A kind of Bacillus fusinus with effect of preventing and controlling cabbage aphids

Abstract

The present invention provides a Bacillus orientalis with the function of preventing and controlling cabbage aphids, and the deposit number thereof is CGMCC No. 28561. The Bacillus orientalis HXBW-2 strain screened by the present invention can alleviate plant pests caused by cabbage aphids, has good insecticidal effects both indoors and outdoors, and has development potential.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganism screening, and particularly relates to a Bacillus thunbergii with the effect of preventing and controlling cabbage aphids. Background Art

[0002] Aphids are a general term for the superfamily Aphidoidea of ​​the order Hemiptera, including all members of the superfamily Aphidoidea. Currently, there are 10 families and about 4,400 species of aphids discovered, most of which belong to the family Aphididae. It has been found that different plants have corresponding aphid species, for example, the pine aphid is the main pest of conifers; the polymorphic hairy aphid of Koelreuteria paniculata mainly harms Koelreuteria paniculata; the yellow apple aphid mainly harms apples, crabapples, papayas, heather, hawthorns, etc.; the host plants of peach aphids are mainly peach, pear, plum, plum, cherry and other Rosaceae fruit trees.

[0003] Cabbage aphid (Brevicoryne brassicae Linnaeus) is an important pest on vegetables, mainly affecting cruciferous plants. Cabbage aphids generally feed on the sap from leaves and stems, stealing nutrients for plant growth, causing the leaves to curl, shrink and deform, slowing down the normal growth of plants.

[0004] At present, the prevention and control of cabbage aphid disease is still mainly chemical control. The extensive use of chemical agents has posed a huge threat to environmental safety and human and animal health. As a result, many chemical pesticides have been restricted or banned. It is urgent to find environmentally friendly, safe and pollution-free control methods. As an environmentally friendly alternative to chemical agents, biocontrol agents have gradually attracted people's attention. Biocontrol bacteria have received widespread attention due to their high safety, easy cultivation and production. It is reported that Verticillium lecanii, Aspergillus niger and Paecilomyces roseus have certain control effects on aphids, but their application to cabbage aphids is not effective. Summary of the invention

[0005] The invention aims to provide a Bacillus orientalis with the function of preventing and controlling cabbage aphids, which can biologically prevent and control cabbage aphids, thereby effectively preventing and controlling the disease losses caused by cabbage aphids.

[0006] The Bacillus toyonensis HXBW-2 strain (Bacillus toyonensis) provided by the present invention has a preservation number of CGMCC No. 28561, a preservation date of September 26, 2023, and a preservation unit of the General Microbiology Center of the China Culture Collection Administration, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides a use of the Bacillus toyota HXBW-2 strain, which is the use in preparing products for preventing and controlling plant pests.

[0008] The plant pests are caused by cabbage aphids.

[0009] The present invention also provides a product for alleviating plant insect pests, wherein the product contains live bacteria and / or fermentation products of the HXBW-2 strain of Bacillus sp.

[0010] The product is a bacterial liquid product.

[0011] The Bacillus toyota HXBW-2 strain screened by the present invention can alleviate plant pests caused by cabbage aphids, has good insecticidal effects both indoors and outdoors, and has development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :Strain colony morphology characteristics;

[0013] Figure 2 :Strain staining characteristics;

[0014] Figure 3 : Phylogenetic tree diagram;

[0015] Figure 4 : Heat map generated from the whole genome of the strain;

[0016] Figure 5 :The situation of aphids 3 days after using microbial agents, where A is before use and B is the result 3 days after use. DETAILED DESCRIPTION

[0017] The present invention extracts 76 pure colonies from the rhizosphere soil of plants that often suffer from aphid diseases, one of which has a strong effect of killing cabbage aphids. The strain is identified as Bacillus toyonensis through 16SrRNA sequencing. The bacteria screened by the method based on the biological preparation of the present invention have a good control effect on cabbage aphids and can be widely used in the control of cabbage aphids.

