CN110846243A - Biocontrol compound microbial inoculum and preparation method and application thereof - Google Patents
Biocontrol compound microbial inoculum and preparation method and application thereof Download PDFInfo
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Abstract
The present invention belongs to the field of plant protecting technologySpecifically discloses a biological control compound microbial inoculum, which comprises Streptomyces KN37(Streptomyces sp.KN37) with the strain preservation number of CGMCC No.13160, trichoderma harzianum (Trichoderma harzianum) with the strain preservation number of CGMCC No.5.1250, Bacillus subtilis (Bacillus subtilis) with the strain preservation number of CGMCC No.1.821, wherein the combined proportion of the Streptomyces KN37, the trichoderma harzianum and the Bacillus subtilis is 1: 1.3-1.4: 0.5-0.6, and the total concentration of viable bacteria is 1 multiplied by 1010～1012CFU/mL. The biocontrol composite microbial inoculum is applied in a drip irrigation mode, has better prevention effect on cotton verticillium wilt, and has the effects of promoting growth and increasing yield of cotton plants.
Description
Technical Field
The invention belongs to the technical field of plant protection, relates to microorganisms and application thereof, and particularly relates to a biocontrol compound microbial inoculum combined by streptomycete KN37, trichoderma harzianum and bacillus subtilis, and application thereof in cotton verticillium wilt prevention and control.
Background
The superior natural conditions of Xinjiang make it have inherent advantages in the production of agricultural products. The planting industry is the main industry in Xinjiang agriculture, accounts for 76% of the total agricultural value, and is a main source for increasing the agricultural income. The crops planted in Xinjiang mainly comprise grains, oil plants, cotton, vegetables, melons and fruits and other economic crops. At present, Xinjiang is the largest cotton production area in China and accounts for more than 83% of the cotton planting area in China. With the increase of cotton planting area, the occurrence of cotton verticillium wilt is more and more common, the disease is very seriously influenced to the cotton planting in large production areas all over the country, and a plurality of cotton fields cause the situation of no harvest due to the verticillium wilt. According to statistics, the loss of ginned cotton caused by diseases in Xinjiang cotton is about 36 million tons every year, and the market price is about 70 million yuan.
Cotton verticillium wilt, known as "cancer of cotton," is a major intractable disease in cotton production and tends to increase year by year. Verticillium wilt of cotton is mainly caused by infection of Verticillium dahliae Kleb. The cotton is gradually expanded after a pale yellow patch appears at the leaf margin and between the leaf veins on the lower leaves of the plants at the early stage of disease incidence, the leaf color is dark green and becomes light, the main veins and the periphery of the main veins are still kept green, palm-shaped mottle appears on the diseased leaves, the mesophyll is thickened, the leaf margin is curled downwards, the leaves gradually fall off from bottom to top, only a few small leaves at the top are left, the bolls are rare, the bolls are cracked in advance, and fine new branches grow at the base parts of the diseased plants at the later stage. The diseased stem was longitudinally split and a light brown discoloring streak was produced on the xylem. The disease can cause the cotton to reduce the yield by 40-60 percent and the cellulose quality to be reduced. The cotton verticillium wilt becomes the most common and serious disease loss in the cotton planting process, and the quality and the yield of cotton are seriously influenced.
At present, the main approaches for preventing and treating cotton verticillium wilt are measures such as breeding disease-resistant varieties, agricultural rotation, chemical prevention and treatment and the like, but the prevention and treatment effects are not ideal. The quality of the existing disease-resistant cotton variety is poor, and the market prospect is not very good. As the microcolonies of the cotton verticillium wilt can survive for more than ten to dozens of years in the soil, the short-term crop rotation cannot achieve the expected purpose. The use of a large amount of chemical pesticides in soil will cause serious problems of limited export of agricultural products, drug resistance, environmental pollution and the like, and is difficult to be a preferable solution for a long time. In this case, biological control becomes the preferred option. The development of novel high-efficiency, broad-spectrum, low-toxicity and environment-friendly bactericides (microbial agents) is always a difficult point and a hotspot in the research of current pesticides. The biological control has good disease control effect, is safe to human, livestock and environment, has no residue, and is safe to natural enemies and beneficial organisms. The biocontrol microbial inoculum which is sold in the market at present and can be used for preventing and treating cotton verticillium wilt mainly comprises bacillus and trichoderma, but the control effect is not ideal.
