CN117683678A - Bacillus eastern with cabbage aphid prevention and control effect - Google Patents

Abstract

The invention provides bacillus eastern with a cabbage aphid prevention function, and the preservation number of the bacillus eastern is CGMCC No.28561. The bacillus megatherium HXBW-2 strain screened by the invention can relieve plant insect damage caused by cabbage aphids, has good insecticidal effect indoors and outdoors, and has development potential.

Description

Bacillus eastern with cabbage aphid prevention and control effect

Technical Field

The invention belongs to the technical field of microorganism screening, and particularly relates to bacillus eastern with cabbage aphid control effect.

Background

Aphids, a collective term for the hemiptera aphid population, include all members of the aphid population, and a total of 10 aphids of the order 4400 species have been found to be present, most of which belong to the aphididae family. It has now been found that different plants have corresponding aphid species, for example pine aphids are the main pests of conifer plants; luan Duotai Aphis pubescens mainly endangers goldenrain tree; the apple yellow aphids mainly harm apples, crabapple, papaya, photinia fraseri, hawthorns and the like; the host plant of the green peach aphid mainly comprises rosaceous fruit trees such as peach, pear, plum, cherry and the like.

Cabbage aphids (Brevicoryne brassicae Linnaeus) are an important pest on vegetables, mainly damaging crucifers. The cabbage aphid generally absorbs plant growth nutrition by sucking juice on leaves and stems to eat, so that the leaf curl, atrophy and deformation of the plant are caused, and the normal growth of the plant is slowed down.

At present, the prevention and treatment of cabbage aphid disease is still mainly chemical prevention and treatment. The use of chemical agents in large quantities poses a great threat to environmental safety and human and animal health, and various chemical pesticides are therefore limited or disabled, and it is urgent to find an environmentally friendly, safe and pollution-free control method. Biocontrol agents are becoming increasingly important as an environmentally friendly alternative to chemical agents. And the biocontrol bacteria are easy to culture and produce due to high safety, and are widely paid attention to. It is reported that verticillium lecanii, aschersonia and paecilomyces roseus have a certain control effect on aphids, but their application to cabbage aphids is poor.

Disclosure of Invention

The invention aims to provide bacillus eastern with the function of preventing and controlling cabbage aphids, which can be used for biologically preventing and controlling cabbage aphids, thereby effectively preventing and controlling disease loss caused by the cabbage aphids.

The preservation number of the bacillus megatherium HXBW-2 strain (Bacillus toyonensis) provided by the invention is CGMCC No.28561, the preservation date is 2023, 9, 26 days, the preservation unit is China general microbiological culture Collection center, and the address is North Chen West Lu No. 1, 3 in the Korean region of Beijing city.

The invention also provides an application of the bacillus eastern HXBW-2 strain in preparing products for preventing and controlling plant insect pests.

The plant insect pest is caused by cabbage aphids.

The invention also provides a product for alleviating plant insect pests, which comprises living bacteria and/or fermentation products of the bacillus eastern HXBW-2 strain.

The product is a bacterial liquid product.

The bacillus megatherium HXBW-2 strain screened by the invention can relieve plant insect damage caused by cabbage aphids, has good insecticidal effect indoors and outdoors, and has development potential.

Drawings

Fig. 1: a bacterial colony morphological characteristic diagram;

fig. 2: strain staining profile;

fig. 3: a phylogenetic tree diagram;

fig. 4: generating a heat map of the whole genome of the strain;

fig. 5: aphid status after 3 days with microbial inoculum, wherein A is before use and B is the result graph after 3 days of use.

Detailed Description

The invention extracts 76 pure colonies from plant rhizosphere soil with frequent aphid diseases, wherein one of the colonies has strong effect of killing cabbage aphids, and the strain is bacillus eastern (Bacillus toyonensis) through 16SrRNA sequencing identification. The bacteria screened by the biological agent-based method has good control effect on cabbage aphids, and can be widely applied to control of cabbage aphids.

