CN107509744B

CN107509744B - Compound biopesticide for preventing and treating wheat stem basal rot and preparation method and application thereof

Info

Publication number: CN107509744B
Application number: CN201710716555.XA
Authority: CN
Inventors: 张洁; 杨丽荣; 夏明聪; 薛保国; 孙润红; 武超; 李佳; 杨艳
Original assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Priority date: 2017-08-18
Filing date: 2017-08-18
Publication date: 2020-11-24
Anticipated expiration: 2037-08-18
Also published as: CN107509744A

Abstract

The invention discloses a compound biopesticide for preventing and treating wheat stem basal rot, a preparation method thereof and application thereof in increasing yield of wheat. The biological pesticide is a compound biological pesticide prepared by uniformly mixing a bacillus subtilis microbial inoculum and shenqinmycin according to a proportion. The biological pesticide is efficient, green and safe, reduces the use of agricultural chemical fertilizers, and is beneficial to the nuisanceless production of wheat crops; the control effect on the wheat stem base rot caused by fusarium pseudograminearum is obviously improved, the yield increasing function is realized, the income of farmers can be increased, and the development and utilization potential is very large.

Description

Compound biopesticide for preventing and treating wheat stem basal rot and preparation method and application thereof

Technical Field

The invention relates to the technical field of pesticide compounding, in particular to a compound biopesticide for preventing and treating wheat stem basal rot, a preparation method thereof and application thereof in wheat yield increase.

Background

The wheat stem basal rot is a soil-borne disease caused by various pathogenic fungi, is commonly called as 'drought foot disease', is increasingly serious in Huang-Huai-Mai region in China in recent years, and brings serious threat to wheat safety production in China. Among them, fusarium graminearum (fusarium graminearum) is a pathogenic bacterium newly found in the stem basal rot of wheat in China in recent years, has strong pathogenicity and fast transmission, is a main pathogenic bacterium of the stem basal rot of wheat at present, seriously affects the yield and quality of wheat, and brings serious economic loss to agricultural production in China. In addition, fusarium pseudograminearum also produces some toxins, which cause serious harm to human and animal health. Although the chemical agent has a certain effect on preventing and treating the wheat stem basal rot, the chemical pesticide has high toxicity and high residue, pollutes the environment, causes pathogenic bacteria to generate drug resistance, seriously threatens the sustainable development of agriculture in China, and is very important for researching and developing environment-friendly biopesticides.

The bacillus subtilis is a biocontrol bacterium with great development and utilization potential and has a wide antibacterial spectrum. At present, the bacterial strain has strong inhibition effect on pathogenic bacteria such as wheat take-all pathogen, wheat sheath blight pathogen, wheat scab pathogen, tomato botrytis cinerea, cucumber botrytis cinerea, apple brown spot pathogen, yam colletotrichum, watermelon wilt pathogen, cucumber phytophthora blight pathogen, cotton red rot pathogen and the like, and has been developed into a biological agent for biological control of wheat take-all. The shenqinmycin is an antibiotic secreted by pseudomonas fluorescens, has strong disease-resistant and growth-promoting effects, is used for preventing and treating wheat take-all and wheat scab at present, but is not found to be used for preventing and treating wheat stem base rot caused by fusarium pseudograminearum.

Disclosure of Invention

The invention aims to solve the technical problem of providing a biopesticide prepared by reducing and compounding bacillus subtilis and shenqinmycin and application thereof in preventing and treating wheat stem basal rot caused by fusarium pseudograminearum and increasing the yield of wheat.

In order to solve the technical problems, the invention adopts the following technical scheme:

a compound biological pesticide for preventing and treating wheat stem basal rot is designed, and contains a bacillus subtilis microbial inoculum and shenqinmycin.

The bacillus subtilis is a bacillus subtilis YB-05 strain with the preservation number of CGMCC NO. 6498.

