CN116114712B - Bactericidal composition containing bacillus amyloliquefaciens and application thereof - Google Patents

Abstract

The invention belongs to the field of agricultural bactericides, and particularly relates to a bactericidal composition containing bacillus amyloliquefaciens, which is characterized by comprising a bacillus amyloliquefaciens WHIG microbial inoculum and fluxapyroxad hydroxylamine; the mass ratio of the bacillus amyloliquefaciens WHIG microbial inoculum to the fluxapyroxad hydroxylamine is 100:1-1:10. The invention utilizes the control strategy of combining biological control and chemical control, not only can reduce the application amount of chemical agents and improve the stability of biological control, but also can achieve the effect of effectively controlling diseases, and the composition shows remarkable synergy.

Description

A kind of bactericidal composition containing bacillus amyloliquefaciens and its application

technical field

The invention belongs to the field of agricultural bactericides, and in particular relates to a bactericidal composition containing Bacillus amyloliquefaciens and an application thereof.

Background technique

Rapeseed belongs to Angiospermae , Dicotyledons, Dilleniidae , Brassicales , Brassicaceae , and Brassica . Rapeseed not only has economic value, but also has ornamental value. It can be used for ornamental purposes when it blooms, and rapeseed oil can be squeezed into edible rapeseed oil after the rape flowers wither, and rapeseed has high medicinal and industrial values. At present, China's rapeseed planting area is the second largest in the world, only after India. Rapeseed is mainly planted in Anhui, Sichuan, Jiangxi, Jiangsu, Henan and other places in China.

Rapeseed is the most important oil crop in my country and one of the four major oil crops in the world. The oil content of its seeds is 33%-50%. It has important economic value and is widely used in medicine, food and industrial raw materials. Rapeseed has a long growth period and a wide variety of diseases. Among them, the most serious damage is Sclerotinia. During the growth period of rapeseed, Sclerotinia sclerotiorum may occur, and the incidence is the most severe during the flowering and fruiting period. The pathogen can infect all parts of the rapeseed plant above the ground, especially the stalks caused the greatest loss after the disease. Most of the pathogens are infected from the broken flowers, which are water-soaked or pale at first, and then the petals fall to cause the disease of stems and leaves. After the stem of the plant is infected with Sclerotinia sclerotiorum, it initially appears as water-soaked lesions, and then the lesions gradually turn brown, sunken inwards, and grow white flocculent hyphae and black rat feces-like sclerotias. When too dry, the diseased part at the base of the stem shrivels up and dies. When the leaves of the plant are infected with Sclerotinia sclerotiorum, the initial manifestations are dark blue water-soaked and irregular spots with concentric ring patterns. When the air becomes humid, the growth rate of the lesions becomes faster, causing the leaves to rot quickly, and then white flocculent hyphae and small black rat droppings-like sclerotia will grow on the lesions. The typical symptoms of rapeseed sclerotinia are white flocculent mold and black rat droppings-like sclerotia.

For a long time, the control of Sclerotinia sclerotiorum mainly relied on chemical control. The application of fungicides has a certain effect on the prevention and control of rapeseed sclerotinia, but the effect is affected by various factors such as the application time, plant development stage and environmental conditions, and the extensive use of chemical agents will not only accumulate chemical pesticide residues, but also affect human health , will destroy the ecological balance and cause environmental pollution. Long-term use of chemical agents will lead to drug resistance of pathogenic bacteria, leading to a decline in the control effect or even failure, which forces people to seek safer and more effective control methods. Rapeseed, as a worldwide oil crop, is widely used in food, medicine, etc. Using biocontrol bacteria to prevent and control diseases can not only reduce the use of chemical pesticides, but also reduce environmental pollution. As an environmentally friendly control technology, it is becoming more and more are becoming more and more popular.

Bacillus amyloliquefaciens is a Gram-positive bacillus, a bacterium closely related to Bacillus subtilis, which is widely distributed in nature. During the growth process, Bacillus amyloliquefaciens can produce a series of metabolites with extensive fungal and bacterial activity, which has broad application prospects in biological control and has gradually become a microorganism with potential for biopesticide development. Bacillus amyloliquefaciens WHIG was isolated from the Rice Disease Control Research Group of the Institute of Plant Protection and Agricultural Product Quality and Safety, Anhui Academy of Agricultural Sciences, and was preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on April 15, 2015. The preservation number is It is CGMCC No.10722. The strain is named WHIG, and the taxonomy is named: Bacillus amyloliquefaciens (refer to CN107136120A).

Flufenapyramide is a new type of pyrazole amide fungicide, which belongs to the succinate dehydrogenase inhibitor (SDHI) fungicide class. It is a pyrazole-4-amide compound with novel structure, high activity and broad fungicide spectrum; However, the current production and use costs of flufenapyramide are relatively high, and there is a great risk of resistance when used alone for a long time.

