CN100549158C - A kind of campelyco and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of campelyco and preparation method thereof, belong to the technical field of utilizing microorganism to prevent and kill off farmland weed.This weedicide is a bacterial classification with curved spore mould (Curvularia Boedijn) Mds0404 bacterial strain CGMCCNO.1827, and the weight percent content of its component and each component is: pure spore 80-95%, chemical herbicide 0-18%, auxiliary 2-5%; Through the preparation of substratum, the activation of bacterial strain Mds0404 and culture of seed liquid, the production of bacterial classification spore cultivate with mixing of collection and each component etc. step be prepared from.Characteristics such as this agent is held concurrently except that weeds such as Herba Setariae Viridis, barnyard grass grass, Herba Eleusines Indicaes to prevent and kill off lady's-grass, has farm crop safety, and especially to monocot crops safety, application adaptability is wide, and is with low cost, pollution-free.Can be applicable in weeds such as lady's-grass harm area.

Description

A kind of biological herbicide and preparation method thereof

technical field

The present invention relates to a technical field of using microorganisms to control farmland weeds, in particular to a biological herbicide for controlling farmland weeds using fungal strains and a preparation method of the herbicide, which is specially used for farmland weeds crabgrass, goosegrass and Prevention and control of barnyardgrass, etc.

Background technique

With people's understanding of chemical herbicide residues, environmental pollution, and the development of drug-resistant and drug-resistant weeds, green food and organic agriculture, the development of biological herbicides to control weeds has become an urgent demand in production, and it has low investment. , short development cycle, strong specificity, environmental safety, no pollution and other advantages. At present, commercialized biological herbicide varieties include DeVine and College developed in the early 1980s and Comperico and Biochon launched in the late 1990s. These herbicides play an important role in controlling relative target weeds. For example, the persistence of DeVine can guarantee that it can be used once, and there is no need to weed again for 2-3 years. There are dozens of bio-herbicide projects that are expected to be commercialized, among which the species of bio-herbicides that control barnyard grass, wild water chestnuts and other weeds have very attractive prospects.

The monocotyledonous weed crabgrass (Diditaria sanguinaris) is one of the 18 kinds of malignant weeds recognized in the world. It is widely distributed in tropical and temperate regions of the world. Grass is also very harmful to the growth of cotton, tobacco, hemp, sugarcane and other seedlings, and it is also a dominant weed in vegetable fields, orchards, lawns and horticultural crop fields. In China, crabgrass damages an area of about 60 million hectares, and the crop loss caused by it accounts for more than half of the total loss of weeds in autumn dry crop fields. The weed has strong reproductive ability, fast growth rate, long growth period, soil consumption, shading, strong drug resistance, and manual and mechanical control is difficult. At present, although chemical control is effective, the long-term use of chemical herbicides causes the generation of resistant weeds and serious environmental pollution, leading to land degradation. Therefore, the development of biological herbicides to control crabgrass can not only effectively control this weed, but also have high environmental safety. At present, about the research of crabgrass fungus herbicide, the U.S. has applied for and utilizes Curvularia intermedia Boedijn bacterial strain (International Mycological Society collects, preservation number is 361688 (MT-5), 361689 (CG-L), 375263 ( MT-6), 375264 (MT-7)) patents for biological control of crabgrass, patent numbers 5,635,444 and 5,952,264. However, when the patent document proposes its use technology and production method, it is only clear that it is relatively safe for soybeans and cotton, but the safety and use of monocot crops such as corn and rice are not mentioned. In addition, domestic Nanjing Agricultural University has applied for a patent (ZL patent No. 01134002.9) for biological control of crabgrass by using the Nakataea Hara strain QZ-2000 (preservation number: CGMCC NO.0640), but its single-strain control effect It is not ideal, and the spores of several individual plants need to be mixed in proportion and then sprayed to achieve a better control effect, which increases the difficulty of production. At the same time, an important feature of fungal herbicides is that they have relatively harsh requirements on environmental conditions. Only when the requirements of the fungal agent for moisture and temperature can be fully met can the effective infection of the fungal herbicide be guaranteed and the It has achieved good control effect, but it may not be able to adapt to the ecological conditions of different places, and it is easy to have a certain regionality. Therefore, in order to solve this problem, the only way to solve this problem is to choose a parasitic species that requires a wide range of environmental conditions when infecting the host and is easy to meet. Fungal strains have broad application prospects.

