CN114058541B - Pesticide-resistant colas-resistant biocontrol bacillus beleisi for wheat sharp eyespot and application - Google Patents

Abstract

The invention discloses Bacillus velesii biocontrol of wheat sheath blight resistant to pesticide kuras and its application, and belongs to the field of agricultural microorganisms. Bacillus velezensis, named NW03, was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, and the deposit number is CGMCC NO.22694. The Bacillus velesi of the present invention has good tolerance to the pesticide kuras, and has a good antagonistic effect on the pathogen of wheat sheath blight, and can be used together with kuras to prevent and control wheat sheath blight, and the control effect is guaranteed. On the basis of achieving pesticide reduction.

Description

Bacillus Veles and its application for the biocontrol of wheat sheath blight resistant to pesticide Kuras

technical field

The invention relates to the technical field of agricultural microorganisms, and more specifically relates to Bacillus velesii for the biocontrol of wheat sheath blight resistant to the pesticide Kuras and its application.

Background technique

Wheat sheath blight, also known as blight blight and sharp eye spot disease, is a pathogenic fungal infection caused by the infection of Ceratobacterium tritici. The leaf sheaths and stems of wheat are easily infected with the disease. As time goes by, the infected parts gradually extend outward, and the onset of the disease is slow before and then urgently. Wheat may suffer from this disease in each growth period, resulting in multiple manifestations such as lodging, rotten buds, withered booting ears, and dead seedlings.

The pesticide Kuras can effectively control wheat sheath blight, protect wheat for a long time, and promote wheat growth, reduce wheat sowing amount and increase yield on a conventional basis. The active ingredients of Kuras are thiamethoxam, fludioxonil, and difenoconazole; they can inhibit pathogenic bacteria, effectively promote the normal growth of crops, and increase crop yield. However, as the problem of pesticide abuse is becoming more and more serious, the drug resistance of pathogenic bacteria is gradually increasing. Continued increase in the use of pesticides will affect the growth of crops, not only cause pesticide pollution, but also affect the quality of crops. Therefore, reducing the amount of pesticides has become an inevitable trend.

The mixed application of biocontrol bacteria and pesticide fungicides can greatly reduce the cost of pesticides, reduce pesticide residues, the negative impact of pesticides on plants, and the safety risks of pesticides to the human body. Effectiveness, ensure the effect of disease prevention and control, and solve the problem of increased resistance of diseases. Therefore, there is an urgent need to provide pathogen antagonists that are compatible with Kuras, explore the possibility of mixing bio-control bacteria and chemical pesticides, and provide an effective way to improve the effect of bio-control bacteria and reduce the production cost of growers.

Contents of the invention

In view of this, the present invention provides wheat sheath blight biocontrol Bacillus Veles that is tolerant to the pesticide Kuras, which has good tolerance to the pesticide Kuras, and has good resistance to the pathogenic bacteria of wheat sheath blight Antagonism, it can be used together with Kuras to prevent and control wheat sheath blight, and reduce the amount of pesticides on the basis of ensuring the control effect.

In order to achieve the above object, the present invention adopts the following technical solutions:

The wheat sheath blight biocontrol Bacillus velezensis resistant to the pesticide Kuras, named NW03, was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms, with the preservation number CGMCCNO.22694, and the preservation address: No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation time is June 11, 2021.

The application of the above-mentioned wheat sheath blight biocontrol Bacillus Veles that is tolerant to the pesticide Kuras in the prevention and treatment of wheat sheath blight.

Further, when preventing and controlling wheat sheath blight, the wheat is applied with the pesticide Kuras and inoculated with the above-mentioned Bacillus velesi.

It can be known from the above technical scheme that the Bacillus Velez NW03 disclosed by the present invention has a strong tolerance to Kuras; when it is shared with Kuras, it can achieve the same effect as that of using Kuras alone when the Kuras is halved. Lass similar control effect, suitable for popularization and application.

Description of drawings

Fig. 1 shows the bacillus that is partly tolerant to the pesticide Kuras obtained by screening;

Figure 2 shows the antagonism effect of part of the screened bacterial strains on wheat sheath blight;

Figure 3 shows the growth of antagonistic strains in the culture medium containing Kuras;

Figure 4 shows the NW03 strain bacterial colony morphology;

Figure 5 shows the phylogenetic tree of NW03 strain.

Detailed ways

The following clearly and completely describes the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Isolation, screening and identification of embodiment 1NW03

1. Test material

The wheat plant samples were taken from the wheat planting field of Hebei Agricultural University Farm, and the samples were collected in April 2019. The pathogenic bacteria of wheat sheath blight was preserved by our laboratory. The wheat variety used in the experiment was Jimai 418, produced in Baoding, without coating.

2. Medium

PDA medium: 200g potato, 20g glucose, 15-20g agar, 1000mL distilled water. Peel the potatoes, cut them into small pieces, add water to boil for 0.5h, filter with double gauze, take the filtrate, add sugar, and add water to make up 1000mL.

