CN108102930B - One inter-species type Curvularia lunata bacterial strain and its application and herbicide - Google Patents

Abstract

The present invention provides an inter-species type Curvularia lunata bacterial strain and its application, it is described between type Curvularia lunata Strain Designation be mt-3, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on December 23rd, 2016, deposit number is CGMCC NO.13376.Type Curvularia lunata between of the invention can be used as the microbial herbicide preparation in the crops such as corn and cotton field.The present invention also provides a kind of herbicides, the herbicidal composition is simple, it is environmental-friendly, application is simple, broad weed-killing spectrum has reached 75% or more to the control efficiency of the weeds such as herba digitariae, herba eleusines indicae, semen euphorbiae, herba setariae viridis, Eclipta prostrata and purslane under the premise of harmless to crops such as corn and cottons, herbicidal effect is quick, is a kind of biological weed killer that can be come into operation for a long time.

Description

A kind of Curvularia intertype strain and its application and herbicide

technical field

The invention belongs to the technical field of microbial engineering and weeding, and in particular relates to a strain of Curvularia intertype and its application and herbicide.

Background technique

Crabgrass (Digitaria sanguinalis) is one of the 18 kinds of malignant weeds recognized in the world. It mainly harms crops such as cotton, corn, sorghum, soybean, tobacco, and peanut. It also often occurs in orchards, vegetables, lawns, and horticultural crop fields. Crabgrass is widely distributed in tropical and temperate regions of the world. It has strong reproductive ability and stress resistance, fast growth rate and long growth cycle. The control of crabgrass mainly depends on chemical methods, but the long-term and large-scale use of chemical herbicides will cause environmental pollution and residual poison and phytotoxicity, and will also lead to the emergence of weed resistance. Therefore, there is an urgent need to explore non-chemical control methods for crabgrass and resistant crabgrass.

Biological herbicides have the characteristics of strong pathogenicity to target weeds and are safe to the environment, so their research and development have attracted more and more attention. At present, there are more than 20 varieties of biological herbicide products that have been successfully commercialized or officially registered in the world, including Biochon, StumpOut, Camperico, bialaphos sodium and Myco-Tech Paste. Among them, in terms of crabgrass research, the strain of Curvularia intermedia has been screened out to have strong pathogenicity, and a patent has been applied for; Ustilago syntherismae has also been reported to have a good control effect; in addition, my country has It is reported that Curvularia eragrostidis has the potential to be developed as a fungal herbicide for controlling crabgrass. However, the herbicidal spectrum of these strains is not wide enough, and the fermentation process is complicated, which is not conducive to popularization and application.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a Curvularia intertype that overcomes crabgrass drug resistance, specifically a Curvularia intertype strain and its application and herbicide.

In order to solve the above technical problems, a strain with strong pathogenicity was isolated and screened from the naturally occurring crabgrass in the cotton field in Hunan, and it was identified from the aspects of morphology and molecular biology, and its biological control The effect and crop safety were evaluated, aiming to provide a basis for the research and development of crabgrass fungal herbicides and solve the problem of resistance to crabgrass in Hunan cotton fields. The technical solution proposed by the present invention is to provide a strain of Curvularia intermedia, which is named mt-3, which was deposited in China Microorganisms on December 23, 2016. The General Microorganism Center of the Preservation Management Committee, the deposit number is CGMCC NO.13376.

Curvularia intertype of the present invention is a kind of crabgrass pathogenic bacterium, and host range is wide, and host range includes crabgrass, goosegrass, daughter of a daughter, foxtail, snakehead sausage and purslane etc., to corn and cotton etc. It is harmless to crops and can be used as microbial herbicide preparation in crop fields such as corn and cotton. Compared with these strains mentioned in the background technology, Curvularia intermedia mt-3 has a wider herbicidal spectrum, and has control effects on weeds such as crabgrass, goosegrass, barnyardgrass, and purslane. The invention is suitable for weeding in cotton fields; in addition, the fermentation process is simple, the formulation is mature, and the invention has a prospect of popularization and application.