The present invention will be described in detail with reference to specific embodiments and drawings.

Example 1: isolation and selection of strains

1.1 isolation of bacterial strains in soil

10g of soil sample (sunlight greenhouse rhizosphere soil with aphid disease all the year round) is weighed, placed into a 250mL sterile triangular flask with 90mL sterile water, and the bottle mouth is sealed by a sealing film. The triangular flask was placed at 25℃and 200rpmmin ^-1 Shake on shaker for 30min. After shaking evenly, diluting the soil suspension with sterile water on an ultra-clean workbench to obtain 10 ^-1 -10 ^-5 And (3) taking 100 mu L of soil suspensions with different concentrations respectively, uniformly smearing the soil suspensions on NA culture medium by using a coater, placing the suspension in a constant temperature incubator at 25 ℃, culturing the suspension for 48 hours in dark, and taking single bacterial colonies for purification after obvious bacterial colonies grow. There were 3 replicates of each concentration of soil suspension.

1.2 bacterial screening with aphid-killing Activity

Single bacterial colonies were picked in 250mL Erlenmeyer flasks (2X 10) containing 100mLNA liquid culture medium ⁹ CFUmL ^-1 )，25℃，150rmin ^-1 Shake on a shaker for 48h under the conditions. Taking out, centrifuging the fermentation liquor in a 10000rpm centrifuge for 10min, taking supernatant, and filtering with a 0.45 μm filter membrane to obtain filtrate for standby. 500. Mu.L of prepared cabbage aphid solution (about 100 cabbage aphids) was added to a 12-well plate, 500, 200, 100. Mu.L of bacterial fermentation solution was added, and 1mL of the fermentation solution was supplemented with sterile water, and after 2-fold, 5-fold, and 10-fold dilutions of the fermentation solution were prepared, the toxicity to the southern cabbage aphid was measured, and after 12 hours the results were examined under a stereoscope. Aphids are stiff and still remain inactive and still do not recover from death after 12 hours of transfer to clear water. NA liquid medium without bacterial liquid is used as control. The test was run in 3 replicates, each replicate containing 3 wells. The aphid mortality and the corrected mortality are calculated as follows:

mortality (%) = number of dead aphids/total number of aphids x 100.

Corrected mortality (%) = (mortality of treated group% -mortality of control group%)/(mortality of 100-control group%)/(mortality of 100)

The aphid killing activities (NA) are classified according to the following criteria, NA is not less than 80%, NA is not less than 50% and not more than 80%, NA is not less than 20% and not more than 50% and NA is not less than 20% respectively are regarded as having strong aphid killing activity, medium aphid killing activity, light aphid killing activity and no aphid killing activity.

76 strains were isolated from rhizosphere soil, of which HXBW-2, HXBW-05, HXBW-27, HXBW-31 and HXBW-53,5 have aphid killing activity. The HXBW-2 fermentation broth with strong aphid killing activity has the mortality rate of 96.3% to aphids at 2-fold dilution concentration. The method comprises the steps of carrying out a first treatment on the surface of the HXBW-27 has moderate aphid killing activity and has a mortality rate of 56.7 percent to aphids; HXBW-05, HXBW-31 and HXBW-53 exhibit weaker aphid killing activity.

Table 1: virulence meter of bacterial fermentation liquor on cabbage aphid

Example 2: identification of strains

2.1 morphological characteristics of the seed

The HXBW-2 strain which is cultured and stored in a pure way is streaked on an NA culture medium flat plate, and is cultured for 48 hours at a constant temperature in a 25 ℃ incubator, and after obvious colonies grow out, the colony morphology, the color, the transparency, the dryness, the surface smoothness and the edge morphology are observed. Fresh bacteria were taken for gram staining and microscopic examination.