NA medium: beef extract 5g, peptone 10g, NaCl 5g, agar 15-20g, distilled water 1000mL, pH 7.0-7.2.

NB medium: beef extract 5g, peptone 10g, NaCl 5g, distilled water 1000mL, pH 7.0-7.2.

3. Cultivation of wheat sheath blight pathogen

After the sterilized PDA medium was melted, the plate was poured, and streptomycin sulfate was added to make the final concentration 40 μg/100mL. Inoculate the bacterial flakes of the pathogenic bacteria of wheat sheath blight on the center of the PDA plate, incubate in the incubator at 26°C for 5-7 days, and wait for the mycelia to cover the plate for later use.

4. Screening of Bacillus tolerant to Kulas pesticide

Wash the roots of wheat seedlings with clean water, blot the water dry with sterile filter paper, sterilize the surface of wheat roots with 75.0% alcohol, grind them in a sterile mortar, put the ground tissue of seedling roots into a sterilized In a 250mL Erlenmeyer flask (contains 100mL sterile water and glass beads in the Erlenmeyer flask), shake it well for 30min, heat it in a water bath at 80°C for 5min, and use the dilution coating method in the mixture containing the pesticide Kuras (final concentration is 100μL /100mL) on the NA plate to isolate and screen the Bacillus resistant to Kurassi. Cultivate overnight at 37°C, and count the strains that are tolerant to Kulas.

After preliminary screening, 106 strains of Bacillus spp. that were able to tolerate Kuras pesticide were screened out from the root tissue of wheat (Fig. 1).

5. Screening of Bacillus against wheat sheath blight

Using pathogenic bacteria as indicator bacteria, the confrontation culture method was used to conduct antagonism test to detect the formation of inhibition zone of the screened strains resistant to kelp, and to screen the strains antagonizing the pathogenic bacteria of wheat sheath blight.

Place the pathogenic bacteria sheet of wheat sheath blight in the center of the PDA plate, pick and screen the strains with a sterile bamboo stick and inoculate them at a distance of 2.5 cm from the pathogenic bacteria sheet, and inoculate 4-5 single colonies evenly on each plate, in a 26°C incubator Cultured upside down for 5-7d. Antagonist bacteria capable of producing obvious inhibition zones were inoculated onto the NA slant, and cultured overnight at a constant temperature of 37°C for later use.

There are 64 strains that have inhibitory effect on the pathogen of wheat sheath blight, and some results are shown in Figure 2; 6 of them have significant antagonistic effect, and the width of the inhibitory zone against the pathogen of wheat sheath blight is more than 1.5cm.

6. Detection of growth characteristics of antagonistic bacteria

The screened strains with significant antagonistic activity against pathogenic bacteria were cultured in NB medium containing Colas (final concentration: 100 μL/100 mL) at 37 °C with constant temperature and shaking, and the absorbance value of each strain at 600 nm was detected every 24 hours. It was 96h, and a control group without adding the pesticide Kulas was set up (the selected bacterial strain was the Bacillus strain Z-5 used for industrial production screened in the laboratory). Record the absorbance value and draw the growth curve. As shown in Figure 3, the concentration of the NW03 strain fermentation liquid reached the maximum value at 72 hours of cultivation, which was higher than that of the control group without addition of Kuras, indicating that the bacterial strain had a strong tolerance to Kuras, and its growth was not affected by Kuras. The inhibition of Lars decreased afterward, and was slightly lower than that of the control group at 96 hours of culture. The growth of the remaining 5 strains was significantly inhibited in the medium containing Kuras.

7. Strain identification

The colony of the NW03 strain grown on the NA plate was round or oval, milky white, moist and smooth on the surface, and raised in the middle (Figure 4). The species of the screened strains were identified according to the method of "Bergey's Bacterial Identification Manual". The 16S rDNA sequences of the screened strains were amplified with universal primers 27f and 1495r. The sequencing results of PCR products were compared by Blast in the NCBI database, and compared with the sequence homology of the type strains, and the phylogenetic tree was constructed with MEGA6.0 software. The results showed that the NW03 strain was Bacillus velezensis ( FIG. 5 ).

Example 2 The control effect of mixed application of antagonistic bacteria and pesticides on wheat sheath blight

Streak inoculate the NW03 strain that grew well in the culture medium containing Kuras on the NA medium, culture at a constant temperature at 37°C overnight, pick a single colony in the NB medium, and culture on a shaker at 37°C for 48 hours. The fermentation broth was centrifuged at 8000rpm for 5min to recover the bacterial cells, suspended and diluted with sterile water to a viable bacterial concentration of about 1×10 ⁸ cfu/mL, and the bacterial count was carried out using a bacterial counting plate.