The isolation method of above-mentioned intertype Curvularia bacterial strain, comprises the steps:

(1) cut tissue block from the disease-healthy junction of the leaf disease of crabgrass natural onset, carry out surface disinfection 3min in the mercuric chloride solution of 0.1% in mass concentration, rinse 3 times with sterile water;

(2) Place the tissue block obtained after washing in the step (1) on a PDA medium for constant temperature culture, and the temperature of the constant temperature culture is 28° C.;

(3) After the colony grows from the constant temperature culture in the step (2), pick the hyphae from the edge of the colony, move it into a new PDA medium for cultivation, and obtain the described Curvularia intertype strain through separation and purification.

Based on a general technical conception, the present invention also provides a kind of application of above-mentioned Curvularia mt-3 in the preparation of herbicides, comprising the following steps: Utilize Curvularia mt-3 to inoculate PD culture fluid (yeast After leaching powder peptone glucose culture solution), carry out shaking culture, vacuum filter the culture solution, and obtain the fermentation broth containing Curvularia intertype after filtering through a microporous membrane, and then use the fermentation broth or the Curvularia intertype in the fermentation broth Prepare said herbicide.

For the above application, preferably, the temperature of the shaking culture is 28°C, the rotation speed of the shaking culture is 150r/min, and the time of the shaking culture is 7 days;

More preferably, the fermentation broth contains 2-100 billion Curvularia spores/gram.

A herbicide, in the form of water preparation, mainly composed of the fermentation liquid of Curvularia intertype mt-3, and the fermentation liquid contains Curvularia intertype spores.

Preferably, the herbicide of the water formulation comprises 95-99% of fermentation broth and 1-5% of Tween 80 in mass fraction, and the fermentation broth contains 20-100 billion Curvularia spores per gram.

More preferably, the fermentation broth is obtained by inoculating Curvularia intertype mt-3 in the PD culture fluid and then undergoing shaking culture, vacuum filtration and microporous membrane filtration; the temperature of the shaking culture is 28°C, The rotational speed of shaking culture is 150r/min, and the time of shaking culture is 7 days.

A herbicide, the dosage form is wettable powder, mainly composed of Curvularia intertype spores of Curvularia intertype mt-3.

Preferably, the herbicide of the wettable powder comprises the following components in mass fraction: Curvularia intertype 70%, disintegrant DK8 3%, wetting agent 1%, white carbon black 3% and kaolin 23% .

More preferably, the intertype Curvularia intertype spores are inoculated into the PD culture medium by Curvularia intertype mt-3, and then subjected to shaking culture, vacuum filtration and microporous membrane filtration to obtain a fermented liquid, and then separated from the fermented liquid ; The temperature of the shaking culture is 28° C., the rotation speed of the shaking culture is 150 r/min, and the time of the shaking culture is 7 days.

The herbicide of the present invention has simple ingredients, is environmentally friendly, simple to apply, and has a wide herbicidal spectrum. Under the premise of being harmless to crops such as corn and cotton, it is effective against crabgrass, goosegrass, daughter of a daughter, foxtail, barnyardgrass, and snakehead. The control effect of intestinal and purslane and other weeds has reached more than 75%, and the weeding effect is quick, and it is a biological herbicide that can be put into use for a long time.

Compared with prior art, the beneficial effect of the present invention is:

1, the intertype Curvularia of the present invention, named after intertype Curvularia mt-3, is a kind of crabgrass pathogenic bacteria, and the host range is wide, and the host range includes crabgrass, goosegrass, daughter of a daughter, foxtail, Channa sausage and purslane, etc. are harmless to crops such as corn and cotton, and can be used as microbial herbicide preparation in fields of crops such as corn and cotton.

2. The herbicide of the present invention has simple ingredients, is environmentally friendly, is simple to apply, and has a wide herbicidal spectrum. Under the premise of being harmless to crops such as corn and cotton, it is effective against crabgrass, goosegrass, daughter of a daughter, foxtail, and snakehead sausage. The control effect of weeds such as purslane and purslane has reached more than 75%, and the weeding effect is quick. It is a biological herbicide that can be put into use for a long time.

Explanation on the preservation of biological materials:

A kind of Curvularia intermedia bacterial strain, described Curvularia intermedia bacterial strain is named as mt-3, and it was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms on December 23, 2016, the The abbreviation of the depository institution is CGMCC, the address of the depository institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.13376.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are For some embodiments of the present invention, those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is the comparison chart of pathogenicity determination of inoculated strain fermentation liquid and sterile PDA culture liquid on crabgrass plant in the embodiment.