The bacillus subtilis preparation is prepared by the following steps:

inoculating a bacillus subtilis YB-05 strain to a LA culture medium, streaking, and culturing for 30-36h at the temperature of 28-32 ℃ until a single colony grows out; selecting a single colony, inoculating the single colony to an LB culture solution, performing shake culture at 30-32 ℃ and 160rpm for 12-16h to prepare a seed solution; inoculating the seed liquid into a fermentation culture medium in an inoculation amount of 2%, performing shake culture at 30-32 ℃ and 160rpm for 48-54h, and diluting the fermentation liquid by 10 times with sterile water.

Preferably, the ratio of the bacillus subtilis microbial inoculum to the 1% by weight of the shenqinmycin is 20-17: 5-1 in terms of volume ratio.

Preferably, the concentration of the shenqinmycin in the compound biological pesticide is 0.5 mg/ml.

Preferably, the colony concentration of bacillus in the compound biological pesticide is 10⁷～10⁸CFU/ml。

A preparation method of a compound biopesticide for preventing and treating wheat stem basal rot is designed, and comprises the following steps:

(1) inoculating bacillus subtilis to LA culture medium, streaking, and culturing at 28-32 deg.C for 30-36h until single colony grows; selecting the single colony to be inoculated into an LB culture solution, performing shake culture at 30-32 ℃ and 160rpm for 12-16h to prepare a seed solution; inoculating the seed solution into a fermentation culture medium in an inoculation amount of 2%, performing shake culture at 30-32 ℃ and 160rpm for 48-54h, and diluting the fermentation liquor by 10 times with sterile water to obtain a bacillus subtilis microbial inoculum;

(2) and uniformly mixing the bacillus subtilis microbial inoculum and the shenqinmycin.

Preferably, the fermentation medium contains 1% sucrose, 1.5% corn flour, 3.5% soybean flour, 0.2% dipotassium hydrogen phosphate, and has a pH of 7.2.

The application of the compound biopesticide for preventing and treating the basal rot of wheat in the yield increase of wheat.

The invention has the beneficial technical effects that:

1. the compound biopesticide has the characteristics of high efficiency, greenness, safety and environmental friendliness, reduces the use of pesticides and fertilizers, is favorable for the pollution-free production of wheat crops, and is favorable for improving the grain quality; the biological pesticide has low cost and saves the expenditure for farmers.

2. The compound biopesticide obviously improves the control effect on the wheat stem basal rot caused by fusarium pseudograminearum.

3. The compound biopesticide has the effect of increasing yield, can increase income of farmers, and has wide market prospect.

Drawings

FIG. 1 is a graph showing the effect of Bacillus subtilis YB-05 on the growth of Fusarium pseudograminearum Fp 001;

FIG. 2 is a graph showing the growth inhibition effect of a fermentation filtrate of Bacillus subtilis YB-05 on Fusarium pseudograminearum Fp 001;

FIG. 3 is a graph showing the effect of the fermentation filtrate of Bacillus subtilis YB-05 on the morphology of Fusarium pseudograminearum Fp001 hyphae;

FIG. 4 is a graph showing the inhibitory effect of different concentrations of shenqinmycin on Fusarium pseudograminearum Fp 001;

FIG. 5 is a graph showing the inhibitory effect of different concentrations of shenqinmycin on Bacillus subtilis YB-05;

FIG. 6 is a graph of the indoor control effect of different biocontrol agents on wheat stem base rot;

FIG. 7 is a field control effect diagram of the compound biopesticide on wheat stem basal rot.

Detailed Description

The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way. The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the raw materials and reagents are all conventional products sold in the market if not specified; the experimental methods are conventional experimental methods unless otherwise specified.

Example 1:

growth effects of bacillus subtilis on fusarium pseudograminearum Fp 00:

(1) the bacillus subtilis is YB-05 strain protected by CN 103074271A;

(2) the fusarium pseudograminearum cake is punched by a puncher and transferred to the center of PDA, the position about 2.5cm away from the fusarium pseudograminearum cake is inoculated with YB-05 strains symmetrically on two sides of the fusarium pseudograminearum Fp001 cake, and plates only inoculated with pathogenic bacteria are used as controls to repeat for 3 times. Culturing in an incubator at 25 ℃, measuring the width of an antibacterial band after the mycelium of a control group grows on a flat plate, measuring the growth diameter of the bacterial colony of the pathogenic bacteria of the wheat stem basal rot, and calculating the growth inhibition rate of the bacterial colony, wherein the calculation formula is as follows:

the bacterial colony growth inhibition ratio (%) (control bacterial colony growth diameter-treated bacterial colony net growth diameter)/control bacterial colony net growth diameter × 100%

