WO2025123647A1 - Bacillus toyonensis having effect of preventing and controlling cabbage aphid - Google Patents

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 kind of Bacillus fusinus with effect of preventing and controlling cabbage aphids Technical Field

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

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.

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.

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

The purpose of the present invention is to provide a Bacillus sp. having the effect of preventing and controlling cabbage aphids. It can biologically control cabbage aphids, thereby effectively preventing and controlling the disease losses caused by cabbage aphids.

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.

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.

The plant pests are caused by cabbage aphids.

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.

The product is a bacterial liquid product.

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

Figure 1: Colony morphology of strains;

Figure 2: Staining characteristics of strains;

Figure 3: Phylogenetic tree;

Figure 4: Heat map of strain whole genome generation;

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

The present invention extracted 76 pure bacterial colonies from the rhizosphere soil of plants that often suffer from aphid diseases, one of which had a strong effect of killing cabbage aphids. The strain was identified as Bacillus toyonensis by 16SrRNA sequencing. The fungus has a good control effect on cabbage aphids and can be widely used in the control of cabbage aphids.

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

Example 1: Isolation and screening of strains

1.1 Isolation of bacterial strains from soil

Weigh 10g of soil sample (rhizosphere soil of solar greenhouse where aphids occur all year round), put it into a 250mL sterile triangular flask filled with 90mL sterile water, and seal the bottle mouth with a sealing film. Place the triangular flask on a shaker at 25℃ and 200rpmmin ^-1 for 30min. After shaking evenly, dilute the soil suspension with sterile water on a clean bench to obtain a suspension with a concentration of ^{10-1-10-5 .} Take 100μL of soil suspension of different concentrations respectively, apply it evenly on NA culture medium with an applicator, place it in a constant temperature incubator at 25℃, and culture it in the dark for 48h. After obvious colonies grow, take a single colony for purification. Set up 3 replicates for each concentration of soil suspension.

1.2 Screening of bacteria with aphidicide activity

Pick a single bacterial colony into a 250mL Erlenmeyer flask containing 100mL NA liquid culture medium (2×10 ⁹ CFUmL ^-1 ), shake on a shaker at 25℃, 150rmin ^-1 for 48h. After taking out, centrifuge the fermentation liquid in a 10000rpm centrifuge for 10min, take the supernatant, and then filter it with a 0.45μm filter membrane to obtain the filtrate for use. Add 500μL of the 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, 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 12h. The aphid body is stiff and motionless, and it is considered dead if it does not recover after being transferred to clean water for 12h. NA liquid culture medium without bacterial liquid is used as a control. The experiment was repeated three times, and each repeat contained three wells. The formula for calculating aphid mortality and corrected mortality is as follows:

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

Corrected mortality rate (%) = (% mortality rate of the treatment group - % mortality rate of the control group) / (100 - The mortality rate of the control group (%) × 100

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.

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.

Table 1: Toxicity of bacterial fermentation broth to cabbage aphids

Example 2: Identification of strains

2.1 Morphological characteristics of strains

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.

It can be observed from Figure 1 that the colonies of the HXBW-2 strain are nearly round, matte white, with wrinkled surfaces and irregular edges. As time goes by, the colonies become larger and show the ability to swim and self-aggregate. The staining results show that HXBW-2 is a Gram-positive short rod-shaped bacterium (Figure 2).

2.2 Physicochemical properties of strains

After the bacteria were cultured on the culture medium for a period of time, various physiological and biochemical indices were measured, including catalase test, Priskaul (V-P) test, starch hydrolysis, gelatin liquefaction, hydrogen sulfide, nitrate reduction, glucose oxidation fermentation and sucrose fermentation test.

As shown in Tables 2 and 3, the strains were positive in arginine, V-P gelatin, sucrose fermentation, and amygdalin tests. In the carbon source utilization experiment, glycerol, ribose, glucose, fructose, mannose, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salvinol, cellobiose, maltose, sucrose, trehalose, starch, and glycogen were positive. According to the Manual of Identification of Common Bacteria Systems, it meets the characteristics of Bacillus.

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

+: positive reaction; -: negative reaction;

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

+: positive reaction; -: negative reaction;

2.3 Strain 16S rRNA gene sequence

The HXBW-2 strain obtained by screening was identified by 16SrDNA sequencing. The sequencing results were searched for similar sequences and a phylogenetic tree was constructed by the Blast alignment system in the NCBI data. As shown in Figure 3, the strain HXBW-2 belongs to the genus Bacillus. Figure 4 shows that the ANI value of the strain HXBW-2 compared with the whole genome data of the model strain Bacillustoyonensis (BCT-7112) is: 98.43%, which is greater than the threshold of 96% for ANI homology judgment. Therefore, the HXBW-2 strain was identified as Bacillustoyonensis.

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.

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

Cabbage aphids collected in the laboratory were used as test pests. Cabbage aphids were taken back with stems and leaves and placed in a petri dish containing paper. Aphids with normal vitality and activity were counted and dead or weak insects were picked out to ensure that the number of aphids in each dish was greater than 100. Cabbage aphids were divided into 7 groups, namely experimental groups (high dose group 2×10 ⁹ cfu/mL, medium-high dose group 1×10 ^{9 cfu} /mL, medium dose group 1×10 ⁸ cfu/mL, medium-low dose group 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 (ddH ₂ O).

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°C to 2×10 ⁹ cfu/mL for later use.

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.

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 2×10 ⁹ , 1×10 ⁹ , 1×10 ⁸ , 4×10 ⁷ , and 2×10 ⁷ cfu/mL, respectively.

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

Table 4: Aphid mortality statistics

The HXBW-2 strain fermentation liquid was counted by dilution plate method, and the concentration of the liquid was 2×10 ⁹ cfu/mL. After the bacterial liquid 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 increased (Table 4), the mortality rate of aphids increased. The mortality rate reached 89.08±2.53% after 48h treatment with a concentration of 2×10 ⁹ cfu/mL, showing good biocontrol potential. When the concentration of HXBW-2 strain was 1×10 ⁸ cfu/mL and above, the 48h mortality rate was higher than 75%. 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%.

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

The HXBW-2 strain was tested for its outdoor efficacy against cabbage aphids. The fermentation liquid was diluted to a concentration of 1×10 ⁸ cfu/mL for outdoor field efficacy testing of aphids. The insect population change was detected for 15 days and the efficacy was calculated.

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.

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:

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

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

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

The results of the outdoor test are shown in Table 5 and Figure 5, and the control ability of HXBW-2 strain on cabbage aphids was further tested. The control results 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 (6)

A Bacillus orientalis, characterized in that the preservation number of the Bacillus orientalis is CGMCC No.28561. Use of the Bacillus subtilis described in claim 1 in the preparation of products for preventing and controlling plant pests. The use according to claim 2, characterized in that the plant pest is a pest caused by cabbage aphids. A product for preventing and controlling plant pests, characterized in that the product contains live bacteria of the Bacillus subtilis according to claim 1. The product according to claim 4, characterized in that the product contains the fermentation product of Bacillus sp. according to claim 1. The product according to claim 4 is characterized in that the product is a bacterial liquid product.