CN114164143B

CN114164143B - Pesticide-resistant biological control bacillus subtilis for wheat root rot and application thereof

Info

Publication number: CN114164143B
Application number: CN202111361759.9A
Authority: CN
Inventors: 张冬冬; 肖嘉文; 刘兆厦; 张学超
Original assignee: Heibei Agricultural University
Current assignee: Heibei Agricultural University
Priority date: 2021-11-17
Filing date: 2021-11-17
Publication date: 2022-10-25
Anticipated expiration: 2041-11-17
Also published as: CN114164143A

Abstract

The invention discloses a wheat root rot biocontrol bacillus subtilis tolerant to pesticide tyrasi and application thereof, belonging to the field of agricultural microorganisms, wherein the bacillus subtilis (Bacillus subtilis) is named as ND06 and is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC NO.22696. The bacillus subtilis has good tolerance to the pesticide Kulas, has good antagonistic action on pathogenic bacteria of the wheat root rot, can be used together with the Kulas for preventing and treating the wheat root rot, and realizes pesticide reduction on the basis of ensuring prevention effect.

Description

Pesticide-resistant biological control bacillus subtilis for wheat root rot and application thereof

Technical Field

The invention relates to the technical field of agricultural microorganisms, in particular to wheat root rot biocontrol bacillus subtilis tolerant to pesticide Kulas and application thereof.

Background

The wheat root rot is a common wheat disease occurring in various wheat producing countries in the world, which damages the whole growth period of wheat, pathogenic bacteria can infect root systems and stem bases of wheat to accelerate leaf senescence, cause plant lodging and withered white ears in severe cases, the yield of light people is reduced by 5-10%, and the yield is reduced by 20-50% in severe cases, thus seriously affecting the yield and quality of wheat.

The pesticide, the colas, can efficiently prevent and control the root rot of wheat, can protect the wheat for a long time, promotes the growth of the wheat, reduces the seeding rate of the wheat on the conventional basis, and improves the yield. The active ingredients of the Kulas are thiamethoxam, fludioxonil and difenoconazole; it can inhibit pathogenic bacteria, promote normal growth of crops, and increase crop yield. However, as the problem of pesticide abuse is getting worse, the drug resistance of pathogenic bacteria is gradually enhanced, and the use of continuously increased pesticide amount can affect the growth of crops, not only cause pesticide pollution, but also affect the quality of crops, so that the reduction of pesticides has been developed into an inevitable trend.

The biocontrol bacteria and the pesticide bactericide are mixed, matched and applied, so that on one hand, the pesticide cost can be greatly reduced, the pesticide residue, the negative influence of the pesticide on plants and the safety risk of the pesticide on human bodies are reduced, on the other hand, the effect of biocontrol bacteria on preventing and treating diseases can be exerted, the effect of preventing and treating diseases is ensured, and the problem of enhancing the drug resistance of the diseases is solved. Therefore, it is urgently needed to provide pathogenic bacteria antagonistic bacteria compatible with the colas, explore the possibility of mixing the biocontrol bacteria and the chemical pesticide, and provide an effective way for improving the action effect of the biocontrol bacteria and reducing the production cost of growers.

Disclosure of Invention

In view of the above, the invention provides a wheat root rot biocontrol bacillus subtilis tolerant to pesticide tyras, which has good tolerance to pesticide tyras, has good antagonistic action on pathogenic bacteria of wheat root rot, can be used together with tyras to control wheat root rot, and realizes pesticide reduction on the basis of ensuring control effect.

In order to achieve the purpose, the invention adopts the following technical scheme:

wheat root rot biocontrol Bacillus subtilis (Bacillus subtilis) which tolerates pesticide Kulas is named as ND06, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.22696 and the preservation address of: xilu No. 1 Hospital No. 3, beijing, chaoyang, with a preservation time of 2021, 06 months and 11 days.

The application of the wheat root rot biocontrol bacillus subtilis tolerant to the pesticide tyrasi in preventing and treating wheat root rot is provided.

Further, in the control of wheat root rot, the pesticide colas is applied to wheat and the above-mentioned bacillus subtilis is inoculated.