The experimental result is shown in figure 1, the inhibition zone of the bacillus subtilis YB-05 on fusarium pseudograminearum Fp001 reaches 14mm, and the colony growth inhibition rate is 75%. Therefore, the strain YB-05 has strong inhibiting effect on fusarium pseudograminearum Fp 001.

Example 2:

the influence of the fermentation filtrate of the bacillus subtilis YB-05 on the growth of fusarium pseudograminearum Fp001 comprises the following steps:

inoculating bacillus subtilis YB-05 in an LB culture medium, culturing at 30 ℃ for 36h at 180r/min, centrifuging at 12000r/min for 5min, taking supernatant, filtering out thalli by using a 0.22 mu m bacterial filter, uniformly mixing obtained fermentation filtrate with a PDA culture medium according to a ratio of 1:10, pouring the mixture into a flat plate, taking the PDA flat plate added with clear water as a control group, repeating for 3 times, inoculating a pseudo-fusarium graminearum cake with the diameter of 0.9cm in the center of the flat plate, culturing for 7d, determining the diameter of a bacterial colony by using a cross method, and calculating the colony growth inhibition rate.

As shown in figure 2, compared with a clear water control, the fermentation filtrate of the Bacillus subtilis YB-05 strain obviously inhibits the growth of fusarium pseudograminearum Fp001, and the inhibition rate reaches 83%. The influence of the fermentation filtrate of the strain YB-05 of the bacillus subtilis on the shape of the fusarium pseudograminearum Fp001 hypha is observed through a microscope, as shown in figure 3, after the fermentation filtrate is treated, the hypha has the obvious malformation phenomena of hypha expansion, hypha thickening, hypha node shortening and the like, and the untreated hypha has smooth surface, uniform thickness and normal growing points. The secondary metabolite in the strain fermentation filtrate has obvious influence on the growth of fusarium pseudograminearum Fp001 hypha, and inhibits the growth of the fusarium pseudograminearum Fp 001.

Example 3:

the preparation method of the bacillus subtilis YB-05 microbial agent comprises the following steps:

(1) strain culture and preparation: inoculating the strain YB-05 to a LA culture medium, streaking, and culturing at 28-32 ℃ for 30-36h until a single colony grows;

(2) preparing a seed solution: inoculating the single colony obtained in the step (1) into an LB culture solution, culturing at 30-32 ℃ and 160rpm under shaking for 12-16h to prepare a seed solution;

(3) preparation of a fermentation medium: adding water into sucrose, corn flour, soybean powder, dipotassium hydrogen phosphate to prepare a fermentation medium containing 1% of sucrose, 1.5% of corn flour, 3.5% of soybean powder and 0.2% of dipotassium hydrogen phosphate, wherein the pH value is 7.2, and autoclaving the fermentation medium at 121 ℃ for 30 min;

(4) liquid fermentation production: inoculating the seed solution prepared in the step (2) into the fermentation culture medium in the step (3) according to the inoculation amount of 2%, performing shake culture at 30-32 ℃ and 160rpm for 48-54h, and collecting the culture solution, wherein the cell biomass is 10⁹-10¹⁰And diluting the CFU/mL by 10 times with sterile water to obtain the YB-05 microbial agent.

Example 4:

the method for determining the inhibition effect of shenqinmycin on fusarium pseudograminearum comprises the following steps of:

1% shenqinmycin and the like are diluted by gradient with sterile water and are mixed with PDA culture medium uniformly to make the final concentrations respectively be 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001 and 0.00005mg/ml, and then pseudo-gramineae fusarium cake with the diameter of 0.9cm is inoculated in the center of a dish, and the control is that PDA is added with equal amount of clear water. After the contrast group hyphae grow over the plate, the colony diameter in each treatment is measured by a cross method, and the inhibition effect of the shenqinmycin with each concentration on the fusarium pseudograminearum is calculated.