Contents of the invention

The object of the present invention is to propose a method aimed at the disadvantages caused by the long-term use of chemical herbicides for malignant weeds-Digits crabgrass, and the defects of existing biological herbicides that are rare in varieties, difficult to adapt to different ecological conditions, troublesome to use, and poor in effect. The species is prepared from fungal strains obtained through screening. It mainly controls crabgrass, and also removes weeds such as foxtail, barnyardgrass, and goosegrass. It is safe for crops, especially for monocotyledonous crops, and has wide application adaptability and low cost. Inexpensive, pollution-free biological herbicide; another object of the present invention is to propose a preparation method of the biological herbicide.

Isolation, screening and identification of pathogenic fungi of the present invention: collect the diseased crabgrass leaves from Yuyao, Zhejiang Province, cut the leaves at the junction of disease and health, carry out surface disinfection with 6% sodium hypochlorite solution, and then place them on PSA medium. Cultivate in an incubator at ℃, isolate and purify the single bacteria after one week, and then carry out the test on the spore production of a single strain and the effect of controlling crabgrass and other monocotyledonous weeds. The microscopic morphology and morphological characteristics of pathogenic bacteria are identified according to the fungal identification manual:

Strain Mds0404: belongs to Fungi Imperfecti, Moniliales, Moniliadeae, Curvularia Boedijn; cultured in PDA containing sucrose, glucose and potassium nitrate substrate grow well in medium, and the colonies are gray-brown or gray-green. When using diammonium hydrogen phosphate and urea as substrates, the colonies are yellow-brown, and when using glycerol and trisodium citrate as substrates, the colonies are colorless; the conidiophores of this fungus differentiate Obvious, solitary or clustered, erect or curved, olive or black, septate, 48-196×4-6um, solitary or occasionally branched at the top, slender; Axial extension, often knee-shaped, new spores are born at the top of new branches born below the previous spore; spore size is 24-36×8-15um, fusiform or elliptic, with 3 septa, the septum is centered or basically centered , straight or curved to one side, the cells at both ends are small and blunt, colorless or light-colored, the two cells in the middle are large, the color is dark conidia brown, broad spindle-shaped, 3 septa, the septum is in the middle, not curved or slightly Curved, symmetrical on both sides, the size is 24-36um*10-18um.

The bacterial strain was deposited in the General Microorganism Center CGMCC of China Microorganism Culture Collection Management Committee on September 30, 2006, and the preservation number is No.1827.

The object of the invention is achieved through the following technical solutions:

A kind of biological herbicide, take Curvularia Boedijn (Curvularia Boedijn) bacterial strain Mds0404 as spore-forming bacterium kind, the component of this herbicide and the weight percent content of each component are:

Pure spores 80-95%

Chemical herbicides 0-18%

Adjuvants 2-5%.

The chemical herbicides are atrazine, alachlor, acetochlor, metolachlor, pretilachlor, isoproturon, chlorotoluron, oxazoyl, diuron, fruit or Yunong music, etc.;

The auxiliary agent is Tween 20, Tween 80, dextran, vegetable oil, cellulose, polyether silicone oil, etc.;

A method for preparing said biological herbicide, carried out as follows:

(1) Preparation of medium: bacterial strain Mds0404 can be activated with PDA medium, PSA medium, bran medium, corn flour medium, starch yeast medium or Czapek medium and induce the production of conidia;

(2) Activation culture of the bacterial strain Mds0404: pick a small amount of preserved mycelium pieces of the fungus and put them in any plate culture medium in step (1), and culture them in the dark at a constant temperature of 28° C. to activate the bacterial strains;

(3) Cultivation of the strain seed solution: pick a little of the bacterial mass cultivated in step (2), insert it into a triangular flask filled with any culture solution (excluding agar) in step (1), 28°C, 150rpm, shake culture 3-5 days until abundant mycelium balls are produced, set aside;

(4) Production, cultivation and collection of strain spores: solid or liquid-solid joint culture methods can be used to inoculate Mds0404 mycelium blocks or liquid-cultured mycelium balls on half-cooked wheat grains under aseptic conditions. Put the eggplant bottle in the dark at 25-28°C for 6-8 days. After the mycelium is covered with solid medium, smash the wheat grains and spread them on a flat plate. Control the humidity to 40-65% for 12 hours The spores can be obtained by irradiating with black light for 2 days at intervals, and the spores can be collected with a spore vibrating sieve machine or a spore collector for later use;

(5) Mix the pure spores, chemical herbicides and adjuvants weighed according to the formula to form a biological herbicide, sub-package, seal, and prepare the product.

