CN1227979C - High efficiency biological weed control bacterial and breeding selection method - Google Patents

Abstract

The present invention relates to the xanthomonascampestris pv. retroflexus of biological weed eliminating bacteria of high efficiency, which belongs to xanthomonas. The strain screening technology uses rhizosphere soil from 5 to 15cm, terrestrial stems and leaves of retroflexus, crab grass, pigweed, moleplant seeds, cassia tora and other weeds for the sampling purpose. In separation, NPC culture media, growth inhibiting tests of chlorella pyrenoidosa and glutamine synthesized enzyme inhibitors are used for screening models, and strains with high weed removing bioactivity and wide weed eliminating spectrums are selected. After tests, the bacterium agent has excellent preventing and eliminating effects on most of broadleaf weeds and partial main grassy weeds in the field at present. The preventing effect of the stem and leaf spraying treatment on the retroflexus, pigweed, cassia tora and water pepper is higher than 85%, and the preventing effects on annual meadowgrass, the crab grass and the moleplant seeds are respectively 75%, 85% and 80%. The present invention is safe for most of lawn grass and seedlings.

Description

A kind of high-efficiency biological herbicide and its breeding method

Technical field

The invention relates to a biological herbicide Xanthomonas campestris pv. retroflexus and a breeding method thereof.

Background technique

Chemical herbicides can effectively control many harmful weeds, but their extensive use has also caused a series of environmental problems, and the development of new chemical herbicides is difficult, with low success rate and high cost. In contrast, microbial herbicides have low toxicity, easy degradation, and good environmental compatibility, and have great development potential.

The research on microbial herbicides began in the 1960s, and a number of commercialized fungal herbicides were launched successively. my country’s Shandong Academy of Agricultural Sciences developed the culture of “Lubao No. 1” Cuscuta spores to control soybean fields. Cuscuta: These fungal herbicides are all spore preparations, with strict requirements for action conditions, high requirements in mass production, formulation, storage and other technologies, have not been widely accepted, and have not produced significant social and economic benefits. In the early 1990s, people turned the research target to bacteria, screened out the bacteria with inhibitory effect from the microbial flora in the root soil of weeds, and obtained the extracellular toxins that make the host pathogenic by culturing. Bacteria have a shorter growth cycle than fungi, simple fermentation process, and easy control of the production process. Their extracellular toxins are not as strict as fungal spores and are easily degraded by soil. They have good application and development prospects and become a new option in the development of microbial herbicides. Big hot spot.

The root bacteria with herbicidal effect are mainly Gram-negative bacteria of the genera Pseudomonas, Erwinia, and Xanthomonas, among which Pseudomonas is the most studied belongs to. The phytotoxin phaseolotoxin produced by Pseudomonas syringae pv. phaseolicola can make the leaves of Kudzu appear chlorosis and cause local necrosis; Green, serious cause necrosis of plant leaves. Gurusiddaiah.S used the crude toxin after preliminary purification of the fermentation broth of the disease-causing Pseudomonas fluorescens strain D7 to control early brome and so on. Johnson B.J. was the first to study the use of Xanthomonas as a biological herbicide. He screened out several strains of Xanthomonas Campestris pv.poannua with herbicidal activity to control the annual bluegrass (Poa annua L. .). It is mainly suitable for lawns, peanut fields, potato fields, roadsides, golf courses and other dry green lands.

Contents of Invention

The object of the present invention is based on the principle of plant ecology, and proposes to separate and obtain the bacterial strain Xanthomonas campestris pathogenic species of Amaranthus retroflexi with higher herbicidal activity from the rhizosphere soil or plant tissue and its selection and breeding of the herbicidal bacterium Methods.