Disclosure of Invention
The invention aims at the current situation that verticillium wilt of cotton fields in Xinjiang at present is serious, and aims to explore and develop a biocontrol compound microbial inoculum for preventing and treating verticillium wilt of cotton. The biocontrol composite microbial inoculum integrates strains such as actinomycetes, fungi, bacteria and the like, and provides a practical and effective solution for preventing and treating cotton verticillium wilt.
In order to achieve the purpose, the invention adopts the technical scheme that:
a biological control compound bacterial agent comprises Streptomyces KN37(Streptomyces sp.KN37) with the strain preservation number of CGMCC No.13160, trichoderma harzianum (Trichoderma harzianum) with the strain preservation number of CGMCC No.5.1250 and bacillus subtilis (Bacillus subtilis) with the strain preservation number of CGMCC No. 1.821. The streptomycete KN37 is an actinomycete separated from soil collected in Xinjiang Kanasi areas by an inventor group, and the fermentation liquor of the streptomycete KN37 recorded in the granted patent ZL201611143553.8 has a good inhibition effect on botrytis cinerea, early blight of tomato, fusarium oxysporum of cotton and leaf mold of tomato. However, the patent ZL201611143553.8 does not prove the control effect on the cotton verticillium wilt through exact field data. In order to expand the biocontrol application of the streptomyces KN37, the patent tries to use the streptomyces KN37 in combination with Trichoderma harzianum and Bacillus subtilis to explore a biocontrol compound microbial inoculum with better control effect on cotton verticillium wilt. The Trichoderma harzianum strain and the Bacillus subtilis strain used in the patent are purchased from the strain collection center of the institute of microbiology of Chinese academy of sciences.
As a preferred embodiment of the biological control compound microbial inoculum, the patent provides a preferred matching use ratio of streptomycete KN37, trichoderma harzianum and bacillus subtilis, the combination ratio of the streptomycete KN37, the trichoderma harzianum and the bacillus subtilis is 1: 1.3-1.4: 0.5-0.6 in terms of mass ratio, and the total concentration of viable bacteria is 1 multiplied by 1010～1012CFU/mL. Test data prove that the biocontrol composite microbial inoculum prepared according to the proportion has the control effect on cotton verticillium wilt higher than 70%.
In addition, the patent also provides a preparation method of the biocontrol compound microbial inoculum, which is formed by mixing the bacterial powder of streptomycete KN37, trichoderma harzianum and bacillus subtilis according to the mass ratio of 1: 1.3-1.4: 0.5-0.6. The total viable bacteria concentration of the biological control compound bacteria agent prepared according to the proportion is 1 multiplied by 10 through measurement10～1012CFU/mL。
As a further preferable preparation method of the biocontrol complex bacterial agent, the preparation method comprises,
respectively inoculating and culturing streptomycete KN37, trichoderma harzianum and bacillus subtilis to obtain bacterial suspensions;
respectively inoculating the bacterial suspensions into fermentation culture solutions, culturing, filtering and drying to respectively obtain bacterial powder of the streptomycete KN37, the trichoderma harzianum and the bacillus subtilis;
and mixing the streptomycete KN37, the Trichoderma harzianum and the bacterial powder of the Bacillus subtilis according to the mass ratio of 1: 1.3-1.4: 0.5-0.6.
As a preferred embodiment of the preparation method of the biocontrol compound microbial inoculum, a culture medium for obtaining the streptomycete KN37 bacterial suspension is a Gao's first culture medium; the culture medium for obtaining the trichoderma harzianum bacterial suspension is a PDA culture medium; the LB culture medium is selected as the culture medium for obtaining the bacillus subtilis suspension; the culture conditions were: the temperature is 32 ℃, the rotation speed is 180rpm, and the shaking culture is carried out for 36 h. The culture medium Gao's-I, PDA culture medium and LB culture medium can be prepared by the ordinary skilled in the art according to the existing technology.
As a preferred embodiment of the preparation method of the biocontrol compound microbial inoculum, the streptomycete KN37, the trichoderma harzianum and the bacillus subtilis suspension are respectively inoculated into a fermentation culture solution according to the mass ratio of 1: 100.