The combination of fig. 4 and table 1 shows that 1, 0.5, 0.1, 0.05 and 0.01mg/ml shenqinmycin completely inhibits the growth of fusarium pseudograminearum hyphae, the inhibition rate reaches 100%, the inhibition rates of 0.005, 0.001, 0.0005 and 0.0001mg/ml shenqinmycin on fusarium pseudograminearum hyphae are all more than 80%, respectively reach 89.44%, 87.78%, 83.33% and 80%, and the inhibition rate of 0.00005mg/ml shenqinmycin on fusarium pseudograminearum also reaches 77.78%. Therefore, the shenqinmycin has stronger inhibiting effect on fusarium pseudograminearum.

TABLE 1 inhibition of Fusarium pseudograminearum by shenqinmycin at different concentrations

Concentration (mg/ml)	Inhibition ratio (%)	Concentration (mg/ml)	Inhibition ratio (%)
				1	100	0.005	89.4
0.5	100	0.001	87.8
				0.1	100	0.0005	83.3
0.05	100	0.0001	80.0
				0.01	100	0.00005	77.8

Example 5:

the safe concentration determination of the shenqinmycin on the strain YB-05 of the bacillus subtilis comprises the following steps:

the method comprises the steps of utilizing sterile water to carry out gradient dilution on 1% shenqinmycin and the like, evenly mixing the shenqinmycin and the like with a PDA culture medium to enable the final concentrations to be 1, 0.5, 0.1 and 0.05mg/ml respectively, pouring plates and airing the plates, selecting a bacillus subtilis YB-05 bacterial colony to mark on the plates with various concentrations, and observing the growth condition of the bacterial strain by taking the culture medium without the shenqinmycin as a control.

As shown in FIG. 5, compared with the control, 1mg/ml of shenqinmycin has a certain inhibition effect on the strain YB-05 of the bacillus subtilis, and 0.5, 0.1 and 0.05mg/ml of shenqinmycin has no obvious inhibition effect on the growth of the strain YB-05 of the bacillus subtilis.

Example 6:

the preparation of the compound biopesticide of the bacillus subtilis YB-05 strain and the shenqinmycin comprises the following steps:

taking the bacillus subtilis YB-05 microbial inoculum of the embodiment 3 and 1% shenqinmycin with the concentration of 0.5mg/ml, and fully and uniformly mixing according to the proportion of 20:3(vt/vt) to obtain the compound biopesticide; measuring the bacterial colony concentration of bacillus subtilis YB-05 in the compound biological pesticide, and measuring the concentration to be 10 by diluting and coating the biological pesticide⁷-10⁸CFU/mL。

Example 7:

the difference from the example 6 is that the ratio of the bacillus subtilis YB-05 to the shenqinmycin calculated by vt/vt is 18: 5.

Example 8:

the difference from the example 7 is that the ratio of the bacillus subtilis YB-05 to the shenqinmycin calculated by vt/vt is 19: 1.

Example 9:

the difference from the example 8 is that the ratio of the bacillus subtilis YB-05 to the shenqinmycin calculated by vt/vt is 17: 2.

Example 10:

compared with the indoor control effect of the bacillus subtilis YB-05 strain, the shenqinmycin and the compound biopesticide on the wheat stem basal rot, the method comprises the following steps:

the experiment is carried out in a greenhouse of agricultural science institute in Henan province, and the matrix material used in the indoor pot culture experiment is the soil: grass carbon: vermiculite 7: 2: 1, and mixing the components in a ratio of 1. Beating a fresh fusarium pseudograminearum Fp001 strain in a culture dish into cakes, boiling millet until the millet is mature, cooling and bottling, inoculating 2 fusarium pseudograminearum cakes into each bottle, culturing in an incubator at 25 ℃ for 7-10 days, and then mixing with soil according to the inoculation amount of 0.5% for use, wherein the tested wheat variety is zheng 366, and the contrast agent is 2% tebuconazole 1 ml/kg. The treatment method is shown in table 2:

table 2 indoor control test treatment method for wheat stem base rot by each experimental group