According to the technical scheme, the bacillus subtilis ND06 disclosed by the invention has a strong tolerance effect on the Kulas, is used together with the Kulas, can achieve a control effect similar to that of the Kulas used alone under the condition that the Kulas is halved, and is suitable for popularization and application.

Drawings

FIG. 1 shows a selection of partially pesticide-resistant Bacillus species;

FIG. 2 shows antagonistic effect of part of the selected strains on Rhizopus cerealis;

FIG. 3 shows the growth of the antagonistic strain in a medium containing Kulas;

FIG. 4 shows the colony morphology of ND06 strain;

FIG. 5 shows a phylogenetic tree of strain ND 06.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 isolation, screening and identification of ND06

1. Test materials

The wheat plant sample is taken from the farm wheat planting field of the university of agriculture in Hebei, and the time for collecting the sample is 2019, month 4. The wheat root rot is preserved in the laboratory. The wheat variety used for the test is Jimai 418, the production place is fixed, and no coating is used.

2. Culture medium

PDA culture medium: 200g of potato, 20g of glucose, 15-20 g of agar and 1000mL of distilled water. Peeling potato, cutting into small pieces, adding water, boiling for 0.5 hr, filtering with double-layer gauze, adding sugar into the filtrate, and adding water to 1000mL.

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

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

3. Culture of wheat root rot pathogenic bacteria

Sterilized PDA medium was thawed and poured out of the plate, and streptomycin sulfate was added to give a final concentration of 40. Mu.g/100 mL. Inoculating the pathogenic bacteria of wheat root rot to the center of a PDA flat plate, and culturing for 5-7 days in an incubator at 26 ℃ until the plate is full of mycelia for later use.

4. Screening of Bacillus resistant to Kulas pesticide

Cleaning wheat seedlings with clear water, draining water with sterile filter paper, sterilizing the surface of wheat plants with 75.0% alcohol, grinding in a sterile mortar, placing ground seedling tissues into a sterilized 250mL conical flask (containing 100mL sterile water and glass beads), shaking and shaking for 30min, heating in a water bath at 80 ℃ for 5min, and separating and screening Bacillus crulas-resistant bacillus on an NA plate containing the pesticide Kulas (100 μ L/100 mL) by dilution coating method. The cells were cultured overnight at 37 ℃ and the selected strain resistant to colas was statistically numbered.

Through preliminary screening, 139 strains of bacillus capable of resisting the colas pesticide are screened from the young wheat seedlings (figure 1).

5. Screening of bacillus for antagonizing wheat root rot

Pathogenic bacteria are used as indicator bacteria, antagonism tests are carried out by adopting an antagonism culture method, the situation that the screened strain which is tolerant to the cool produces a bacteriostatic zone is detected, and the strain which antagonizes the rhizoctonia solani is screened.

Inoculating the wheat root rot pathogenic bacteria to a PDA inclined plane, and culturing at a constant temperature of 28 ℃ for 7d. Adding sterile water to the inclined surface of the pathogenic bacteria, and scraping the pathogenic bacteria by using a sterile glass rod to suspend the spores. Sterile water was used to adjust the number of spores in the suspension to a concentration of 1X 10 ⁶ mL, spore number was counted by a hemocytometer. 10mL of the pathogen spore suspension and 100mL of the PDA medium were mixed and poured onto a plate. After solidification, selecting the screened strains by using a sterile bamboo stick and inoculating the strains to a pathogenic bacteria flat plate, and carrying out inverted culture in an incubator at 26 ℃ for 5-7 d. And inoculating antagonistic bacteria capable of generating an obvious inhibition zone to the NA slant for constant-temperature culture at 37 ℃ overnight for later use. The strains with the inhibiting effect on the rhizoctonia solani comprise 73 strains, 6 strains have obvious antagonistic effect, and the width of the inhibition zone on the rhizoctonia solani is more than 1.5cm (figure 2).