As a preferred embodiment of the preparation method of the bio-control complex microbial inoculum, the formula of the fermentation culture solution is as follows: 10g/L of millet, 10g/L of soybean meal, 10g/L of glucose and 3g/L, NaCl2g/L, CaCO g/of peptone31g/L, and the pH value is 7.2-7.4; the fermentation culture conditions are as follows: the temperature is 32 ℃, the rotating speed is 280rpm, the pH value is 7.0-7.5, the dissolved oxygen content is 20 percent, and the ventilation volume is 5-7 m3The tank pressure is 0.05-0.1 KPa.
In addition, the application of the biocontrol compound microbial inoculum in preventing and treating cotton verticillium wilt is provided. The test data shows that the biocontrol compound microbial inoculum is applied in a drip irrigation mode in the cotton growth process, and the control effect on the cotton verticillium wilt reaches 76.79%.
In addition, the application of the biocontrol compound microbial inoculum in increasing the yield of cotton is provided. Test data show that the biocontrol composite microbial inoculum has a good growth promoting effect on cotton, and the increase rate of the biocontrol composite microbial inoculum on the plant height and fresh weight of the cotton exceeds 35 percent.
Compared with the prior art, the invention has at least the following beneficial effects or advantages:
(1) the invention provides a biocontrol compound microbial inoculum containing streptomyces KN37, trichoderma harzianum and bacillus subtilis, wherein the mass ratio of the streptomyces KN37 to the trichoderma harzianum to the bacillus subtilis is 1: 1.3-1.4: 0.5-0.6. The biocontrol compound microbial inoculum prepared according to the proportion has better prevention and treatment effect on cotton verticillium wilt, and the experimental data shows that the biocontrol compound microbial inoculum is applied in a drip irrigation mode in the growth process of cotton, and the prevention and treatment effect on the cotton verticillium wilt reaches 76.79%.
(2) The active ingredients of the biocontrol compound microbial inoculum are streptomyces KN37, trichoderma harzianum and bacillus subtilis, so that the biocontrol compound microbial inoculum is safe to the environment and avoids negative effects caused by chemical pesticides.
(3) The biocontrol compound microbial inoculum also has the effects of promoting growth and increasing yield, the biocontrol compound microbial inoculum with the concentration of 0.6g/L has the strongest promotion effect on the plant height and fresh weight of cotton, reaches 35.47 percent and 35.02 percent, can reduce the expenditure of farmers and improve the income of the farmers.
Drawings
FIG. 1 is a curved surface diagram of the addition amounts of Trichoderma harzianum and Bacillus subtilis in an example of screening the optimal mixture ratio of the biocontrol compound microbial inoculum.
FIG. 2 is a contour line of addition amounts of Trichoderma harzianum and Bacillus subtilis in an optimal proportioning screening example of the biocontrol compound microbial inoculum.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following examples.
Example 1: preparation of biocontrol compound microbial inoculum
This example provides a preferred preparation method of a biocontrol complex microbial inoculum. The microbial active components of the biocontrol compound microbial inoculum comprise streptomyces KN37, trichoderma harzianum and bacillus subtilis. Inoculating streptomycete KN37 to a Gao's I culture medium, inoculating trichoderma harzianum to a PDA culture medium, inoculating bacillus subtilis to an LB culture medium, and performing shake culture for 36h under the culture conditions of 32 ℃ of temperature and 180rpm of rotation speed to respectively obtain streptomycete KN37 bacterial suspension, trichoderma harzianum bacterial suspension and bacillus subtilis bacterial suspension. The three bacterial suspensions are respectively inoculated into a 50L fermentation tank filled with fermentation culture solution according to the mass ratio of 1: 100. The formula of the fermentation culture solution is as follows: 10g/L of millet, 10g/L of soybean meal, 10g/L of glucose and 3g/L, NaCl2g/L of peptone、CaCO31g/L, and the pH value is 7.2-7.4; the fermentation culture conditions are as follows: the temperature is 32 ℃, the rotating speed is 280rpm, the pH value is 7.0-7.5, the dissolved oxygen content is 20 percent, and the ventilation volume is 5-7 m3The tank pressure is 0.05-0.1 KPa. Culturing for 4-5 days, filtering, and spray drying to obtain three kinds of bacteria powder, wherein each gram of bacteria powder contains 9 × 1013～1017And (4) each thallus. And mixing the streptomycete KN37, the Trichoderma harzianum and the bacterial powder of the Bacillus subtilis according to the mass ratio of 1: 1.3-1.4: 0.5-0.6 to prepare the biocontrol compound microbial inoculum.