[0018] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0019] Example 1: Isolation and screening of strains

[0020] 1.1 Isolation of bacterial strains from soil

[0021] Weigh 10 g of soil sample (rhizosphere soil in a solar greenhouse where aphids occur all year round) and put it into a 250 mL sterile triangular flask filled with 90 mL sterile water. Seal the flask with a sealing film. Place the triangular flask at 25 °C and 200 rpm. -1 After shaking evenly, dilute the soil suspension with sterile water on a clean bench to obtain 10 -1 -10 -5Take 100 μL of soil suspension of different concentrations, apply it evenly on NA medium with an applicator, place it in a 25℃ constant temperature incubator, and culture it in the dark for 48 hours. After obvious colonies grow, take a single colony for purification. Set up 3 replicates for each concentration of soil suspension.

[0022] 1.2 Screening of bacteria with aphidicide activity

[0023] Pick a single bacterial colony and place it in a 250 mL Erlenmeyer flask containing 100 mL NA liquid medium (2 × 10 9 CFUmL -1 ), 25℃, 150rmin -1 Shake on a shaker under the same conditions for 48 hours. After taking out, centrifuge the fermentation liquid in a 10000rpm centrifuge for 10 minutes, take the supernatant, and then filter it with a 0.45μm filter membrane to obtain the filtrate for use. Add 500μL of the pre-prepared cabbage aphid solution (containing approximately 100 cabbage aphids) to a 12-well plate, and then add 500, 200, and 100μL of bacterial fermentation liquid respectively, and make up to 1mL with sterile water. Prepare the fermentation liquid into 2-fold, 5-fold, and 10-fold dilutions, and determine the toxicity to southern cabbage aphids. Check the results under a stereoscope after 12 hours. The aphid body is stiff and motionless, and it is considered dead if it does not recover after being transferred to clean water for 12 hours. NA liquid culture medium without bacterial solution was used as a control. The experiment was repeated 3 times, and each repeat contained 3 wells. The formula for calculating aphid mortality and corrected mortality is as follows:

[0024] Mortality (%) = number of dead aphids / total number of aphids × 100.

[0025] Corrected mortality rate (%) = (% mortality rate of treatment group - % mortality rate of control group) / (100 - % mortality rate of control group) × 100

[0026] The aphidicide activity (NA) was classified according to the following standards: NA ≥ 80%, 50% ≤ NA < 80%, 20% ≤ NA < 50% and NA < 20% were respectively regarded as having strong aphidicide activity, moderate aphidicide activity, weak aphidicide activity and no aphidicide activity.

[0027] A total of 76 strains were isolated from the rhizosphere soil, of which HXBW-2, HXBW-05, HXBW-27, HXBW-31 and HXBW-53, 5 strains had aphidicide activity. The one with strong aphidicide activity was HXBW-2, whose fermentation liquid had a 96.3% aphid mortality rate at 2-fold dilution concentration. HXBW-27 had moderate aphidicide activity, with a 56.7% aphid mortality rate. HXBW-05, HXBW-31 and HXBW-53 showed weak aphidicide activity.

[0028] Table 1: Toxicity of bacterial fermentation broth to cabbage aphids

[0029]

[0030] Example 2: Identification of strains

[0031] 2.1 Morphological characteristics of strains

[0032] The pure cultured HXBW-2 strain was streaked on a NA medium plate and cultured in a 25°C incubator for 48 hours. After obvious colonies grew, the colony morphology, color, transparency, dryness, surface smoothness and edge morphology were observed. Fresh bacteria were taken for Gram staining and microscopic examination.

[0033] Depend on Figure 1 The colonies of HXBW-2 strain were observed to be nearly round, matte white, with wrinkled surface and irregular edges. The colonies became larger over time, showing swimming and self-aggregation abilities. The staining results showed that HXBW-2 was a Gram-positive short rod-shaped bacterium ( Figure 2 ).