The pot experiment was divided into 4 groups, namely the blank control group CK0, the pathogenic bacteria group CK1, the pesticide treatment group NY and the bacterial agent plus pesticide group JY, with 5 replicates in each group. Take a flower pot with a diameter of 12.9 cm and a height of 12.4 cm, and sow 12 wheat seeds evenly in each pot. The soil for planting wheat is taken from the wheat planting field of the Hebei Agricultural University farm. The blank control group was irrigated with water without inoculation of pathogenic bacteria and antagonistic bacteria. The pathogen group was inoculated with wheat sheath blight pathogen, and wheat seeds were placed on pathogenic bacteria sheets (7 mm in diameter), and one wheat seed was placed on each bacterial sheet. The method of inoculating pathogenic bacteria in the pesticide treatment group and the bacterial agent plus pesticide group was the same as that of the pathogenic bacteria group. In the pesticide treatment group, the wheat seeds were seed-dressed according to the dose of 100kg seeds inserted into 600mL Kuras; in the bacterial agent plus pesticide group, the pesticide was halved, and 20mL of NW03 bacterial suspension was poured, incubated at room temperature, and the control effect was investigated after 20 days of planting . The detection of plant growth status is carried out by plant, and the sampling amount of each group is 20 plants, and then the average value is calculated.

Grading standards for the incidence of wheat sheath blight:

Grade 0: The whole plant is disease-free;

Grade 1: Less than 1/2 of the outer leaf sheath turns brown;

Grade 2: more than 1/2 of the outer leaf sheath is brown;

Grade 3: Obvious eye-like lesions appear at the base of the stem.

Disease index=[∑(number of diseased plants at all levels×representative value)/total number of investigated plants×representative value of the most serious disease level]×100.

Prevention and treatment effect=[(control group disease index-treatment group disease index)/control group disease index]×100%.

The results are shown in Table 1. Compared with the pesticide treatment group NY and the blank control group CK0, the difference in root length, root weight, above-ground height and above-ground weight of the bacterial agent plus pesticide group JY was not significant, but was significantly different from that of the pathogenic bacteria group CK1. The control effect of JY group on wheat sheath blight was 78.4%, which was slightly higher than that of NY group, but the difference was not significant. It shows that under the condition of halving the application of pesticides, the application of fermentation liquid of NW03 strain can effectively prevent and control the occurrence of wheat sheath blight, which has a control effect similar to that of applying a sufficient amount of kolas, and achieves the condition of reducing the application of pesticides. The purpose of effective prevention and control of disease occurrence.

Table 1 Control effect of mixed application of Kuras and NW03 strains on wheat sheath blight

CK0 is the blank control group, CK1 is the pathogen group, NY is the pesticide treatment group, and JY is the bacterial agent plus pesticide group.

Example 3 Field test detection NW03 bacterial strain reduces the use of pesticide Kuras to control the effect of wheat sheath blight

Select a plot with severe and uniform occurrence of wheat sheath blight in the previous year, with an area of about 666.7m ² , and divide it into 3 small plots, each of which is about 222.2m ² . Pesticide group. For the pesticide group, the seeds were dressed according to the dose of 6mL kulas pesticide: 1kg seeds, the seeds and pesticides were put into a strong plastic bag, the mouth was tied tightly, and the pesticides and wheat seeds were mixed evenly by turning them upside down. In the bacterial agent plus pesticide group, the pesticide was halved, and NW03 bacterial agent with a live bacterial content of about 1.0×10 ¹⁰ cfu/mL was added for seed dressing, and the inoculation amount was 10 mL/kg of seeds. Mechanical sowing is adopted, and the management method is the same as that of ordinary fields.

In the wheat milk ripening stage, take 5-point sampling method to sample, take 3 consecutive rows at each point, and take 0.4m in each row, and investigate and count the plant height and average grain number per ear (30 plants at each random sampling point, 150 plants per group of samples) ), weigh the 1000-grain weight after the seeds are dried, and measure the output per square meter by single harvesting. The results are shown in Table 2.

Table 2 Control effect of NW03 strain and pesticide Kuras on wheat sheath blight at milky stage

The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant difference at P<0.05 level by LSD test.

The investigation on the control effect of sheath blight at the milky stage of wheat showed that compared with the control group, the plant height and yield of the bacterial agent plus pesticide group increased significantly, the wheat plant height increased from 76.24cm to 87.03cm, and the wheat yield increased from 603.2g/m ² increased to 778.96g/m ² , the effect was remarkable, indicating that the application of the bacterial agent prepared by the NW03 strain could effectively reduce the use of the pesticide Kuras, but the control effect on wheat sheath blight was not reduced. Compared with the pesticide group and the bacterial agent plus pesticide group, the wheat plant height and yield both increased, but the difference was not significant.

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (3)

1. Wheat sheath blight biocontrol Bacillus velezensis resistant to the pesticide Kuras, characterized in that it is named NW03, and is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation number is CGMCC NO.22694. 2. the application of the wheat sheath blight biocontrol Bacillus velei that is tolerant to the pesticide Kuras of claim 1 in the control of wheat sheath blight. 3. The application according to claim 2, characterized in that, Wheat was applied with the pesticide Kuras and inoculated with the Bacillus velesii.

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