Bacillus toyonensis having effect of preventing and controlling cabbage aphid

Abstract

a type of Bacillus toyonensis having the effect of preventing and controlling cabbage aphids, which has a preservation number of CGMCC No. 28561. The present invention can alleviate plant pest damage caused by cabbage aphids, and has a good insecticidal effect both indoors and outdoors.

Description

A Bacillus sp. with the effect of preventing and controlling cabbage aphids Technical Field

[0001] The invention belongs to the technical field of microbial screening, and particularly relates to a Toyobacillus sp. having the effect of preventing and controlling cabbage aphids. Background Art

[0002] Aphids are a general term for all members of the superfamily Aphidoidea in the order Hemiptera. Currently, there are approximately 4,400 species of aphids belonging to 10 families, most of which belong to the family Aphididae. Different plant species have been found to harbor specific aphid species. For example, the pine aphid is a major pest of conifers; the polymorphic hairy aphid primarily harms Koelreuteria paniculata; the yellow apple aphid primarily harms apples, crabapples, papayas, heathers, and hawthorns; and the peach aphid's host plants primarily include peaches, pears, plums, apricots, and cherries, all members of the Rosaceae family.

[0003] Cabbage aphids (Brevicoryne brassicae Linnaeus) are a significant pest of vegetables, primarily in the cruciferous family. They feed by sucking sap from leaves and stems, stealing nutrients from the plant, causing leaves to curl, shrink, and deform, slowing normal plant growth.

[0004] Currently, the control of cabbage aphid disease is still primarily based on chemical control. The extensive use of chemical agents poses a significant threat to environmental safety and human and animal health, leading to restrictions or bans on the use of many chemical pesticides. The search for environmentally friendly, safe, and pollution-free control methods is urgent. Biocontrol agents are increasingly attracting attention as an environmentally friendly alternative to chemical agents. Biocontrol bacteria, in particular, are gaining widespread attention due to their safety, ease of cultivation, and production. While Verticillium lecanii, Ascophyllum spp., and Paecilomyces roseum have been reported to have some control effects against aphids, their effectiveness against cabbage aphids has been limited.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a Bacillus sp. that has the effect of preventing and controlling cabbage aphids, which can biologically control cabbage aphids, thereby effectively preventing and controlling the disease losses caused by cabbage aphids.

[0007] The Bacillus toyonensis HXBW-2 strain (Bacillus toyonensis) provided by the present invention has a deposit number of CGMCC No. 28561, a deposit date of September 26, 2023, and a depository unit of the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides a use of the Bacillus toyotii HXBW-2 strain, which is use in preparing products for preventing and controlling plant pests.

[0009] The plant pests are caused by cabbage aphids.

[0010] The present invention also provides a product for alleviating plant pests, wherein the product contains live bacteria and / or fermentation products of the Toyo Bacillus sp. HXBW-2 strain.

[0011] The product is a bacterial liquid product.

[0012] The Toyo Bacillus sp. 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

[0013] Figure 1: Colony morphology of the strain;

[0014] Figure 2: Staining characteristics of strains;

[0015] Figure 3: Phylogenetic tree;

[0016] Figure 4: Heat map of strain genome generation;

[0017] Figure 5: Aphid situation 3 days after using microbial agents, where A is before use and B is the result 3 days after use. DETAILED DESCRIPTION

[0018] The present invention extracted 76 pure bacterial colonies from the rhizosphere soil of plants frequently affected by aphids. One of these strains showed strong efficacy against cabbage aphids. 16S rRNA sequencing confirmed that the strain was Bacillus toyonensis. The bacteria screened using the present invention's biological preparation method exhibited excellent control efficacy against cabbage aphids and can be widely used in cabbage aphid control.

