background Art

[0002] Rice planthoppers belong to the Delphacidae family of the order Homoptera, commonly known as midge bugs, fire midge bugs, and clicking bugs, and damage crops such as rice by sucking the sap of plants. There are mainly three types of planthoppers that damage rice in China: the brown planthopper (Nilaparvata lugens Stal), the white-backed planthopper (Sogatella furcifera (Horvath)), and the small brown planthopper (Laodelphax striatellus Fallén). Among them, the brown planthopper causes the most serious damage, followed by the white-backed planthopper. On September 15, 2020, rice planthoppers were included in the list of first-class crop pests and diseases by the Ministry of Agriculture and Rural Affairs. Chemical control methods are usually used to control rice planthoppers, such as buprofezin, imidacloprid and other agents. Chemical control uses the biological activity of pesticides to reduce harmful biological populations below the level of economic loss. It has a killing effect on harmful organisms and a regulatory and inhibitory effect on their growth and development. However, long-term use will pollute the environment and cause phytotoxicity.

[0003] Considering the deficiencies of chemical control, in recent years, biological control has gradually been adopted to control various pests including rice planthoppers, such as Metarhizium anisopliae. The essence of biological control is to utilize the mutual relationship between species and use one organism to inhibit another organism. Therefore, the environmental pollution is relatively small. Biological control can effectively protect natural enemies, play a continuous role in disaster control, is safe for humans and livestock, and pests are not likely to develop resistance. Usually, the disadvantages are low efficiency, slow insecticidal effect, and the drug effect is not as fast as chemical pesticides. Therefore, there is an urgent need to develop a biological product and method that can effectively control rice planthoppers.

[0004] Chinese Patent CN103525729(A) discloses the strain Rhodovulum sulfidophilum IV-1, which is deposited in the China Center for Type Culture Collection (abbreviated as CCTCC), with the deposit number CCTCC NO: M2012369. The address of the depository is located at Wuhan University, China, and the deposit date is September 20, 2012. The Rhodovulum sulfidophilum strain IV-1 has the following main characteristics: G-, the individual morphology is egg-shaped rod, V-P reaction is positive, methyl red reaction is negative, H2S is positive, indole reaction and catalase reaction are positive, it can grow in 3% NaCl by mass concentration, the optimal growth temperature is 30-35 °C, the optimal growth pH value is 6.5-7.5, the characteristic absorption peaks of the bacterial cells are 385 nm, 525 nm, 585 nm, 805 nm and 865 nm, the length of the 16S rDNA sequence is 1338 bp, and the accession number in Genbank is KF682218. It is reported that its bacterial liquid can promote the growth of rapeseed efficiently, non-toxic and environmentally friendly, and also improve the germination rate of rapeseed seeds. The paper "Research and Application of 200 million CFU / mL Rhodovulum sulfidophilum HNI-1 Suspension" by Fu Wei et al. (Chinese Journal of Biological Control, 36(6) 866-868, December 2020) discloses its application in tomato root-knot nematodes and tomato mosaic virus disease. For the control of tomato root-knot nematodes, irrigation of the roots is carried out during tomato transplantation, 2-3 times per season, with a dosage of 400-600 mL / 667m 2 ; for the control of tomato mosaic virus disease, spraying is selected before or at the initial stage of the disease, 2-3 times per season, with a dosage of 180-240 mL / 667m 2 . No phytotoxicity was observed after use; it has low toxicity or no effect on predatory natural enemies and parasitic natural enemies. According to reports, the action mechanism of Rhodovulum sulfidophilum HNI-1 includes: the metabolites have nematocidal active substances, such as 5-aminolevulinic acid, one of the metabolic active substances, which has good toxicicidal effects on plant parasitic nematodes. In addition, after being absorbed by plants, 5-aminolevulinic acid can promote the synthesis of chlorophyll in plants, increase plant photosynthesis, promote plant growth and healthy seedlings, thereby enhancing the resistance of plants to nematodes. Summary of the Invention

[0005] The object of the present invention is to provide a method for controlling rice planthoppers based on Rhodovulum sulfidophilum preparations. Another object of the present invention is to provide the application of Rhodovulum sulfidophilum preparations in controlling rice planthoppers.

[0006] The present invention is realized through the following technical solutions: A method for controlling rice planthoppers, characterized in that a Thiobacillus thiooxidans preparation is applied to the rice growth environment, such as the soil, water body, areas around plants and other areas where rice planthoppers are active. The Thiobacillus thiooxidans preparation is made by inoculating the Thiobacillus thiooxidans strain IV-1 into a culture medium for cultivation. Preferably, the Thiobacillus thiooxidans preparation is made by inoculating the Thiobacillus thiooxidans strain IV-1 into a liquid culture medium for cultivation. The Thiobacillus thiooxidans preparation is a suspension, and the bacterial concentration in the suspension is 100 million to 500 million CFU, that is, 1×10 8 cfu / mL to 5×10 8 cfu / mL, preferably 1×10

[0007] cfu / mL to 3×10 Specific embodiments

[0008] Example 1 Preparation of Thiobacillus thiooxidans suspension

[0009] The preparation method specifically includes the following steps:

[0010] a. The cryopreserved strain of Thiobacillus thiooxidans strain IV-1 is cultured according to the plate culture method until red colonies appear. A double-layer MSYN solid medium is used, the pH value is controlled at 7.0 - 7.2, the culture temperature is controlled at 30℃ - 35℃, and the light condition is controlled at 2000 - 3000 Lx.