The method that above-mentioned Curvularia strain Mds0404 herbicide is used for controlling weeds is:

The aqueous suspension of pure conidia of Curvularia strain Mds0404 at a concentration of 3-50g/L can be used for foliar spraying or seed treatment of weeds; Suspension liquid; or the mixture of conidia and adjuvant mixed with 5-20% conventional dosage of chemical herbicides for water suspension; or used in combination with pathogenic strains isolated from other weeds. All are conventional techniques mastered by those skilled in the art.

Compared with the prior art, the present invention has the beneficial effects as follows:

1. Strain Mds0404 is safe to seedlings of wheat, soybean, cotton, cowpea, sunflower, corn, peanut, rice and other crops. It is partially infected to weeds such as foxtail, barnyard grass and goosegrass, but it is serious to crabgrass Infect, the control effect after one week is more than 85%, and the host range of this bacterium is relatively single-minded, and agricultural production is safe (see table 2); Therefore adopt Curvularia crabgrass fungi agent of the present invention not only to be used for preventing and eradicating soybean, cotton , peanut and other dicotyledonous crop field weeds can also be used to control weeds in monocotyledonous crop fields such as rice, corn and wheat, which is obviously superior to conventional control that can hardly choose between monocotyledonous crops and monocotyledonous weeds Chemical herbicides for monocotyledonous weeds are therefore superior to the prior art US patents.

2. When used in conjunction with related adjuvants, the control effect of the fungal agent on the target weed crabgrass can reach 85%-100%, especially for plants below the age of 3 leaves.

3. The bacterial strain Mds0404 obtained by screening in the present invention is used as a production species for the preparation of bacterial agents. It has wide adaptability to culture conditions and can grow at 10-40°C and pH 5-10. The raw materials of the culture medium are easy to obtain and the cost is low. Commonly used agricultural basic products can be mass-produced, and the spore yield is high (2.0×10 ⁷ spores/gram dry weight of wheat kernels); as a susceptible fungal agent for weed control, it has a wide range of disease-causing conditions in the field, 10 The spores can germinate at -40°C, and the inactivation temperature is 55°C, which is easy to infect the control objects. Studies have shown that this fungus has great potential to be developed into a commercial biological herbicide, which can be used to control weed crabgrass in most crop fields.

4. The biological herbicide of the present invention utilizes the living organism itself and comes from the environment in which it is used. Its degradation products are all recyclable organic matter and will not cause pollution due to use. Therefore, it is environmentally safe and can be used to produce green or organic agricultural products.

Therefore, the present invention utilizes Curvularia sp. strain Mds0404 to develop a biological herbicide to control crabgrass. This bacterial strain is highly safe to main crops, does not infect gramineous crops and broad-leaved plants, and has a wide range of applications. Therefore, this biological herbicide has a very significant effect. economic and social benefits.

Detailed ways

The present invention will be further described in detail through examples below, but this does not constitute any limitation to the present invention.

Embodiment 1: (screening of preventing and controlling crabgrass efficient strain Mds0404)

From the diseased plants of crabgrass crabgrass in the wild that naturally occurred in Yuyao, Zhejiang Province, the diseased leaves of crabgrass crabgrass were collected, and the pathogenic fungi were isolated and cultured with PDA medium. Several fungal strains were isolated from them, and their pathogenicity was tested. The following test: pick the single spores of each strain under the microscope, inoculate them on PDA medium respectively, and cultivate them in the dark at 28°C to observe the colony characteristics of the pure culture of pathogenic fungi; and inoculate each colony disc with a diameter of 6mm on the In the center of the PDA flat plate, measure the diameter of each bacterial colony after 7 days; each bacterial strain is formulated into a concentration of 1 × ¹⁰⁶ /ml spore suspension and sprayed on 2 leaves and 1 heart crabgrass plant, after 4 days of treatment, observe the spore suspension of each bacterial strain Pathogenic properties (see Table 1), select the Curvularia strain Mds0404 with the strongest morbidity and mortality therefrom, and preserve; the scabs of the diseased plant leaves of crabgrass crabgrass are dot-shaped at the beginning, and then expand into flat ovals of different sizes Or fusiform lesions, the center of the lesions is brown, the periphery is purple or purple red, there is a yellow halo on the periphery, and there are necrotic lines at both ends. In severe cases, multiple lesions combine, causing the entire leaf to shrivel and curl; Black fruiting bodies can be seen in the center of crabgrass lesions infected by this strain, and a small amount of conidia under the same culture conditions can be seen in diseased leaves.