[0019] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0020] Example 1: Isolation and screening of strains

[0021] 1.1 Isolation of bacterial strains from soil

[0022] Weigh 10g of soil sample (rhizosphere soil of a solar greenhouse where aphids are common), place it in a 250mL sterile triangular flask filled with 90mL of sterile water, and seal the flask with a sealing film. -1 After shaking evenly, dilute the soil suspension with sterile water on a clean bench to obtain 10 -1 -10 -5For suspensions of different concentrations, take 100 μL of each soil suspension and evenly apply it to NA medium using an applicator. Place the suspension in a 25°C constant temperature incubator in the dark for 48 hours. After obvious colonies grow, single colonies are collected for purification. Three replicates are set for each soil suspension concentration.

[0023] 1.2 Screening of bacteria with aphidicide activity

[0024] 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 following 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. After preparing the fermentation liquid into 2-fold, 5-fold, and 10-fold dilutions, the toxicity to southern cabbage aphids was determined, and the results were examined under a stereoscope after 12 hours. Aphids are stiff and motionless, and are considered dead if they do not recover after being transferred to clean water for 12 hours. NA liquid culture medium without bacterial liquid 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:

[0025] Mortality (%) = number of dead aphids / total number of aphids × 100.

[0026] Corrected mortality rate (%) = (% mortality rate of the treated group - % mortality rate of the control group) / (100 - % mortality rate of the control group) × 100

[0027] Aphidicidal activity (NA) was classified according to the following standards: NA ≥ 80%, 50% ≤ NA < 80%, 20% ≤ NA < 50% and NA < 20% were considered as strong aphidicidal activity, moderate aphidicidal activity, weak aphidicidal activity and no aphidicidal activity, respectively.

[0028] A total of 76 strains were isolated from the rhizosphere soil, of which five, HXBW-2, HXBW-05, HXBW-27, HXBW-31, and HXBW-53, showed aphidicide activity. HXBW-2 exhibited strong aphidicide activity, with a 96.3% aphid mortality rate at a two-fold dilution. HXBW-27 exhibited moderate aphidicide activity, with a 56.7% aphid mortality rate. HXBW-05, HXBW-31, and HXBW-53 exhibited weaker aphidicide activity.

[0029] Table 1: Toxicity of bacterial fermentation broth to cabbage aphids

[0030] Example 2: Identification of strains

[0031] 2.1 Morphological characteristics of strains

[0032] Streak pure cultured HXBW-2 strains onto NA medium plates and incubate in a 25°C incubator for 48 hours. Once distinct colonies have grown, observe their morphology, color, transparency, dryness, surface smoothness, and edge morphology. Collect fresh bacteria for Gram staining and microscopic examination.

[0033] As shown in Figure 1, the colonies of the HXBW-2 strain are nearly round, matte white, with wrinkled surfaces and irregular edges. As time goes by, the colonies grow larger and show the ability to migrate and self-aggregate. The staining results show that HXBW-2 is a Gram-positive short rod-shaped bacterium (Figure 2).

[0034] 2.2 Physicochemical properties of the strain

[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, Priscaul (VP) test, starch hydrolysis, gelatin liquefaction, hydrogen sulfide, nitrate reduction, glucose oxidation and fermentation, and sucrose fermentation tests.

[0036] As shown in Tables 2 and 3, the strain tested positive for arginine, VP gelatin, sucrose fermentation, and amygdalin. In carbon source utilization tests, it also tested positive for glycerol, ribose, glucose, fructose, mannose, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salvinol, cellobiose, maltose, sucrose, trehalose, starch, and glycogen. According to the Manual for Systematic Identification of Common Bacteria, this strain conforms to the characteristics of Bacillus.

[0037] Table 2: Physiological and biochemical characteristics of strain HXBW-2 – enzyme activity and carbon source oxidation

[0038] +: positive reaction; -: negative reaction;

[0039] Table 3: Carbon source utilization information of strain HXBW-2

[0040] +: positive reaction; -: negative reaction;

[0041] 2.3 Strain 16S rRNA gene sequence

[0042] The HXBW-2 strain obtained by screening was identified by 16S rDNA sequencing. The sequencing results were then used to search for similar sequences using the NCBI Blast alignment system and construct a phylogenetic tree. The phylogenetic tree in Figure 3 shows that strain HXBW-2 belongs to the genus Bacillus. Figure 4 shows that a comparison of the full genome data of strain HXBW-2 with that of the model strain Bacillus oyonensis (BCT-7112) yielded an ANI value of 98.43%, exceeding the 96% threshold for identifying homologous strains. Therefore, strain HXBW-2 was identified as Bacillus oyonensis.