The method for the biological herbicide of the present invention is as follows:

Take samples from the rhizosphere 5-15cm of soil, above-ground stems and leaves of weeds such as Amaranthus reflexae, crabgrass, quinoa, Cassia, and other weeds, rinse 1-3 times in 200ml sterile water, and put 100ml 0.01-2.0% Tween- Shake the flask at 400-600rpm in 80 solution for 10min, make 10-20 times serial dilution with sterile phosphate buffer (5-20Mm K ₃ PO ₄ -KH ₂ PO ₄ , 0.05-2M NaCl, PH6.4-8.2), take Spread 0.1-0.3ml on the NPC medium plate, incubate at 20-32°C for 20-30h to observe the results, select colored colonies, store them on nutrient agar, and further isolate and purify the single colony screened out: ①Bacteriostatic test , connect a ring of bacteria to be tested with an inoculation needle, puncture and inoculate it on an E. coli plate, incubate in an incubator at 20-32°C for 20-30h, and observe the antibacterial plaque; ② For the growth inhibition experiment of Chlorella pyrenoidosa, the The solution was inoculated into the Erlenmeyer flask, the initial concentration was 8×10 ³ -8×10 ⁶ cells/ml; the treatment was added to the test solution, and the control was not added, 20-30°C, 3000-6000lux illuminance, continuous light and 100-200rpm Rotate and shake culture for 2-7 days, take the culture medium as a reference, measure the absorbance value (optical path 1cm) at the maximum absorption wavelength of 680nm, and measure the inhibition rate; Amide and the regular medium with glutamine at a concentration of 0.05-10% are coated with a Bacillus subtilis bacterial suspension plate, and the antibacterial effect is observed, and the strains with fast colony growth, good antibacterial effect and high biological activity are selected. .

The selected strain with high-efficiency biological herbicidal activity is Xanthomonas campestris pv.retroflexus, which has an inhibition zone of 15-30mm for E. Strong, the inhibition to Chlorella pyrenoidosa is 60-90% when the fermentation broth is diluted 10-30 times.

The population characteristics of the Xanthomonas campestris pathogenic species of Xanthomonas campestris obtained by the method of the present invention: the colony is round on the solid plate, with a whole margin, smooth and moist surface, translucent and yellow. The diameter of the colony is 5-6mm/24h; when cultured statically in the liquid medium, a sterile film is formed, the cells are stable, and the bacterial solution becomes turbid and precipitates.

Individual morphology: short rod-shaped cells, 1.5-2.0×0.5-1.0um, no spores, polar flagella, motile, negative Gram stain, with lipid granules.

Physical and chemical characteristics: organic chemical can be heterotrophic, catalase reaction produces gas, strictly aerobic, does not fix nitrogen, and can use one-carbon compounds as the only carbon source. Can produce water-insoluble carotenoids, oxidase reaction is negative, can not reduce nitrate. It can hydrolyze glucose and gelatin, but not starch. The growth temperature is 8-50°C, and the optimum growth temperature is 25-35°C; it can grow at pH 4-9, and the optimum pH is 5.5-7.5.

According to the above characteristics, the bacterium belongs to the genus Xanthomonas (Xanthomonas) in the eighth edition of "Bergey's Handbook of Bacteriology for Identification". Deposited, the deposit number is: CGMCC NO.0902. The culture characteristics, individual characteristics and physiological and biochemical characteristics of the strain are shown in Tables 1 and 2.

Prepare herbicide with high-efficiency biological herbicide of the present invention:

The isolated Xanthomonas campestris pathogenic strain of Amaranthus retroflexi is cultured on a slant in a conventional solid medium, rejuvenated, and cultured at 25-30°C for 9-24h; then seed culture is carried out, and 2 /3 volume of liquid medium, the formula of liquid medium is: beef extract 0.5-2.2%, peptone 0.2-4%, glucose 0.2-4%, NH4H2PO4 0.05-0.5%, NaCl 0.1-2.0%, make up the volume with water ; Adjust the initial pH to 6.0-7.4, sterilize at 121-125°C for 20-30min, inoculate with 1% inoculum after cooling, and incubate at 25-30°C for 12-32h.

Fill the liquid culture medium of 2/3 volume in the fermentation tank, the formula of liquid culture medium is the same as the formula of seed liquid culture medium, inoculate with the inoculum size of 6-12%, and ventilation rate is 1: 0.6-1.5 (V: V), Cultivate at 25-30°C for 54-120 hours; the bacteria content in the bacterial fermentation broth is 10 ^5-9 cfu/ml; add additives, such as 0.5-5% Tween 80 or ammonia silicon, to obtain herbicides; directly package into products.

The application of high-efficiency biological herbicide of the present invention:

The high-efficiency biological herbicide fungus agent of the invention is used for preventing and controlling broadleaf weeds and gramineous weeds in lawn grasses, woody seedlings and herbaceous seedlings.