From FIG. 1, it is observed that HXBW-2 strain colonies are nearly round, have matt white color, have irregular surface folds and edges, become larger with time, and show swimming and self-aggregation capabilities; the staining results indicated that HXBW-2 bacteria were gram-positive short bacilli (FIG. 2).

2.2 physicochemical Properties of Strain

After bacteria are cultured on a culture medium for a period of time, various physiological and biochemical index measurements, a contact enzyme test, a Priscol (V-P) measurement, starch hydrolysis, gelatin liquefaction, hydrogen sulfide, nitrate reduction, glucose oxidation fermentation and sucrose fermentation tests are carried out.

As can be seen from tables 2 and 3, the strains were positive in the arginine, V-P gelatin, sucrose fermentation, and amygdalin assay. In the carbon source utilization experiment, the results of glycerin, ribose, glucose, fructose, mannose, N-acetyl-glucosamine, amygdalin, arbutin, esculin, saligenin, cellobiose, maltose, sucrose, trehalose, starch, glycogen and the like were positive. According to

The handbook for identifying common bacterial systems accords with the characteristics of bacillus.

Table 2: bacterial strain HXBW-2 physiological and biochemical characteristics-enzyme activity and carbon source oxidation table

+: a positive reaction; -: a negative reaction;

table 3: carbon source utilization information table of strain HXBW-2

+: a positive reaction; -: a negative reaction;

2.3 Strain 16SrRNA Gene sequence

And (3) carrying out 16SrDNA sequencing identification on the HXBW-2 strain obtained by screening, and searching similar sequences and constructing a phylogenetic tree through Blast comparison systems in NCBI data according to the sequencing result. As can be seen from the phylogenetic tree of FIG. 3, the strain HXBW-2 belongs to the genus Bacillus. FIG. 4 shows that the comparison of the genome-wide data of the HXBW-2 strain with the Bacillus stearotonensis (BCT-7112) model strain gives ANI values of: 98.43% and the result was greater than 96% of the threshold value for ANI identity judgment, thus identifying the HXBW-2 strain as Bacillus eastern (Bacillus).

The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.28561 and a preservation date of 2023, 9 and 26; preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

Example 3: HXBW-2 indoor insecticidal effect on aphids

Cabbage aphids collected in a laboratory are used as test pests. The stem and leaf of cabbage aphid is retrieved and placed in a culture dish containing paper, and aphids with normal vitality and activity are counted and dead or weak aphids are selected, so that the number of aphids in each dish is ensured to be more than 100. Cabbage aphids were divided into 7 parts, respectively experimental groups (high dose group 2X 10 ⁹ cfu/mL, medium and high dose group 1X 10 ⁹ cfu/mL, medium dose group 1X 10 ⁸ cfu/mL, medium and low dose group 4X 10 ⁷ cfu/mL, low dose group 2X 10 ⁷ cfu/mL), control group (bio group 2X 10) ⁹ cfu/mL) and a blank group (ddH) ₂ O)。

Purchasing the finished Bacillus easternensis (No. bio 118045) lyophilized powder from strain library, activating it according to the application method on instruction, culturing at 30deg.C to 2×10 ⁹ cfu/mL is reserved.

After 10 times, 20 times, 50 times and 100 times of the original liquid of the HXBW-2 strain fermentation liquor is diluted, an indoor quantitative fungus spraying and disinfestation test is carried out by using the original liquid and the diluted liquid, tween 20 with the total volume of 1 per mill is added as a dispersing agent, a certain amount of surviving aphids are taken to be moisturized and fostered on leaves of an indoor culture dish, 100 heads of each dish are used, the strain fermentation liquor with different concentrations is evenly sprayed on the bodies of cabbage aphids until the surfaces of the bodies are moist, finished products of the Bacilluseastern bacillus are used as a control group, water is used as a blank control group, 3 times of the steps are repeated, after the culture dish is sealed by using a preservative film, dozens of small holes are stamped by needles to ensure air circulation, and the death rate of the aphids of 2 hours, 8 hours, 24 hours and 48 hours is recorded.