Processing number	Name of process
		1	YB-05 microbial inoculum 30ml/kg seed dressing
2	Seed dressing with 1% shenqinmycin 2ml/kg
		3	YB-05 and 1% shenqin20ml/kg seed dressing of mycin 19:1(vt/vt) biopesticide
4	Seed dressing treatment of 2% tebuconazole 1ml/kg
		5	CK

After inoculation, the potted plants are regularly observed and watered, the roots of the wheat are washed by water after 30 days, the disease incidence is investigated, and the disease index and the disease prevention effect are calculated according to the disease incidence.

Grading standard:

0: no symptoms; 1: browning blastoderm nodes; 2: blastoderm nodes, mediterranean stems or leaves\37559; 3; the hypocotyl nodes, the underground stems or the leaf pins are all browned; 4: browning the underground stem, leaf pin and lower stem; 5; the stems have the symptoms of dry rot and rot; 6; plant death due to stalk rot; 7: seedling withering or seed decay.

Calculating disease index and disease prevention effect according to the following formula:

the results are shown in fig. 6 and table 3, the bacillus subtilis YB-0530ml/kg seed dressing treatment and 1% shenqinmycin 2ml/kg seed dressing treatment have better control effects on the wheat stem basal rot, and the control effects respectively reach 48.1% and 54.5%; in addition, the bacillus subtilis YB-05 and 1% shenqinmycin are compounded in a reduction ratio of 19:1 for 20ml/kg for seed dressing treatment, the disease is the lightest, the prevention and treatment effect is the best, the prevention and treatment effect reaches 58.9%, and the prevention and treatment effect exceeds the prevention and treatment effects of single-dose treatment and control medicament 2% tebuconazole 1ml/kg for seed dressing treatment. Therefore, the bacillus subtilis YB-05 microbial inoculum and 1% shenqinmycin are compounded, so that the control effect on the wheat stem basal rot is obviously improved, and the development and utilization values are very high.

TABLE 3 indoor control effect of YB-05, shenqinmycin and its built biological pesticide on wheat stem base rot

Numbering	Name of process	Index of disease condition	Control effect (%)
				1	YB-05 microbial inoculum 30ml/kg seed dressing	40.1b	48.1
2	Seed dressing with 1% shenqinmycin 2ml/kg	35.2bc	54.5
				3	YB-05 and 1% shenqinmycin 19:1 are compounded into 20ml/kg seed dressing	31.8c	58.9
4	Seed dressing treatment of 2% tebuconazole 1ml/kg	33.5c	56.7
				5	CK	77.3a	/

Example 11:

the field control effect of the bacillus subtilis YB-05 and 1% shenqinmycin compound biopesticide on wheat stem basal rot comprises the following contents:

test site: wu\38495, Kyodo city, Henan province, Wangzhong county, wheat is planted in test plots all the year round, the stem base rot is severe, base fertilizer and top dressing are applied according to the conventional method, and other bactericides are not used in the whole growth period except the test.

Wheat variety: short seed resistance of 58, and seeding rate of 15 kg/mu.

Test time: 2016-10-2017-6 months

And (3) test treatment: the treatment is carried out by mixing 30ml/kg of bacillus subtilis YB-05 microbial inoculum, 2ml/kg of 1% shenqinmycin, 20ml/kg of compound biological pesticide of the bacillus subtilis YB-05 microbial inoculum and 19:1(vt/vt) of 1% shenqinmycin, mixing 2ml/kg of the seed suspension agent of 4.8% cornstarch in water, and carrying out 3 times of treatment without any treatment on the seeds in a blank control.

The efficacy survey is divided into 3 times, namely a seedling stage before winter, a jointing stage and a grouting stage. And (3) adopting a five-point sampling method in each cell, taking 25 seedlings in each point, digging out the plants with roots, keeping the root systems complete, carrying out grading investigation according to grading standards in different periods, and calculating the prevention and treatment effect according to disease indexes.