Table 1 The characteristics of the colony of crabgrass pathogenic fungi and the comparison of their pathogenicity to crabgrass

Embodiment 2: (measurement of bacterial strain Mds0404 host range)

Determination of the host range clearly shows that strain Mds0404 is safe to crops such as cotton, sunflower, soybean, cowpea, peanut, corn, wheat and rice; barnyardgrass, goosegrass and foxtail are infected, while crabgrass is a serious infection (see Table 2) , the control effect after one week is more than 85%.

Table 2 Determination of the host range of Curvularia crabgrass strain Mds0404

Note: NS means no response; LS means infection; HS means serious infection

Embodiment 3: (a kind of biological herbicide of the present invention and preparation method 1 thereof)

Take by weighing 83 grams of pure spores of bacterial strain Mds0404, 15 grams of atrazine, and 2 grams of Tween, and mix them into 100 grams of biological herbicide. The preparation method of this agent is carried out according to the following steps:

(1) Preparation of culture medium: bacterial strain Mds0404 can be activated and induce the production of conidia with any of the following medium:

PDA medium: 150g potato, 10g glucose, 18g agar, 1L water;

PSA medium: 150g potato, 10g sucrose, 18g agar, 1L water;

Bran medium: 50g bran, 18g agar, 1L water;

Corn flour medium: corn flour 50g, agar 18g, water 1L;

Starch yeast medium: starch 10g, yeast powder 2g, agar 18g, water 1L;

Czapek medium: KNO ₃ 2g; K ₂ HPO ₄ 1g; KCl 0.5g; MgSO ₄ .7H ₂ O 0.5g;

FeSO ₄ 0.01g; sucrose 30g; agar 18g; water 1L.

(2) Activation culture of bacterial strain Mds0404: pick a small amount of preserved mycelia pieces into the PDA plate culture medium in step (1), culture in constant temperature and dark at 28° C., and activate the bacterial strain;

(3) Cultivation of the strain seed liquid: pick a little bacterial block cultivated in step (2), insert it into a triangular flask filled with step (1) PD culture solution (PDA medium without agar), at 28°C, 150rpm, shaking culture for 3-5 days until the bacterial solution produces abundant mycelium balls, set aside;

(4) Production, cultivation and collection of bacterial strain spores: solid or liquid-solid joint culture methods can be used; under sterile conditions, the mycelial balls of Mds0404 mycelium block or liquid culture are inoculated in half-cooked wheat grains Put the eggplant bottle in the dark at 25-28°C for 6-8 days. After the mycelium is covered with solid medium, smash the wheat grains and spread them on a flat plate, and control the humidity to 40%-65%. 12 Hour interval black light (20W, vertical light distance is 25cm) light for 2 days, can get spores, after drying, collect spores with spore vibrating screen machine or spore collector, and set aside;

(5) The pure spores, atrazine, and Tween weighed according to the formula are mixed into a biological herbicide, subpackaged, sealed, and the product is ready-made.

Embodiment 4: (biological herbicide of the present invention and preparation method 2 thereof)

Take 95 grams of pure spores of bacterial strain Mds0404 and 5 grams of vegetable oil according to the formula, and mix them into 100 grams of biological herbicide; the preparation method of the agent is the same as in Example 3.

Embodiment 5: (biological herbicide of the present invention and preparation method 3 thereof)

Weigh 90 grams of pure spores of bacterial strain Mds0404, 7 grams of alachlor, and 3 grams of polyether silicone oil according to the formula, and mix them into 100 grams of biological herbicide; the preparation method of the agent is the same as in Example 3.

Embodiment 6: (pure spore control effect test on crabgrass)

Bacterial strain Mds0404 was cultivated in PDA medium at 28°C for 8 days in the dark, brushed off the surface hyphae, irradiated with a black light for 12 hours at intervals for 1 day, rinsed with sterile water and filtered through gauze, collected the filtrate and examined the concentration of spores under a microscope (1 × 10 ⁶ individuals/ml), spray the filtrate with a hand-held sprayer, inoculate on the crabgrass at the stage of 1 leaf, 1 heart to 3 leaves, 1 heart, moisturize for 12 hours, and after 5 days of treatment, the control effect of the grass reaches more than 90%. . The pathogenic symptoms of this spore inoculated on crabgrass are: purple-red lesions appear on the leaves at first, dot-shaped, and then expand to nearly round or spindle-shaped brown lesions, which are connected into pieces when the number is large, and finally the leaves are large or whole. The slices are dry. There are transparent round spots on the leaves of young plants, and the whole plant will die soon in severe cases. Black fruiting bodies are visible on diseased leaves. It was observed that the spores on the surface of diseased leaves were the same as the spores of Curvularia sp. Mds0404 sprayed. The onset symptoms under the above experimental conditions are similar to natural diseases.