The combination of biological control and chemical control can not only reduce the application amount of chemical agents, improve the stability of biological control, but also achieve the effect of effective disease control. In the prior art, Bacillus amyloliquefaciens WHIG and Related reports on the combination use of flufenapyramide. The applicant found that the combination of Bacillus amyloliquefaciens WHIG and flufenapyramide has a very good synergistic effect on Sclerotinia sclerotiorum.

Contents of the invention

The invention utilizes the control strategy of combining biological control and chemical control, which can not only reduce the application amount of chemical agents, but also improve the stability of biological control, and the composition exhibits a significant synergistic effect.

Technical scheme of the present invention is:

A bactericidal composition comprising Bacillus amyloliquefaciens, characterized in that it comprises Bacillus amyloliquefaciens WHIG bacterial agent and flufenapyl hydroxylamide; the mass ratio of the Bacillus amyloliquefaciens WHIG bacterial agent and flufenapyroxylamide is 100:1-1:10; Bacillus amyloliquefaciens WHIG is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, the preservation number is CGMCCNo.10722; the concentration of Bacillus amyloliquefaciens WHIG is 1×10 ⁶ -1×10 ¹⁰ cfu/mL.

Preferably, the concentration of the Bacillus amyloliquefaciens WHIG agent is 1×10 ⁷ -1×10 ⁹ cfu/mL; the mass ratio of the Bacillus amyloliquefaciens WHIG agent and flufenapyramide is 80:1-10: 1. More preferably, the concentration of the Bacillus amyloliquefaciens WHIG agent is 1×10 ⁸ cfu/mL; the mass ratio of the Bacillus amyloliquefaciens WHIG agent to flufenapyramide is 80:1-10:1.

The bactericidal composition also includes pesticide auxiliary material components, which are prepared into pesticide preparations. The pesticide preparation is a suspension concentrate, a wettable powder, and a water-dispersible granule. The adjuvant is selected from conventional adjuvant components in the field, and is prepared by a conventional preparation method.

The fungicidal composition can be used for preventing and controlling crop diseases. A preferred crop disease is Sclerotinia sclerotiorum.

Adopt above-mentioned technical scheme, the present invention has following beneficial effect:

The invention utilizes the prevention and control strategy combining biological control and chemical control, which can not only reduce the application amount of chemical agents, improve the stability of biological control, but also achieve the effect of effectively controlling diseases.

When the mass ratio of WH1G bacterial suspension to flufenapyramide is 80:1-10:1, the composition exhibits a synergistic effect on Sclerotinia sclerotiorum, not only can effectively reduce the dosage of a single agent, It can also delay the emergence of bacterial resistance.

Detailed ways

The present invention will be further explained below in combination with specific embodiments.

Example 1: Indoor Toxicity Test of Compound Agents to Rapeseed Sclerotinia

1.1 Test materials

Biocontrol bacteria: Bacillus amyloliquefaciens WH1G, Institute of Plant Protection and Agricultural Product Quality Safety, Anhui Academy of Agricultural Sciences. Use improved NA medium (beef extract 1.0g, peptone 5.0g, yeast extract 5.0g, NaCl 5.0g, sucrose 10.0g, agar 20.0g, distilled water 1000mL, pH 6.8-7.0), in a 28°C incubator nourish. Prepare a suspension of Bacillus amyloliquefaciens WH1G with a concentration of 1×10 ⁸ cfu/mL.

Sclerotinia sclerotiorum was isolated, screened, identified and preserved by the Institute of Plant Protection and Agricultural Product Quality Safety, Anhui Academy of Agricultural Sciences.

Flufenapyramide, commercially available.

1.2 Test method

The mycelial growth rate method was used. On the basis of the pre-experiment, PDA plates containing different gradient concentrations of drugs were prepared, and the Sclerotinia sclerotiorum bacteria block (diameter 5mm) was placed in the center of the plate containing the drug, and placed in a 28°C incubator for cultivation. A control was set up, and each treatment was repeated 4 times. When the control colony covered 2/3 of the petri dish, the diameter of the colony of each treatment was measured by the cross method, and the average inhibition rate was calculated.

Average inhibition rate = [(average colony diameter of the control group-average colony diameter of the treatment group)/(average colony diameter of the control group-5.0)]×100%.

Convert the mycelia growth inhibition rate into the inhibition probability value (y), and convert the drug concentration into the concentration logarithm (x), and draw the toxicity regression line according to the concentration logarithm as the abscissa and the probability value as the ordinate, to obtain the single agent and Toxicity regression equation of its mixture to bacteria, and calculate EC ₅₀ value and correlation coefficient r value.

According to Sun Yunpei's method, the toxicity index of each drug and the co-toxicity coefficient (CTC value) of the mixture were calculated, that is, CTC ≤ 80 was antagonism, 80<CTC<120 was additive, and CTC ≥ 120 was synergistic.