6. Growth characteristic detection of antagonistic bacteria

The screened strains with remarkable antagonistic activity to pathogenic bacteria are subjected to constant-temperature shaking culture at 37 ℃ in an NB culture medium containing Kulas (the final concentration is 100 mu L/100 mL), the light absorption value of each strain at 600nm is detected every 24h, the culture time is 96h, and a control group without adding pesticide Kulas is arranged (the strains are selected as bacillus strains Z-5 which are screened in the early stage of a laboratory and are used for industrial production). And recording the good light absorption value, and drawing a growth curve. As shown in FIG. 3, the concentration of the fermentation broth of ND06 strain reached the maximum value at 72h of culture, which was close to the control group, indicating that the strain has strong tolerance to Kulas and has good growth in the presence of Kulas. Correspondingly, the growth of the remaining 5 strains was significantly inhibited in the medium containing coolas.

7. Identification of strains

The colony of the ND06 strain growing on the NA plate is round or oval, brownish yellow, has wrinkles on the surface, is sunken in the center, is crater-shaped, and has darker convex parts (figure 4). The species identification of the selected strains was carried out according to the method of Bergey's Manual of identification of bacteria. The 16S rDNA sequence of the selected strain was amplified using the universal primers 27f and 1495 r. The sequencing result of the PCR product is subjected to Blast comparison in an NCBI database, sequence homology comparison with a model strain is carried out, and MEGA6.0 software is used for constructing a phylogenetic tree. The results showed that the ND06 strain was Bacillus subtilis (FIG. 5).

Example 2 prevention and treatment of wheat root rot by Mixed application of antagonistic bacteria and pesticide

The ND06 strain with good growth condition in the culture medium containing the Kulas is streaked and inoculated in an NA culture medium, and is cultured overnight at constant temperature of 37 ℃, and a single colony is picked up in an NB culture medium and is cultured for 48h by shaking in a shaking table at 37 ℃. Centrifuging the fermentation liquid at 8000rpm for 5min to recover thallus, suspending and diluting with sterile water to viable bacteria concentration of about 1 × 10 ⁸ cfu/mL, bacterial count was performed using bacterial count plates.

4 groups of pot experiment are set, namely a blank control group CK0, a pathogenic bacterium group CK1, a pesticide treatment group NY and a bactericide and pesticide group JY respectively, and each treatment is set to be 5 times. A flowerpot with the diameter of 12.9cm and the height of 12.4cm is taken, and 12 wheat seeds are uniformly sown in each flowerpot. The blank control group was irrigated with water without inoculation of pathogenic bacteria and antagonistic bacteria. The pathogenic bacteria group is inoculated with wheat root rot pathogenic bacteria, the wheat seeds are placed on pathogenic bacteria tablets (the diameter is 7 mm), 1 wheat seed is placed on each pathogenic bacteria tablet, and the soil for planting wheat is taken from a farm wheat planting field of Hebei agriculture university. The mode of inoculating pathogenic bacteria by the pesticide treatment group and the bacteria agent and pesticide adding group is the same as that of the pathogenic bacteria group. The pesticide treatment group is used for dressing wheat seeds according to the dosage of inoculating 100kg of seeds into 600mL of coolas; and the group of the microbial inoculum and the pesticide is half-reduced in pesticide application, 20mL of ND06 bacterial suspension is irrigated, the mixture is cultured at room temperature, and the control effect is investigated after 20 days of planting. The plant growth status was checked on a plant-by-plant basis, with 20 plants per group being sampled and the average calculated.

The severity grading standard of the wheat root rot is as follows:

stage 0: the disease is not developed;

level 1: the outer leaf sheath of the stem base of the wheat plant turns black brown, and the length of the scab is less than 1/2 of the leaf sheath;

stage 2: the outer leaf sheath of the stem base of the wheat plant turns into black brown, and the length of the disease spot is more than 1/2 of the length of the leaf sheath;

and 3, level: the leaf sheath in the stem base of the wheat plant turns black and brown, and the length of the lesion is less than 1/2 of the leaf sheath;

4, level: the leaf sheath in the stem base of the wheat plant turns black and brown, and the length of the lesion is more than 1/2 of the leaf sheath;

and 5, stage: the roots rot and the plants die.