Said lawn grasses are tall fescue, bermudagrass, horseshoe gold, ryegrass, zoysia and the like. Seedlings are camphor, cedar, magnolia, osmanthus, cedar, Chinese pagoda tree, boxwood, privet, safflower magnolia, golden leaf Michelia, wisteria, Lingxiao and so on. The broad-leaved weeds and grass weeds are mainly amaranth, purslane, amaranth, shepherd’s purse, barnyardgrass, foxtail, goosegrass, crabgrass, bluegrass, kangaroo, barnyardgrass, quinoa, Cocklebur, shepherd's purse, pig's cattail, mother-in-law, field bindweed, hollow lotus seed grass, yellow punt, cotton wool sorrel leaves, broken rice shepherd's, Cyperus cyperus, etc.

Method of use, dosage and time: apply by spraying, the dosage is 500-1000L/ha; the best use period is when the weeds germinate to the five-leaf stage, generally in March and April in the first half of the year, and in September in the second half of the year , October; the bacterial agent can be applied once or twice, and the second application is one to four weeks after the first application.

Temperature, humidity and light condition tests show that the bacteria agent prepared by the biological herbicide fungus of the present invention has relatively loose requirements on the environment, and the temperature is 10-35 °C, and the humidity is more than 20%. The pathogenicity is good; the pathogenicity is generally shown 1 week after application, and can last for more than 4 weeks. There is no residue, no pathogenicity, and it is safe for the environment. The bacterial agent can also enrich soil microorganisms. At the same time, the bacterial agent is also suitable for large-scale production, has simple effective strains, simple process, low cost and convenient use.

Description of drawings

Fig. 1 is a process flow diagram of preparing bacterial agent from the isolated Xanthomonas campestris pathogenic species.

Detailed ways

The method for selecting and breeding biological herbicides is further illustrated below by examples, and the concrete steps are as follows:

Sampling from the 10cm soil, above-ground stems and leaves of Amaranth retroflexus weeds, remove the excess soil at the roots of the weeds, rinse twice in 200ml sterile water, drop into 100ml 1.0% Tween-80 solution, shake the flask at 500rpm for 10min, and use Sterile phosphate buffer (6MmK ₃ PO ₄ -KH ₂ PO ₄ , 0.2MNaCl, PH7.2) was serially diluted 10 times, and 0.1ml was spread on the NPC medium plate. The NPC medium plate refers to Davis’ minimum Add penicillin, novobiocin, and cycloheximide to the culture medium, culture at 24°C for 22 hours to observe the results, select colored colonies, and store them on nutrient agar. Further isolate and purify the single colony screened out: ①Antibacterial test, connect a ring of bacteria to be tested with an inoculation needle, puncture and inoculate it on an E. The bases were conventional medium. ② Chlorella pyrenoidosa growth inhibition experiment, the algae liquid was inoculated into the Erlenmeyer flask, the initial concentration was 8×10 ³ cells/ml. The fermentation broth of the bacteria to be tested was added to the treatment, and the control was not added. The 22°C, 3000lux light intensity, continuous light and 100rpm rotary shaking culture were cultivated for 3 days. Taking the culture broth as a reference, the absorbance value was measured at the maximum absorption wavelength of 680nm (optical path 1cm), and the inhibition was measured. Rate. ③ Screening of glutamine synthetase inhibitor model, spread Bacillus subtilis bacteria suspension plate on conventional medium without glutamine and with 2% glutamine added, and observe the antibacterial effect. Thereby, the bacterial strains with fast bacterial colony growth, good antibacterial effect and high biological activity were selected.

The selected bacterial strain with high-efficiency biological herbicidal activity is Xanthomonas campestris pv.retroflexus. The inhibition to Chlorella pyrenoidosa is more than 70% when the fermentation broth is diluted 30 times.

The application example of the high-efficiency biological herbicide bacterium prepared with the biological herbicide of the present invention is as follows:

Embodiment 1: Amaranthus retroflexus

Spray fungal agents at the stage of 4 true leaves from the budding of Amaranthus retroflexus seeds, and the dose is 500L/ha, which shows a good control effect (see Table 3). New leaves wilted after 1 day, old leaves began to wilt after 2 days, leaves basically withered after 4 days, and the control effect reached more than 90%. After 3 weeks, the control effect decreased slightly, and part of the stems and leaves of Amaranthus reflexae began to recover, but the growth was poor, and it had no effect on the protected plants.