Grading standards of field seedling stage and green turning and jointing stage:

level 0: the whole plant has no browning symptoms; level 1: the plant coleoptile or the ground stem is browned; and 3, level: browning of the primary leaf sheath; and 5, stage: second leaf sheath browning; and 7, stage: the third scab of the plant is browned or the whole stem is browned and softened; and 9, stage: the plant died.

Grading standard in grouting period:

level 0: the whole plant has no browning symptoms; level 1: browning the first stem node part of the aerial part; and 3, level: the first stem node of the overground part is completely browned or the second stem node is partially browned; and 5, stage: the second stem node of the overground part is completely browned or the third stem node is partially browned; and 7, stage: the third stem node of the overground part is completely browned or the part above the third stem node is browned, but the plant does not die; and 9, stage: the plants died.

In addition, the yield is measured in the harvest period, 5 points are sampled in each cell, and 1m is taken in each point²And (3) measuring the yield of the wheat plants, converting the yield per hectare and calculating the yield increase rate.

The results are shown in fig. 7 and table 4, the control effect of the bacillus subtilis YB-05 and 1% shenqinmycin 19:1(vt/vt) compound biopesticide in the seedling stage of wheat is the best, and reaches 62.1%, exceeds 56.2% of the control effect of the Minidan seed dressing treatment and the control effect of the single-dose treatment, and also reaches 57.6% and 43.1% respectively in the jointing stage and the adult stage of wheat, and both are higher than the control effects of the single-dose and Minidan seed dressing treatments.

TABLE 4 field control effect of pesticides in each experimental group on wheat stem base rot caused by fusarium pseudograminearum

The influence of the bacillus subtilis YB-05 and 1% shenqinmycin 19:1(vt/vt) compound biopesticide on the yield of wheat is measured in the harvest period of wheat, and as shown in Table 5, the average yield per mu of compound biopesticide treatment is 631.1kg, which is 11.3% higher than that of a control, and is higher than 608.8kg and 599.2kg of YB-05 fungicide seed dressing treatment and shenqinmycin seed dressing treatment. Therefore, the bacillus subtilis YB-05 and 1% shenqinmycin 19:1(vt/vt) compound biopesticide not only has relatively stable control effect on the wheat stem basal rot, but also has a certain yield increasing effect and has very great development and utilization values under the field condition.

TABLE 5 Effect of pesticides of each experimental group on wheat yield in the field

Name of process	Average yield per mu (kg)	Contrast increase and decrease (%)
			YB-05 seed dressing	608.8±67.1	7.4
Shenqinmycin seed dressing	599.2±33.6	5.7
			The compound biological pesticide of the embodiment	631.1±20.3	11.3
Pellet for wheat	595.1±52.7	5.0
			Control	566.9±58.0	/

While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.

Claims

1. A compound biological pesticide for preventing and treating wheat stem basal rot is characterized by comprising a bacillus subtilis microbial inoculum and shenqinmycin with a volume ratio of 19: 1;

the bacillus subtilis is a bacillus subtilis YB-05 strain with the preservation number of CGMCC NO. 6498;

the bacillus subtilis microbial inoculum is prepared by the following method:

inoculating the bacillus subtilis YB-05 strain to a LA culture medium, streaking, and culturing for 30-36h at the temperature of 28-32 ℃ until a single colony grows out; selecting the single colony to be inoculated into an LB culture solution, performing shake culture at 30-32 ℃ and 160rpm for 12-16h to prepare a seed solution; inoculating the seed solution into a fermentation culture medium by the inoculation amount of 2%, performing shake culture at 30-32 ℃ and 160rpm for 48-54h, and diluting the fermentation liquor by 10 times with sterile water;

the concentration of the shenqinmycin in the compound biological pesticide is 0.5 mg/mL;

the fermentation medium contains 1% of sucrose, 1.5% of corn flour, 3.5% of soybean flour and 0.2% of dipotassium hydrogen phosphate, and the pH value is 7.2.

2. The preparation method of the compound biopesticide for preventing and treating wheat stem basal rot, disclosed by claim 1, is characterized by comprising the following steps of:

3. The application of the compound biopesticide for preventing and treating wheat stem basal rot in wheat yield increase of claim 1.