Example 2: screening of optimum mixture ratio of biocontrol compound bacteria
Streptomyces KN37 was used as a quantitative test, Trichoderma harzianum and Bacillus subtilis were used as variables, and CCD (Central Composite Design) response surface test Design was performed by using Design Expert 8.0 software as shown in Table 1. Taking 100mg streptomycete KN37 bacteria powder, respectively adding corresponding Trichoderma harzianum bacteria powder and Bacillus subtilis bacteria powder according to the test design shown in Table 2, uniformly mixing, dissolving in 1L water, and respectively carrying out the potted plant efficacy test of cotton verticillium wilt.
TABLE 1 selected variables and parameter levels in CCD test design
TABLE 2 CCD test design and results
And (3) carrying out statistical analysis on the result by adopting a response surface analysis method to obtain a multi-item fitting regression model as follows: 64.9760+12.7395x1－4.9812x2－0.7550x1x2－8.1749x1 2－5.5524x2 2After the variance analysis is carried out on the model, the regression equation model can be shown to better describe the relationship (P) between each factor and the response value<0.0001), model mismatching terms are not significant (P is 0.0648), and the mismatching square sum of the regression equation is mainly caused by accidental factors such as experimental errors. Correlation coefficient R of model20.9786, indicating that the model better describes the results of the test. Comprehensive analysis shows that the model can be used for analyzing and predicting the influence of the addition of the trichoderma harzianum and the bacillus subtilis on the control effect.
From regression equation model analysis of variance (Table 3), Trichoderma harzianum (P)<0.0001) was the main effect. As can be seen from FIGS. 1 and 2, the effects of Trichoderma harzianum and Bacillus subtilis on control effect were both increased and then decreased, and thus the highest values were present. The optimal ratio of trichoderma harzianum to bacillus subtilis is obtained by Design Expert 8.0 software as follows: trichoderma harzianum added in 136mg (x)10.72), the amount of bacillus subtilis added was 56mg (x)2-0.88), the theoretical control effect is 70.50%. According to the analysis results, 100mg of streptomycete KN37 strain powder, 136mg of trichoderma harzianum strain powder and 56mg of bacillus subtilis are uniformly mixed and then dissolved in 1L of sterile water to carry out a cotton verticillium wilt pot drug effect test, and the result shows that the control effect of the formula on cotton verticillium wilt reaches 71.43 percent and is not greatly different from the theoretical control effect. Therefore, the optimal mixture ratio of the compound microbial inoculum is as follows: streptomycete KN37, Trichoderma harzianum and Bacillus subtilis in the ratio of 1 to 1.3-1.4 to 0.5-0.6, and the total concentration of viable bacteria is 1 x 1010～1012CFU/mL。
TABLE 3 regression model analysis of variance
Example 3: potted plant test for cotton verticillium wilt
The cotton seedlings with consistent growth vigor in the 5-leaf stage are taken as test crops, the biocontrol compound microbial inoculum prepared in the embodiment is diluted to a certain concentration (0.2, 0.4 and 0.6g/L), 50mL of root irrigation is taken, and after 5 days, 10mL of cotton verticillium wilt germ spore suspension (10 mL) is irrigated to the root7CFU/mL), continuously culturing, irrigating 50mL of biocontrol compound microbial inoculum at every 5d, investigating disease index, plant height and fresh plant weight when blank control is fully attacked, and calculating control effect.
As control agents, 10 million viable spores/gram wettable powder of Bacillus subtilis (Baoding, Biochemical technologies, Ltd.) and 3 million CFU/gram wettable powder of Trichoderma harzianum (Bewy, Ltd., USA) were used. The treatment and results of the cotton verticillium wilt pot test are shown in Table 4.