Example 2: Cassia

Spraying fungicides at the stage of cassia seed germination to the fourth round of true leaves, the dose is 500L/ha, shows a good control effect. After 2 days, the new leaves began to wilt, and after 3 days, some old leaves began to wilt, and after 7 days, the leaves basically withered, and the control effect reached more than 85%. After 3 weeks, the control effect declined, and a small part of the stems and leaves of Cassia Cinnamomi began to recover, but the growth was extremely poor, and did not affect the protected plants.

Example 3: Small quinoa

The antibacterial agent was sprayed at the stage of budding and 4 true leaves of the seeds of quinoa, with a dose of 500L/ha, and the control effect was good. After 5 days, the leaves basically withered, and the control effect reached more than 85%. After 3 weeks, the control effect decreased slightly, but it had been controlled below the economic threshold.

Embodiment 4: bluegrass

When bluegrass seeds sprout from white to 3 leaves, the fungal agent is sprayed at a dose of 500L/ha, and the control effect is better. After 4 days, most of the leaves withered, and the control effect reached 75%. After 2 weeks, the protective effect decreased.

Embodiment 5: crabgrass

When the seeds of crabgrass germinate from white to 4 true leaves, the fungicide is sprayed at a dose of 500L/ha, which shows a good control effect. The new leaves wilted after 1 day, the old leaves wilted after 2 days, and the leaves basically withered after 4 days. The control effect reached more than 85%, and the control effect began to decline after 3 weeks.

Example 6: Tall Fescue

The bacterial agent was sprayed after the 2-leaf stage of tall fescue germination, and the dosage was 900L/ha, which showed better safety (see Table 4). There is no phytotoxicity above the 3-leaf stage, and the plants at the 2-leaf stage are slightly dwarfed, and they can recover after 1 week, and it is safer to use low doses. Therefore, it is safer to apply pesticides after the tall fescue grows to the 2-leaf stage in the field.

Example 7: Golden Leaf Michelia

Spray the bacteria agent after the golden leaf Michelia germinates and grow to more than 4cm, and the dose is 900L/ha, which shows better safety (see Table 5). Growth was inhibited by spraying at 3-4cm, but recovered quickly. Therefore, it is safe to apply pesticides in the field after Michelia jinye grows to a height of 4.5cm.

Embodiment 8: safflower magnolia

After the safflower magnolia sprouts and grows to more than 5cm, the fungal agent is sprayed at a dose of 900L/ha, which is relatively safe.

Table 1. Culture characteristics and individual characteristics of pathogenic variants of Xanthomonas campestris retroflexus Solid plate colony characteristics size 5-6mm shape round surface structure smooth and moist edge Entire optical characteristics translucent Lawn color yellow produce pigment have liquid culture Biofilm none turbid have precipitation have individual characteristics width (um) 0.5-1.0 length (um) 1.5-2.0 Gram stain Negative athleticism have flagellum Extreme > 1 root Liposome staining have

Table 2. Physiological and biochemical characteristics of pathogenic variants of Xanthomonas campestris Catalase + Oxidase - Glucose oxidation fermentation test + gelatin hydrolysis + starch hydrolysis - L-valine + beta-alanine + DL-Arginine + Nitrate reduction - Grow at 41°C + Determination of arginine dihydrolase - Production of Fluorescent Pigments -

Table 3 Bacteria control effect on weeds of different leaf ages (inhibition rate of fresh weight %) weed name leaf age 1 2 3 4 5 Amaranthus retroflexus 98.1 90.5 86.6 82.4 45.2 Cassia 100.0 93.4 84.1 81.3 50.7 Quinoa 100.0 94.3 88.6 84.2 67.8 Polygonum 95.6 86.4 80.1 71.8 39.6 car front 98.2 91.4 86.3 69.4 41.8 sorrel 94.0 88.7 79.4 71.4 52.6 bluegrass 95.0 87.7 78.5 63.4 41.7 crabgrass 100.0 96.3 90.5 86.2 66.4 Daughter 94.7 86.7 74.5 65.7 44.9 goosegrass 90.4 80.0 67.5 53.3 37.7