Embodiment 7: (spore+auxiliary agent removes crabgrass effect test)

According to the method of Example 4, firstly prepare the spore suspension, then add the auxiliary agent Tween, vegetable oil, cellulose, starch or polyether silicone oil to treat crabgrass, and the result is that the speed and efficiency of weeding are obviously improved. (See Table 3).

Table 3 The effect of adjuvant on Mds0404 inoculation effect

Auxiliary Strain control effect (%) Fresh weight control effect (%) Without additives 45.36c 52.98c Grade A Cellulose 91.53a 98.20a TW-20 86.79b 93.12b TW-80 93.19a 97.36a Polyether silicone oil 94.24a 99.26a

Data in each column are significantly different from different lowercase letters (P≤0.05).

Embodiment 8: (the chemical herbicide of spore+adjuvant+5-20% routine dosage removes crabgrass effect test)

According to the method of Example 3 or 5, first prepare the spore suspension with auxiliary agent added, then add atrazine, alachlor, acetochlor, metolachlor, and trichlor in a conventional dosage of 5-20% Ammonium, isoproturon, chlorotoluron, dioxazone, diuron, Guoer or Yu Nongle, evenly sprayed on crabgrass at the 2.5-3.5 leaf age, moisturizing for 12 hours and no moisturizing treatment 4 days after the treatment, the crabgrass of the moisturizing group was controlled by 100%, and the crabgrass of the non-moisturizing group was controlled by 70-90% (see Table 4).

Table 4 Effect of atrazine and Mds0404 mixed inoculation

Embodiment 9: (pure spore control effect test of crabgrass seed)

After the culture dish autoclaving that is covered with filter paper is set, put 50 crabgrass seeds in every dish, the spore suspension that the spore concentration produced by the method of embodiment 6 reaches (1×10 ⁶ pieces/ml) is applied to cultivation dish, 2 ml per dish, repeated 5 times; in addition, the crabgrass seeds in the aseptic water treatment dish were used as a control; after 4 days, the control crabgrass seeds germinated and grew normally, while the crabgrass seeds treated with the bacterial solution were immediately germinated. All infected died.

Claims (3)

1. Curvularia Boedijn Mds0404 strain, whose strain preservation number is CGMCCNO.1827. 2. A biological herbicide, characterized in that the herbicide takes Curvularia Boedijn (Curvularia Boedijn) Mds0404 bacterial strain CGMCC NO.1827 as bacterial species, and the weight percent content of its components and each component is: pure spores 80-95 %, chemical herbicide Atrazine 0-18%, auxiliary agent Tween 20, Tween 80, vegetable oil, cellulose or polyether silicone oil 2-5%. 3. A method for preparing the biological herbicide according to claim 2, characterized in that the method is carried out in the following steps: (1) Preparation of medium: bacterial strain Mds0404 can be activated with PDA medium, PSA medium, bran medium, corn flour medium, starch yeast medium or Czapek medium and induce the production of conidia; (2) Activation culture of the bacterial strain Mds0404: pick a small amount of preserved mycelium pieces of the fungus and put them in any plate culture medium in step (1), and culture them in the dark at a constant temperature of 28° C. to activate the bacterial strains; (3) Cultivation of bacterial strain seed liquid: pick a little bacterial block cultivated in step (2), insert it into any triangle flask containing step (1) without agar culture solution, 28°C, 150rpm, shaking culture 3- 5 days until abundant mycelium balls are produced, set aside; (4) Production, cultivation and collection of strain spores: using a solid or liquid-solid joint culture method, under sterile conditions, inoculate Mds0404 mycelium blocks or liquid-cultured mycelium balls on eggplants with half-cooked wheat grains Place in the bottle and culture in the dark at 25-28°C for 6-8 days. After the mycelium is covered with solid medium, smash the wheat grains and spread them on the flat plate, control the humidity to 40-65%, and keep the interval of 12 hours After 2 days of black light irradiation, spores can be obtained, and the spores can be collected with a spore vibrating sieve machine or a spore collector for later use; (5) Mix the pure spores, chemical herbicides and adjuvants weighed according to the formula to form a biological herbicide, sub-package, seal, and prepare the product.