[0034] 2.2 Physicochemical properties of strains

[0035] After the bacteria were cultured on the culture medium for a period of time, various physiological and biochemical indices were measured, including catalase test, Priscal (VP) test, starch hydrolysis, gelatin liquefaction, hydrogen sulfide, nitrate reduction, glucose oxidation fermentation and sucrose fermentation tests.

[0036] As shown in Table 2 and Table 3, the strain showed positive reactions in arginine, VP gelatin, sucrose fermentation, and amygdalin tests. In the carbon source utilization experiment, the results of glycerol, ribose, glucose, fructose, mannose, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salvinol, cellobiose, maltose, sucrose, trehalose, starch, and glycogen were positive.

[0037] Manual of Systematic Identification of Common Bacteria, consistent with the characteristics of Bacillus.

[0038] Table 2: Physiological and biochemical characteristics of strain HXBW-2 – enzyme activity, carbon source oxidation table

[0039]

[0040]

[0041] +: positive reaction; -: negative reaction;

[0042] Table 3: Carbon source utilization information of strain HXBW-2

[0043]

[0044]

[0045] +: positive reaction; -: negative reaction;

[0046] 2.3 Strain 16S rRNA gene sequence

[0047] The HXBW-2 strain obtained by screening was identified by 16S rDNA sequencing. The sequencing results were searched for similar sequences using the Blast alignment system in the NCBI data and a phylogenetic tree was constructed. Figure 3 The phylogenetic tree showed that strain HXBW-2 belonged to the genus Bacillus. Figure 4 It can be seen that the ANI value of the strain HXBW-2 was 98.43% when the whole genome data was compared with the model strain Bacillustoyonensis (BCT-7112), which was greater than the ANI threshold of 96% for isotype judgment. Therefore, the strain HXBW-2 was identified as Bacillustoyonensis.

[0048] The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 28561 and the deposit date September 26, 2023; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0049] Example 3: Indoor insecticidal effect of HXBW-2 on aphids

[0050] Cabbage aphids collected in the laboratory were used as test pests. The cabbage aphids were taken back with their stems and leaves and placed in a petri dish containing paper. The aphids with normal vitality and activity were counted and dead or weak aphids were picked out to ensure that the number of aphids in each dish was greater than 100. The cabbage aphids were divided into 7 groups, namely the experimental group (2×10 9 cfu / mL, medium and high dose groups 1×10 9 cfu / mL, medium dose group 1×10 8 cfu / mL, medium and low dose groups 4×10 7 cfu / mL, low dose group 2×10 7 cfu / mL), control group (bio group 2×10 9 cfu / mL) and blank control group (ddH2O).

[0051] Purchase the finished product of Bacillus toyota (No. bio118045) freeze-dried powder from the strain library, activate it according to the instructions, and culture it at 30℃ until 2×10 9 cfu / mL for future use.

[0052] After the HXBW-2 strain fermentation liquid was diluted 10 times, 20 times, 50 times, and 100 times, the original bacterial liquid and the diluted liquid were used for indoor quantitative spraying insecticidal test, and 1‰ of Tween 20 was added as a dispersant. A certain number of surviving aphids were placed on the leaves of indoor culture dishes to keep them moist, with 100 aphids per dish. The fermentation liquid of the strains at different concentrations was evenly sprayed on the cabbage aphids until the surface of the insect body was moist. The finished product of Bacillus thuringiensis was used as the control group, and water was used as the blank control group. The test was repeated 3 times. After spraying, the culture dishes were sealed with plastic wrap, and a dozen small holes were poked with a needle to ensure air circulation. The aphid mortality rates at 2h, 8h, 24h, and 48h were recorded.