The phytotoxicity situation (fresh weight inhibition rate %) of table 4 inoculum to different leaf ages turfgrass lawn grass name leaf age 1 2 3 4 tall fescue 45.7 15.6 6.3 3.4 Bermudagrass 51.3 20.4 5.5 0 Gold Horseshoe 66.7 17.3 9.1 3.6 rye grass 53.4 19.3 -1.3 2.7

The phytotoxicity situation (fresh weight inhibition rate %) of table 5 bacterial agent to different plant height seedlings lawn grass name Plant height (cm) 2 3 4 5 Jin Ye smiles 55.7 31.6 7.8 4.4 Emei smiling 56.3 40.4 22.5 4.9 Red Flower Magnolia 62.7 47.6 27.6 6.6

Claims (4)

1. a biological weed control bacterium is characterized in that this bacterium is the pathogenic mutation (Xanthomonas campestris pv.retroflexus) of xanthomonas campestris Amaranthus retroflexus, and deposit number is: CGMCC NO.0902.

2. biological weed control bacterium according to claim 1 is characterized in that the form of this bacterium and physiological characteristic and condition of culture are:

1) bacterium colony is rounded on solid plate, full edge, and smooth surface is moistening, and is translucent, yellow, colony diameter is 5-6mm/24h; Do not have mycoderm during static cultivation in the liquid medium within and form, cytotostatic, bacterium liquid become muddy also has precipitation to produce, and cell is a rod-short, 1.5-2.0 * 0.5-1.0um, and no gemma, polar flagella, the Albert'stain Albert feminine gender is removed from office in motion, and the class fat granule is arranged;

2) organic chemoheterotrophy, the catalase reaction aerogenesis, strict aerobic, fixing nitrogen can not utilize one-carbon compound as unique carbon source, can produce non-water-soluble carotenoid, oxydase reaction feminine gender, the nitrate that can not reduce can hydrolyzation of glucose, gelatin, can not hydrolyzed starch;

3) growth temperature is 8-50 ℃, and pH is 4-9.

3. biological weed control bacterium according to claim 2 is characterized in that growth temperature is 25-35 ℃, and pH is 5.5-7.5.

4. the selection of biological weed control bacterium according to claim 1, it is characterized in that from Amaranthus retroflexus, lady's-grass, lamb's-quarters, moleplant seed or Cassia tora weeds rhizosphere 5-15cm soil, terrestrial stem, blade sampling, rinsing is 1-3 time in the 200ml sterile water, 400-600rpm shakes a bottle 10min in the input 100ml 0.01-2.0% Tween-80 solution, with the sterile phosphate buffer solution of PH6.4-8.2, composition is 5-20Mm K ₃PO ₄-KH ₂PO ₄, 0.05-2MNaCl makes 10-20 times of serial dilution, get 0.1-0.3ml and coat the NPC culture medium flat plate, cultivate the 20-30h observed result for 20-32 ℃, select coloured bacterium colony, be stored on the nutrient agar, the single bacterium colony that just sifts out is further separated purifying: 1. bacteriostatic test, connect ring bacterium to be measured with transfer needle, percutaneous puncture-inoculation is to the Escherichia coli flat board, and 20-32 ℃ of incubator cultivated 20-30h, observing antibacterial spot, is 15-30mm to colibacillary inhibition zone; 2. chlorella pyrenoidosa (Chlorella pyrenoidosa) growth inhibition experiment is inoculated into algae liquid in the conical flask, and initial concentration is 8 * 10 ³-8 * 10 ⁶Individual cell/ml; Handle and add liquid to be measured, contrast does not add, 20-30 ℃, 3000-6000lux illuminance continuous light and 100-200rpm rotational oscillation are cultivated 2-7d, with the culture fluid is reference, under maximum absorption wavelength 680nm, measure light absorption value, light path is 1cm, surveys inhibiting rate, and the zymotic fluid dilution 10-30 that is suppressed at of chlorella pyrenoidosa is doubly had 60-90% inhibition effect down; 3. glutamine synthetase inhibitor model discrimination, be coated with bacillus subtilis bacterial suspension flat board not adding glutamine and added on the conventional medium of glutamine that concentration is 0.05-10% respectively, observe fungistatic effect, it is fast therefrom to select colony growth, good antimicrobial effect, the bacterial strain that biologically active is high.

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