Fig. 2 is the comparison chart of the pathogenicity determination of the whole plant of crabgrass spraying strain fermentation liquid and sterile PDA culture liquid in the embodiment.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

Unless otherwise defined, all technical terms used hereinafter have the same meanings as commonly understood by those skilled in the art. The terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the protection scope of the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

Example:

A kind of Curvularia intermedia bacterial strain, described Curvularia intermedia bacterial strain is named as mt-3, and it was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms on December 23, 2016, the The abbreviation of the depository institution is CGMCC, the address of the depository institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.13376.

1. Isolation of pathogenic fungi

The naturally diseased leaves of crabgrass in Hunan cotton field were collected, and the symptoms of plant disease were recorded. Cut tissue pieces from the junction of disease and health, sterilize the surface in 0.1% mercuric chloride solution for 3 minutes, rinse with sterile water for 3 times, place on PDA medium, and cultivate at a constant temperature of 28°C. Pick part of the hyphae from the edge, transfer them to a new PDA plate for culture, and obtain a strain of Curvularia intermedia after separation and purification, named mt-3, which was deposited in China Microbiology on December 23, 2016 The General Microorganism Center of the Preservation Management Committee, the deposit number is CGMCC NO.13376.

2. Pathogenicity assay of strain mt-3

The pathogenicity of strain mt-3 to crabgrass leaves was determined by in vitro method. The colony mycelium blocks that have been cultivated for 7 days were punched out with a puncher, and inoculated into PD culture solution (100 mL/bottle), and three pieces of mycelium blocks with a diameter of 9 mm were inoculated in each bottle, and cultured at 28 °C and 150 r/min with shaking. After 7 days, the culture solution was vacuum filtered, and then filtered with a microporous membrane to obtain a fermentation solution. Spray and inoculate onto the crabgrass plant (Treantment) at the stage of 1 leaf, 1 heart to 2 leaves with a micro-sprayer, and the inoculation amount is 20 mL/pot. With sterile PDA culture medium as the control (CK), each treatment was repeated 3 times. Slight lesions appeared on the leaves 3 days after inoculation, and a large number of hyphae grew from the hyphae blocks, which gradually infected around the leaves, and the leaves began to turn yellow. After 5 days, brown spots appeared on the leaves, which gradually expanded, and the leaves turned black and rotted (Figure 1). Two days after the whole plant was sprayed with the fermentation broth, the resistant crabgrass plants showed disease symptoms, and the leaves turned yellow and chlorotic, and gradually wilted after 5 days, and finally the whole plant withered and died (Figure 2).

In order to further determine the pathogenicity of the strain mt-3 to crabgrass at different growth stages, we used the mycelium block inoculation method to inoculate 1 leaf, 1 heart to 2 leaves, 3 leaves to 3 leaves, 1 heart, and 4 leaves to 1 leaf, respectively. The inoculation was carried out at the heart-5 leaf stage. 1 pot was treated, and 10 crabgrass plants with consistent growth were kept in each pot. As shown in Table 1, 5 days after the mycelium fermentation liquid was inoculated with crabgrass plants of different growth stages, the resistant horses at the stages of 1 leaf, 1 heart to 2 leaves, 3 leaves to 3 leaves, 1 heart, and 4 leaves, 1 heart to 5 leaves The incidence rate of Tang plants was as high as 100%, 95% and 88.33%, and the average fresh weight control effect reached 82.78%, 79.24% and 63.37% respectively. most sensitive.

Table 1 Pathogenicity of strain mt-3 to crabgrass at different growth stages

Note: The values in the table are the mean ± standard error, and different letters after the data in the same column indicate significant differences at the 0.05 level.

3. Determination of the host range of strain mt-3

In addition to crabgrass, we also prepared to test weeds goosegrass, daughter, foxtail, channa sausage and purslane, which were treated with cotton and corn seedlings at the same time, and fermented with mt-3 at the three-leaf one-heart stage The liquid was sprayed and kept moist for 48 hours, and the plants that were not applied with the mt-3 fermentation liquid were used as a control to investigate the pathogenesis of the plants that were applied with the mt-3 fermentation liquid. The results are shown in Table 2. The strain mt-3 was asymptomatic (NS) to the test crop cotton, and moderately susceptible (MS) to the test weeds Goosegrass, Stephania and Setaria. Lesions appeared on the leaves, and the plant height and growth were inhibited in the later stage. It showed a slight reaction (LS) to snakehead sausage and purslane, and a small amount of smaller lesions appeared on the leaves.