[0053] The concentration of the bacterial solution was counted by the dilution plate method, and the concentrations of the original bacterial solution and the dilution solution were recorded as follows: 2×10 9 , 1×10 9 , 1×10 8 , 4×10 7 , 2×10 7 cfu / mL.

[0054] Mortality rate (%) = number of dead insects (heads) × 100 / total number of insects treated (heads)

[0055] Table 4: Aphid mortality statistics

[0056]

[0057]

[0058] The HXBW-2 strain fermentation broth was counted by dilution plate method, and the concentration of the broth was 2×10 9 cfu / mL. After the bacterial solution was evenly sprayed on the surface of cabbage aphids, the death of aphids was observed. The surface of aphids was lightly touched with a brush. If there was no obvious physiological reaction, it indicated that the aphids were dead. As time went by (Table 4), the higher the aphid mortality rate, the higher the 2×10 9 cfu / mL after 48 h of treatment, the mortality rate reached 89.08±2.53%, showing good biocontrol potential. 8 cfu / mL and above, the 48h mortality rate was higher than 75%, and the lower the concentration, the lower the mortality rate. The mortality rate of the finished product control group (bio group) at 2h, 8h, 24h, and 48h was lower than that of the low-dose group of the experimental group, and the mortality rate after 48h was only 51%.

[0059] Example 4: Outdoor insecticidal effect of HXBW-2 on aphids

[0060] The HXBW-2 strain was used to test its efficacy against cabbage aphids in an outdoor test. The fermentation liquid was diluted to a concentration of 1×10 8cfu / mL was used to test the aphid control efficacy in outdoor fields, and the insect population changes were detected for 15 days and the control efficacy was calculated.

[0061] The treatment settings were as follows: Treatment 1 was spraying the bacterial solution once; Treatment 2 was spraying the bacterial solution twice (the same treatment was repeated 5 days after the first spraying); and the control was sprayed with an equal amount of sterile water.

[0062] Before the test, the number of cabbage aphids on the roses was adjusted to be as close as possible. The 24 roses were randomly divided into 3 groups with equal numbers and different plots. Leaves of the same size and age were selected from each pot of seedlings as the test objects, randomly sorted 1 to 24, and marked with labels. The number of aphids on each branch was counted and then treated. The number of aphids was observed and recorded regularly, and the control effect was calculated as follows:

[0063] Insect population reduction rate (%) = (number of live insects before application - number of live insects after application) / number of live insects before application × 100%

[0064] Control effect (%) = (insecticidal reduction rate in treated area - insecticidal reduction rate in control area) × 100 / (1 - insecticidal reduction rate in control area)

[0065] Table 5: Statistics of aphid population reduction rate and control effect

[0066]

[0067] The results of the outdoor tests are shown in Table 5 and Figure 5 , and further tested the control ability of HXBW-2 strain against cabbage aphids. The results of the control effect of treatment 1 and treatment 2 showed that both had a high insect population reduction rate, and the control effect of treatment 2 was higher than that of treatment 1. In the first 5 days after treatment, the insect population reduction rate showed an increasing trend. The control effect of treatment 1 began to decline after 5 days, while treatment 2 showed a better control effect after supplementation. Comparing the differences between the two can further predict that the timeliness of HXBW-2 and the optimal control period are about 5 days, and then when using the drug in the field, supplementation must be made after 5 days to obtain a higher control effect.

Claims

1. A Bacillus sp., characterized in that The Bacillus toyota ( Bacillus toyonensis )’s deposit number is CGMCC No.28561.

2. Use of the Bacillus orientalis according to claim 1 in the preparation of a product for preventing and controlling pests caused by cabbage aphids.

3. A product for preventing and controlling pests caused by cabbage aphids, characterized in that: The product contains live bacteria of Bacillus subtilis according to claim 1.

4. The product according to claim 3, characterized in that The product also contains the fermentation product of Bacillus subtilis according to claim 1.

5. The product according to claim 3, characterized in that The product is a bacterial liquid product.

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