Counting the concentration of bacterial liquid by adopting a dilution coating flat plate method, and recording the concentration of the original bacterial liquid and the concentration of the dilution liquid as follows: 2X 10 ⁹ 、1×10 ⁹ 、1×10 ⁸ 、4×10 ⁷ 、2×10 ⁷ cfu/mL。

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

Table 4: aphid mortality data statistics

The stock solution of HXBW-2 strain fermentation broth is counted by a dilution coating flat plate method, and the concentration of the stock solution is 2 multiplied by 10 ⁹ cfu/mL. After the bacterial liquid is uniformly sprayed on the surface of cabbage aphid, the death condition of the aphid is observed, the body surface of the aphid is lightly touched by a writing brush, and no obvious physiological reaction indicates that the aphid dies. Over time (Table 4), the higher the mortality rate of aphids, 2X 10 ⁹ After 48 hours of treatment with cfu/mL concentration, the death rate reaches 89.08 +/-2.53 percent, the good biocontrol potential is shown, and the concentration of HXBW-2 strain is 1 multiplied by 10 ⁸ At cfu/mL and above, the death rate is higher than 75% in 48 hours, and the lower the concentration is, the lower the death rate is. The mortality rate of the finished control group (bio group) is lower than that of the experimental group low-dose group at 2h,8h,24h and 48h, and the mortality rate is only 51% after 48h.

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

Outdoor control experiment is carried out on cabbage aphid of HXBW-2 strain, and the concentration of fermentation liquor is diluted to 1X 10 ⁸ cfu/mL is used for outdoor field control effect detection of aphids, and the control effect is calculated by detecting the change of the insect quantity of 15 d.

The process is set as follows: the treatment 1 is spraying the bacterial liquid for 1 time; treatment 2 was 2 times of spraying the bacterial liquid (the same treatment was performed again after 5d of the first spraying), and the control was sprayed with an equal amount of sterile water.

Before the test, the quantity of cabbage aphids on China rose is regulated to be as similar as possible, and the cabbage aphids are randomly and equally divided into 3 groups from 24 China rose plants, and different cells are set. Selecting leaves with the same size and leaf age as detection objects from each seedling pot, randomly sequencing the leaves to 1-24, marking the leaves by using a label plate, counting the number of aphids on each branch, and then processing the leaves. The number of aphids is observed and recorded at regular time, and the control effect is calculated, and the calculation method is as follows:

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

Control effect (%) = (treatment area reduction rate-control area reduction rate) ×100/(1-control area reduction rate)

Table 5: aphid mouth decline rate and prevention effect statistics table

The results of the outdoor tests are shown in Table 5 and FIG. 5, and the ability of HXBW-2 strain to control cabbage aphids is further examined. The control effect results of the treatment 1 and the treatment 2 show that the two have higher rate of reduction of insect population, and the control effect treatment 2 is higher than the treatment 1. 5d before treatment, the rate of the reduction of the insect population shows a growing trend, the control effect starts to decline after 5 days in treatment 1, and the better control effect is shown after the medicine supplement in treatment 2. The difference between the two can be further predicted to be about 5 days in the timeliness of HXBW-2 and the optimal control time period, and then the drug must be supplemented after 5 days when the field is used for obtaining higher control effect.

Claims (6)

1. The bacillus eastern is characterized in that the preservation number of the bacillus eastern is CGMCC No.28561.

2. Use of bacillus eastern claim 1 for the preparation of a product for controlling plant pests.

3. The use according to claim 2, wherein the plant pest is caused by cabbage aphids.

4. An article of manufacture for controlling plant pests, said article of manufacture comprising a viable bacterium of bacillus eastern claim 1.

5. The article of claim 4, wherein the article comprises a fermentation product of bacillus eastern of claim 1.

6. The article of claim 4, wherein the article is a bacterial fluid article.