Co-toxicity coefficient (CTC) = measured toxicity index (ATT) of the mixture/theoretical toxicity index (TTI) of the mixture.

Actual Toxicity Index (ATI) = standard drug EC ₅₀ / tested drug EC ₅₀ × 100

Theoretical toxicity index (TTI) = toxicity index of agent A × percentage content of A in the mixture + toxicity index of agent B × percentage content of B in the mixture.

1.3 Test results

It can be seen from Table 1 that the EC ₅₀ value of WH1G bacterial suspension against S. sclerotiorum was 20.68 mg/L; the EC ₅₀ value of flufenapyramide against S. sclerotiorum was 0.19 mg/L. When the mass ratio of WH1G bacterial suspension to flufenapyramide was 80:1-10:1, it showed synergistic effect on Sclerotinia sclerotiorum, and it showed additive effect under other ratio conditions. When the composition exhibits a synergistic effect, it can effectively reduce the dosage of a single agent and slow down the generation of disease resistance.

Table 1 Indoor toxicity test of compound agents against Sclerotinia sclerotiorum

Remarks: 1mL in the bacterial suspension configuration is calculated as 1g.

Embodiment 2: The pot test of compound medicament to Sclerotinia sclerotiorum

2.1 Test materials

Biocontrol bacteria: Bacillus amyloliquefaciens WH1G, Institute of Plant Protection and Agricultural Product Quality Safety, Anhui Academy of Agricultural Sciences. Use improved NA medium (beef extract 1.0g, peptone 5.0g, yeast extract 5.0g, NaCl 5.0g, sucrose 10.0g, agar 20.0g, distilled water 1000mL, pH 6.8-7.0), in a 28°C incubator nourish. Prepare a suspension of Bacillus amyloliquefaciens WH1G, with a bacterial content of 1×10 ⁸ cfu/mL.

Rapeseed Sclerotiorum ( S.sclerotiorum ) was isolated, screened, identified and preserved by the Institute of Plant Protection and Agricultural Product Quality and Safety, Anhui Academy of Agricultural Sciences.

Flufenapyramide, commercially available.

2.2 Test method

The agents of different concentrations were evenly sprayed on the surface of rapeseed leaves, and 24 hours later, the surface of the leaves was inoculated with Sclerotinia sclerotiorum bacteria. Set controls. Each treatment was repeated 10 plants, and the experiment was repeated 3 times. Placed in an incubator to moisten the culture. The disease incidence was investigated 7 days after the inoculation, the severity of the disease was recorded, and the disease index was calculated.

Grading criteria for disease severity:

Grade 0, no disease;

Grade 1, the diseased area accounts for less than 5% of the total leaf area;

Grade 3, the diseased area accounts for 5%-10% of the total leaf area;

Grade 5, the diseased area accounts for 11%-30% of the total leaf area;

Grade 7, the diseased area accounts for 31%-50% of the total leaf area;

Grade 9, the diseased area accounts for more than 50% of the total leaf area.

Disease index = ∑ [(Number of diseased plants at all levels × value of relative disease level) / (Total number of plants under investigation × value of highest disease level)] × 100

Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100

2.3 Test results

It can be seen from Table 1 that the control effect of WH1G bacterial suspension alone on rape sclerotinia is 50.9%; the control effect of flufenapyramide alone on rape sclerotinia is 59.4%. The combined use of WH1G bacterial suspension and flufenapyramide can significantly improve the control effect of the agent on Sclerotinia sclerotiorum, and has a significant synergistic effect.

Table 2 Pot test of compound agents on Sclerotinia sclerotiorum

Remarks: 1mL in the bacterial suspension configuration is calculated as 1g.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention are considered to be within the protection scope of the present invention.

Claims (4)

1. A bactericidal composition containing bacillus amyloliquefaciens, which is characterized by comprising bacillus amyloliquefaciens WHIG bacterial agent and fluxapyroxad hydroxylamine; the mass ratio of the bacillus amyloliquefaciens WHIG microbial inoculum to the fluxapyroxad hydroxylamine is 80:1-10:1;

the bacillus amyloliquefaciens WHIG is preserved in China general microbiological culture collection center (CGMCC No. 10722);

bacillus amyloliquefaciens WHIG microbial inoculum concentration 1×10 ⁸ cfu／mL。

2. The bactericidal composition of claim 1, further comprising a pesticidal adjuvant component, wherein the bactericidal composition is formulated as a pesticidal formulation.

3. The bactericidal composition of claim 2, wherein the pesticide formulation is a suspension, a wettable powder, or a water dispersible granule.

4. Use of a fungicidal composition according to any one of claims 1-3 for controlling crop diseases, wherein the crop disease is sclerotium disease of colza.