[0011] b. The single colony in the red colonies obtained in step a is inoculated into a serum bottle and cultured to the logarithmic growth phase with the seed medium of Thiobacillus thiooxidans strain IV-1. The seed medium uses MSYN liquid medium, the pH value is controlled at 7.0 - 7.2, the culture temperature is controlled at 30 - 35℃, and the light condition is controlled at 2000 - 3000 Lx.

[0012] c. The bacterial liquid obtained after culturing in step b is inoculated into a production bottle at an inoculation amount of not less than 1% (preferably 1 - 5%) of the total volume of the production medium, and cultured to the logarithmic growth phase with the production medium of Thiobacillus thiooxidans strain IV-1. The production medium uses MSYN liquid medium, the pH value is controlled at 7.0 - 7.2, the culture temperature is controlled at 30 - 35℃, and the light condition is controlled at 2000 - 3000 Lx.

[0013] d. Collect the off-white viscous liquid culture product obtained in step c, remove impurities, and then divide and package it to obtain a suspension of Rhodovulum sulfidophilum. After serial dilution, culture it and perform colony counting. Appropriately dilute it to adjust the number of bacteria per unit volume of products from different batches to the set specification, such as 200 million CFU.

[0014] The total process time of the above preparation method is generally about 16 - 20 days. Colony counting after the end of the culture shows that the number of bacteria can reach more than 500 million per ml.

[0015] Example 2: Test on controlling rice planthoppers

[0016] This test refers to "Pesticide Field Efficacy Test Guidelines (Part 1)" GB / T 17980.4 - 2000, Part 4: Insecticides for Controlling Rice Planthoppers, and is carried out strictly in accordance with Announcement No. 2570 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China, the test plan, and relevant SOPs.

[0017]

[0018]

[0019] The test plot is set in a certain village in Changsha City, Hunan Province. Rice is planted in the test plot all year round. The irrigation and drainage are convenient. The soil has medium fertility, the soil pH is 6.1, and the organic matter content is 35.68 g / kg.

[0020] The test crop is rice, with the variety Taiyou 390, an indica three-line hybrid rice. The whole growth period is about 118.5 days. It was transplanted on June 18, 2021, and the transplanting specification is 20 cm × 20 cm.

[0021] The test target is rice planthopper (Nilaparvata lugens Stal).

[0022] Test method: Set 5 treatments, and the area of each treatment plot is 20 m 2 , and the field plots are arranged in a randomized block design. Ridges are set between plots, and a 50-cm buffer zone is set between replicates. There are 4 replicates.

[0023] Treatment number Agent treatment Dosage of preparation 1 200 million CFU Rhodovulum sulfidophilum HNI-1 suspension 100 mL / mu 2 200 million CFU Rhodovulum sulfidophilum HNI-1 suspension 150 mL / mu 3 200 million CFU Rhodovulum sulfidophilum HNI-1 suspension 200 mL / mu 4 8 billion spores / g Metarhizium anisopliae CQMa421 wettable powder 300 g / mu 5 Blank control /

[0024] According to the dosage of the medicament and the plot area in the treatment design, calculate the dosage of the sample preparation and the amount of the liquid medicine for each plot. Set the safety factor to 2, that is, the amount of the medicament preparation and the amount of the liquid medicine added to the sprayer during the application of each plot are 200% of the theoretical dosage. Weigh the test samples directly with an electronic balance / pipette, place them in a measuring cup, make up the volume to the amount of the liquid medicine required for the plot, and stir evenly with a glass rod. The actual sample volume and the amount of the liquid medicine taken for each plot are shown in the following table.

[0025] Treatment number Name of test sample Sampling volume of plot sample Volume of prepared liquid in plot (mL) 1 Test agent 6.0 mL 1800 2 Test agent 9.0 mL 1800 3 Test agent 12.0 mL 1800 4 Control agent 18.0g 1800 5 Blank control / 1800

[0026] Use the Singapore Lignon HD400 knapsack hand sprayer to apply the pesticide once during the occurrence period of the low-instar nymphs of rice planthoppers. The growth stage of the rice at the time of pesticide application is the booting stage. The water consumption is 450 liters per hectare. Conduct a base number survey before applying the pesticide, and investigate the control effect 1 time each at 3 days, 7 days, 14 days, and 21 days after applying the pesticide, for a total of 5 investigations. Use the parallel jumping method to investigate 10 points in each plot, with 2 clusters at each point. Pat the rice clusters and count the number of planthoppers floating on the water surface between the rice clusters, and calculate the reduction rate to statistically analyze the control effect. At 7 days after applying the pesticide, observe the growth of rice in each plot by visual inspection. During the test period, the rice in the test chemical treatment plots grew normally, and no phytotoxicity was found; nor were any adverse effects of the test chemical on wild organisms, fish, and beneficial insects found, which was basically in line with the reported situation.