[0043] The strain is deposited in the General Microbiology Center of China 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.

[0044] Example 3: Indoor insecticidal effect of HXBW-2 on aphids

[0045] Cabbage aphids collected in the laboratory were used as test pests. The aphids were retrieved along 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: 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).

[0046] The finished product of Bacillus toyota (No. bio118045) freeze-dried powder was purchased from the strain library, activated according to the instructions, and cultured at 30℃ until 2×10 9 cfu / mL for future use.

[0047] 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 strain at different concentrations was evenly sprayed on the cabbage aphids until the surface of the insect body was moist. The finished product of Bacillus orientalis 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 dish was sealed with plastic wrap and a dozen small holes were poked with a needle to ensure air circulation. The aphid mortality rate was recorded at 2h, 8h, 24h, and 48h.

[0048] 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.

[0049] Mortality rate (%) = number of dead insects (heads) × 100 / total number of insects treated (heads)

[0050] Table 4: Aphid mortality statistics

[0051] 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 evenly spraying the bacterial solution on the surface of cabbage aphids, observe the death of the aphids. Use a brush to touch the aphid surface. If there is no obvious physiological reaction, it means the aphids are dead. As time increases (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. The HXBW-2 strain concentration was 1×10 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 experimental group, and the mortality rate after 48h was only 51%.

[0052] Example 4: Outdoor insecticidal effect of HXBW-2 on aphids

[0053] The HXBW-2 strain was tested for its efficacy against cabbage aphids in an outdoor test. The fermentation liquid was diluted to a concentration of 1×10 8 cfu / 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.

[0054] 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.

[0055] Before the experiment, the number of cabbage aphids on the roses was adjusted to be as similar as possible. Twenty-four rose plants were randomly divided into three equal groups, each with different plots. Leaves of consistent size and age were selected from each pot of seedlings as test subjects. These leaves were randomly ranked 1 to 24 and labeled with labels. The number of aphids on each branch was counted before treatment. The number of aphids was regularly observed and recorded, and the control efficacy was calculated as follows:

[0056] Insect population reduction rate (%) = (number of live insects before application - number of live insects after application) / number of live insects before application × 100%

[0057] Control effect (%) = (insecticidal population reduction rate in the treated area - insecticidal population reduction rate in the control area) × 100 / (1 - insecticidal population reduction rate in the control area)

[0058] Table 5: Statistics of aphid population reduction rate and control effect

[0059] The results of the outdoor experiment are shown in Table 5 and Figure 5. The HXBW-2 strain's ability to control cabbage aphids was further tested. A comparison of the control effects of Treatments 1 and 2 revealed that both treatments achieved high population reduction rates, with Treatment 2 showing a higher control efficacy than Treatment 1. During the first five days after treatment, the population reduction rate showed an increasing trend. The efficacy of Treatment 1 began to decline after five days, while Treatment 2 demonstrated a stronger control effect after the application of the supplemental treatment. This comparison further suggests that the timeliness of HXBW-2's effectiveness and the optimal control window are approximately five days. Consequently, supplemental treatment should be applied five days later for greater efficacy in field applications.

Claims

1. A Bacillus sp., characterized in that The deposit number of the Bacillus toyota is CGMCC No.28561.

2. Use of the Bacillus subtilis according to claim 1 in the preparation of products for preventing and controlling plant pests.

3. The use according to claim 2, characterized in that The plant pests are pests caused by cabbage aphids.

4. A product for controlling plant pests, characterized in that: The product contains live bacteria of Bacillus subtilis according to claim 1.

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

6. The article according to claim 4, characterized in that The product is a bacterial liquid product.

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