Disease index = [ Σ (number of diseased plants at each stage × representative value)/total number of investigated plants × representative value of the most serious stage of disease attack ] × 100.

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

Data analysis was performed using SPSS statistics 21 statistical software, one-way analysis of variance using ANOVA, and multiple comparisons using the Duncan's method.

The results are shown in Table 1. The bacterial agent and pesticide group JY has no obvious difference compared with the pesticide treatment group NY and the blank control group CK0 in root length, root weight, overground height and overground weight and has obvious difference with the pathogenic bacteria group CK 1. The control effect of JY group on wheat root rot is up to 74.6%, slightly higher than NY group, but the difference between them is not significant. The application of the ND06 strain fermentation liquor can effectively prevent and treat the occurrence of wheat root rot under the condition that the pesticide is half applied, thereby achieving the similar prevention and treatment effect as the full application of the Kulas and achieving the purpose of effectively preventing and treating the occurrence of diseases under the condition that the pesticide is not applied.

TABLE 1 prevention and control of wheat root rot by Mixed application of Kulas and ND06 strains

CK0 is a blank control group, CK1 is a pathogenic bacteria group, NY is a pesticide treatment group, and JY is a microbial inoculum and pesticide group.

Example 3 field test to examine the Effect of ND06 Strain in reducing the use of pesticide Kulas for controlling wheat root rot

Selecting a land with serious and uniform wheat root rot in the last year, wherein the area of the land is about 666.7m ² Divided into 3 small blocks, each block is about 222.2m ² Respectively set as a control group, an agricultural medicine group and a bactericide and an agricultural medicine group. Pesticide groups were as per 6mL of colas pesticide: 1kg of seeds are mixed, the seeds and the pesticide are added into a firm plastic bag, the opening is tightly bound, and the pesticide and the wheat seeds are uniformly mixed by turning upside down. The microbial inoculum and pesticide composition is applied by halving the pesticide, and viable bacteria content is about 1.0 × 10 ¹⁰ The ND06 microbial inoculum of cfu/mL is used for seed dressing, and the inoculation amount is 10mL/kg of seeds. Adopts mechanical seeding and the management mode is the same as that of the common field.

In the wheat milk stage, sampling is carried out by adopting a 5-point sampling method, 3 continuous lines are taken at each point, 0.4m is taken at each line, the plant height and the average grain number per spike are surveyed and counted (30 plants are taken at each random sampling point, 150 plants are sampled at each group), the seeds are weighed after being dried, the yield per square meter is measured by single threshing and single harvesting, and the results are shown in a table 2.

TABLE 2 prevention and control effect of mixed application of ND06 strain and pesticide-Kulas on wheat root rot during wheat milk stage

The data in the table are mean ± standard deviation. The different letter designations after the same column of data were significantly different at a level of P <0.05 by the LSD method.

The investigation on the control effect of the root rot in the milk stage of the wheat shows that compared with a control group, the plant height and the yield of a fungicide and pesticide group are obviously increased, the plant height of the wheat is increased from 79.66cm to 87.13cm, and the yield of the wheat is increased from 602.15g/m ² Increased to 778.54g/m ² The effect is obvious, and the application of the microbial inoculum prepared by the ND06 strain can effectively reduce the use of the pesticide, namely the colas, without reducing the control effect on the wheat root rot. Compared with the fungicide and pesticide group and the pesticide group, the plant height and yield of the wheat are increased, but the difference is not obvious.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to 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

1. Wheat root rot biocontrol bacillus subtilis (Bacillus subtilis) resistant to pesticide KulasBacillus subtilis) The strain ND06 is characterized in that the strain ND06 is preserved in China general microbiological culture Collection center (CGMCC) at 11 th of 2021 and 06 th of month, and the preservation number is CGMCC NO.22696.

2. The use of the pesticide-resistant Bacillus subtilis for biocontrol of wheat root rot of colas as claimed in claim 1 for controlling wheat root rot,

the pesticide, coolas, was applied to wheat and the bacillus subtilis was inoculated.