[0027] According to the "Pesticide Field Efficacy Test Guidelines (Part 2)" GB / T 17980.4 - 2004, the control effect calculation method is as follows:

[0028]

[0029]

[0030] The results of the field efficacy test of the test chemical and the comparative chemical against rice planthoppers are shown in the following table.

[0031]

[0032] Note: The control effect (%) in the table is the average value of each repetition; data with the same letter in the same column are not significantly different.

[0033] Analysis of variance method: Use the DPS Duncan's new multiple range method (DMRT).

[0034] The test results showed that 3 days after application, the control efficacy of the test agents with the active ingredient dosages of 100, 150, and 200 mL / mu against rice planthoppers were 52.5%, 55.5%, and 56.3% respectively, and the control efficacy of the control agent treatment was 43.5%. 7 days after application, the control efficacy of each test agent treatment against rice planthoppers was significantly improved. The control efficacy of each dosage treatment of the test agents was between 73.8% and 76.3%, which was higher than the control efficacy of 69.0% of the control agent treatment. 14 days after application, the control efficacy of each test agent treatment against rice planthoppers reached the highest level. The control efficacy of the test agents against rice planthoppers were 91.3%, 93.3%, and 94.3% respectively, and the control efficacy of the control agent treatment was 90.8%. The results of variance analysis showed that there was no significant difference in the control efficacy between the test agent dosage of 100 mL / mu and the control agent treatment, while the control efficacy of the test agent dosages of 150 and 200 mL / mu was significantly higher than that of the control agent. 21 days after application, the control efficacy of the test agents with dosages of 100, 150, and 200 mL / mu against rice planthoppers was between 90.4% and 93.1%, and the control efficacy of the control agent treatment was 89.7%. The results of variance analysis showed that there was no significant difference in the control efficacy between the test agent dosage of 100 mL / mu and the control agent treatment, while the control efficacy of the test agent dosages of 150 and 200 mL / mu was significantly higher than that of the control agent treatment.

[0035] Thus, it can be seen that the suspension agent of Rhodovulum sulfidophilum HNI-1 has a fast onset of action in controlling rice planthoppers. The control efficacy can reach 52.5% 3 days after application; the control efficacy against rice planthoppers reaches the highest level about 14 days after application, exceeding 94% at most; the control efficacy against rice planthoppers slightly decreases 21 days after application, but still exceeds 94%. It can be seen that its persistence is relatively good. Generally speaking, it has excellent control efficacy, which is comparable to that of commercial biological agents.

Claims

1. A method for controlling rice planthoppers, characterized in that Apply the Thiobacillus rhodophilus preparation to the rice growth environment during the occurrence period of the low-instar nymphs of the rice planthopper; the Thiobacillus rhodophilus preparation is prepared by inoculating the Thiobacillus rhodophilus strain IV-1 into a culture medium and culturing it; the preservation number of the Thiobacillus rhodophilus strain IV-1 is CCTCC NO: M2012369.

2. The method for controlling rice planthoppers according to claim 1, characterized in that The Thiobacillus rhodophilus preparation is prepared by inoculating the Thiobacillus rhodophilus strain IV-1 into a liquid culture medium and culturing it.

3. The method for controlling rice planthoppers according to claim 1, characterized in that The Thiobacillus rhodophilus preparation is a suspending agent, and the bacterial concentration in the suspending agent is 100 million - 500 million CFU / mL.

4. The method for controlling rice planthoppers according to claim 3, characterized in that The bacterial concentration of the Rhodovulum sulfidophilum preparation suspension is 1 - 3 х 10 8 CFU / mL.

5. The method for controlling rice planthoppers according to claim 4, characterized in that The cell concentration of the Rhodovulum sulfidophilum preparation suspension is 2х10 8 CFU / mL.

6. The method for controlling rice planthoppers according to claim 5, characterized in that The dosage of the Thiobacillus rhodophilus preparation suspending agent is 100 - 150 mL / mu, and the application method is to spray evenly after diluting with 30 - 45 L / mu of water.

7. The method for controlling rice planthoppers according to claim 2, characterized in that The total process culture time of the Thiobacillus rhodophilus preparation is 16 - 20 days.

8. Application of the Rhodovulum sulfidophilum preparation in controlling rice planthoppers during the occurrence period of low-instar nymphs of rice planthoppers, wherein the Rhodovulum sulfidophilum preparation is prepared by inoculating the Rhodovulum sulfidophilum IV-1 strain with the preservation number of CCTCC NO: M2012369 into a liquid medium for cultivation.

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