The pot experiment results (Table 4) show that the control effects of the biocontrol compound microbial inoculum with the concentrations of 0.2, 0.4 and 0.6g/L on cotton verticillium wilt are 58.87%, 72.54% and 88.36% respectively, which are superior to the control effects of control medicaments of trichoderma harzianum microbial inoculum and bacillus subtilis microbial inoculum. The biocontrol compound microbial inoculum, the trichoderma harzianum microbial inoculum and the bacillus subtilis microbial inoculum all have certain promotion effects on the growth of cotton seedlings, wherein the biocontrol compound microbial inoculum with the concentration of 0.6g/L has the strongest promotion effects on the plant height and fresh weight of cotton, and the promotion effects reach 35.47% and 35.02%.
Table 4, the potted plant control effect of the biocontrol compound microbial inoculum on cotton verticillium wilt
Example 4: field efficacy test of cotton verticillium wilt
In 143 th team of military team of Xinjiang construction, cotton field with uniform soil shape, uniform fertility and serious verticillium wilt of cotton is selected for drip irrigation, and the bactericide is mixed into the cotton field for dripping into soil. The experiment was divided into 4 groups and treated as follows:
processing one: mixing 3 kg/mu of the biocontrol compound microbial inoculum into water when the second water drop begins in the beginning of the sixth month, wherein the water drop amount is 20m each time3Dripping 5 times every 10d per mu.
And (5) processing: mixing 3 kg/mu of Trichoderma harzianum WP into water at the beginning of the second water drop of the first six months, wherein the water drop amount is 20m3Dripping 5 times every 10d per mu.
And (3) treatment III: mixing 3 kg/mu of Bacillus subtilis WP with water at the beginning of the second water drop of 20m each time3Dripping 5 times every 10d per mu.
And (4) treatment: and (5) clear water control.
Each test cell adopts a five-point sampling method, 40 plants are sampled at each point, the verticillium wilt disease level is investigated, the disease index, the morbidity and the control effect are recorded and counted, and the grading standard and the calculation method are as follows:
grading standard of cotton verticillium wilt:
level 0: cotton plants are healthy, have no diseased leaves and grow normally;
level 1: less than 25% of cotton plants have leaf disease, become yellow and wilting;
and 2, stage: the cotton plants are infected by 25-50% of leaves, and become yellow and wilted;
and 3, level: 50-75% of cotton plants have leaf diseases and become yellow and wilted;
4, level: more than 75% of cotton plants have leaf diseases or the cotton plants die.
Disease index [ case number of disease-grade leaves × disease grade)/total number of leaves × highest disease grade ] × 100
Control group disease index-treatment group disease index)/control group disease index x 100
After harvesting the cotton field, recording the yield of the cotton field, and calculating the yield increase rate by the following calculation method:
yield increase (%) (yield of seed cotton in treated group-yield of seed cotton in control group)/yield of seed cotton in control group
The field control effect of each treatment on cotton verticillium wilt is shown in table 5. The yield-increasing effect of each treatment on cotton is shown in table 6.
TABLE 5 field control of cotton verticillium wilt for each treatment
TABLE 6 field yield enhancement of cotton by each treatment
The results of the prevention effect in table 5 show that the biocontrol compound microbial inoculum, trichoderma harzianum WP and bacillus subtilis WP can reduce the incidence of cotton verticillium wilt, the incidence of the cotton verticillium wilt after the biocontrol compound microbial inoculum is used is reduced to 13.28 percent and is obviously lower than the treatment group of trichoderma harzianum WP and bacillus subtilis WP, and the control effect of the biocontrol compound microbial inoculum on the cotton verticillium wilt can reach 76.79 percent and is obviously higher than the treatment group of the trichoderma harzianum WP and the bacillus subtilis WP. Table 6 shows that the field yield increasing effect shows that after the biocontrol compound microbial inoculum is used, the yield of cotton reaches 423.84 kg/mu, the yield is increased by 33.15 percent, and the yield is obviously higher than that of a Trichoderma harzianum WP and Bacillus subtilis WP treatment group.
The present invention has been further described with reference to the examples, but the present invention is not limited to the above-described embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (9)
1. A biological control compound bacterial agent is characterized by comprising Streptomyces KN37(Streptomyces sp. KN37) with the strain preservation number of CGMCC No.13160, trichoderma harzianum (Trichoderma harzianum) with the strain preservation number of CGMCC No.5.1250 and bacillus subtilis (Bacillus subtilis) with the strain preservation number of CGMCC No. 1.821.