Table 2 Determination of the host range of strain mt-3

4. Determination of the control effect of bacterial strain mt-3

Using metolachlor EC as the control agent, a field test was carried out in the cotton field of Hanshou County in 2016. After the strain mt-3 was fermented, cotton seedlings were transplanted 7 days later, and the fermentation liquid of the strain mt-3 was used to prepare the fermentation liquid The method is the same as above, and the fermented liquid contains 2-100 billion Curvularia spores/gram. The concentration of bacterial strain mt-3 fermented liquid is 500, 600, 800, 1200ml per mu, and metolachlor is selected as contrast agent, and the results are shown in table 3. The results show that the control effect on total grass reaches more than 75%, especially The control effect on purslane and goosegrass reaches more than 80%, and the control effect on barnyard barnyardgrass reaches 83%. .

Table 3 Determination of control effect of bacterial strain mt-3

The fermented liquid can be mixed with Tween 80 to make a herbicide according to the ratio of 95-99% of fermented liquid and 1-5% of Tween 80 in mass fraction, and the fermented liquid contains 2-100 billion Curvularia interspores per gram ; also the intertype Curvularia in the fermented liquid can be isolated, by mass parts Curvularia intertype 70%, disintegrating suspending agent DK8 3%, wetting agent 1%, white carbon black 3% and kaolin 23% The proportioning of making herbicide, the preparation method of fermented liquid is the same as above. Cotton seedlings 7 days after transplanting are sprayed with herbicide, and the control effects on weeds are all above 75%.

Claims (10)

1. a kind of Curvularia intermedia bacterial strain, it is characterized in that, described Curvularia intermedia (Curvularia intermedia) bacterial strain is named after Curvularia intermedia mt-3, and it was preserved in China on December 23, 2016 The General Microbiology Center of the Microbiological Culture Collection Management Committee, the preservation number is CGMCC NO.13376. 2. a kind of application of intertype Curvularia as claimed in claim 1 in the preparation of herbicide, is characterized in that, comprises the following steps: utilizes intertype Curvularia mt-3 to inoculate in PD nutrient solution and carry out shaking culture , the culture solution is vacuum filtered, and the fermentation solution containing Curvularia intertype is obtained after filtering through a microporous membrane, and then the herbicide is prepared by using the fermentation solution or the Curvularia intertype in the fermentation solution. 3. The application according to claim 2, characterized in that the temperature of the shaking culture is 28° C., the rotation speed of the shaking culture is 150 r/min, and the time of the shaking culture is 7 days. 4. The application according to claim 2 or 3, characterized in that, the fermented liquid contains 2-100 billion curvularia spores/gram. 5. A herbicide, characterized in that, the dosage form is an aqueous solution, mainly composed of the fermentation liquid of Curvularia intertype according to claim 1, containing Curvularia intertype spores in the fermentation liquid. 6. The herbicide according to claim 5, characterized in that, the herbicide comprises 95-99% of the fermented liquid and 1-5% of Tween 80 in mass fraction, and the fermented liquid contains 20-100 billion Curvularia type spores/g. 7. according to the described herbicide of claim 5 or 6, it is characterized in that, described fermented liquid is through shock culture, vacuum filtration and microporous membrane filtration after being inoculated in PD nutrient solution by Curvularia intertype mt-3 It is prepared later; the temperature of the shaking culture is 28° C., the rotation speed of the shaking culture is 150 r/min, and the time of the shaking culture is 7 days. 8. A herbicide, characterized in that the dosage form is a wettable powder, mainly composed of Curvularia intertype spores of Curvularia intertype according to claim 1. 9. The herbicide according to claim 8, characterized in that, the herbicide comprises each component of the following mass fractions: Curvularia intertype 70%, disintegrating agent DK8 3%, wetting agent 1%, Silica 3% and Kaolin 23%. 10. according to the described herbicide of claim 8 or 9, it is characterized in that, the intertype Curvularia spores are inoculated in the PD nutrient solution by the intertype Curvularia after shaking culture, vacuum filtration and microporous membrane filtration The fermentation broth is obtained and then separated from the fermentation broth; the temperature of the shaking culture is 28° C., the rotation speed of the shaking culture is 150 r/min, and the time of the shaking culture is 7 days.