2. The biocontrol composite microbial inoculum according to claim 1, wherein the combination ratio of the streptomycete KN37, the trichoderma harzianum and the bacillus subtilis is 1: 1.3-1.4: 0.5-0.6, and the total concentration of viable bacteria is 1 x 1010～1012CFU/mL。
3. A preparation method of a biocontrol compound microbial inoculum is characterized by being formed by mixing bacterial powder of streptomycete KN37, Trichoderma harzianum and Bacillus subtilis according to a mass ratio of 1: 1.3-1.4: 0.5-0.6.
4. The method for preparing the biocontrol complex bacterial agent as claimed in claim 3, comprising,
respectively inoculating and culturing streptomycete KN37, trichoderma harzianum and bacillus subtilis to obtain bacterial suspensions;
respectively inoculating the bacterial suspensions into fermentation culture solutions, culturing, filtering and drying to respectively obtain bacterial powder of the streptomycete KN37, the trichoderma harzianum and the bacillus subtilis;
and mixing the streptomycete KN37, the Trichoderma harzianum and the bacterial powder of the Bacillus subtilis according to the mass ratio of 1: 1.3-1.4: 0.5-0.6.
5. The method for preparing the biocontrol complex bacterial agent as claimed in claim 4, wherein,
the culture medium for obtaining the streptomycete KN37 bacterial suspension is a Gao's first culture medium;
the culture medium for obtaining the trichoderma harzianum bacterial suspension is a PDA culture medium;
the LB culture medium is selected as the culture medium for obtaining the bacillus subtilis suspension;
the culture conditions were: the temperature is 32 ℃, the rotation speed is 180rpm, and the shaking culture is carried out for 36 h.
6. The preparation method of the biocontrol compound microbial inoculum according to claim 4, wherein the streptomycete KN37, trichoderma harzianum and bacillus subtilis bacterial suspensions are respectively inoculated into a fermentation culture solution according to the mass ratio of 1: 100.
7. The method for preparing the biocontrol complex bacterial agent as claimed in claim 4 or 6, wherein,
the formula of the fermentation culture solution is as follows: 10g/L of millet, 10g/L of soybean meal, 10g/L of glucose, 3g/L, NaCl2g/L, CaCO31g/L of peptone and 7.2-7.4 of pH value;
the fermentation culture conditions are as follows: the temperature is 32 ℃, the rotating speed is 280rpm, the pH value is 7.0-7.5, the dissolved oxygen content is 20%, the ventilation volume is 5-7 m3/h, and the tank pressure is 0.05-0.1 KPa.
8. The use of the biocontrol compound bacterial agent as defined in claim 1 in cotton verticillium wilt control.
9. The use of the biocontrol complex bacterial agent as defined in claim 1 for increasing cotton yield.
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| CN113122477A (en) * | 2021-04-25 | 2021-07-16 | 金禾佳农（北京）生物技术有限公司 | Compound microbial preparation, preparation method and application |
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| CN113122477A (en) * | 2021-04-25 | 2021-07-16 | 金禾佳农（北京）生物技术有限公司 | Compound microbial preparation, preparation method and application |
| CN113264801A (en) * | 2021-06-22 | 2021-08-17 | 江苏富寿康农业生物技术有限公司 | Preparation method of bio-organic fertilizer |
| CN114456012A (en) * | 2022-01-14 | 2022-05-10 | 西安理工大学 | Low-yield composite biological organic bacterial fertilizer for cotton fields and preparation method thereof |
| CN114868770A (en) * | 2022-06-25 | 2022-08-09 | 石河子大学 | Agricultural composition containing bactericide and gamma-polyglutamic acid |
| CN114931149A (en) * | 2022-06-25 | 2022-08-23 | 石河子大学 | Axillary bud inhibition composition containing bacteria agent and preparation method thereof |
| CN114868770B (en) * | 2022-06-25 | 2024-03-19 | 石河子大学 | Agricultural composition containing microbial inoculum and gamma-polyglutamic acid |
| CN114931149B (en) * | 2022-06-25 | 2024-04-16 | 石河子大学 | Axillary bud inhibition composition containing microbial inoculum and preparation method thereof |
| CN115960788A (en) * | 2022-12-31 | 2023-04-14 | 石河子大学 | Special microbial inoculum for preventing and treating cotton verticillium wilt |
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