CN1973631A - Biocontrol fungus for preventing and controlling plant mycosis and its prepn process - Google Patents

Abstract

The invention belongs to the technical field of biological pesticides. The content includes four aspects: (1) the isolation and identification of Acinetobacter baumannii LCH0606 strain (Acinetobacter baumannii LCH0606); (2) the preparation of Baumannii liquid and its application in the control of plant diseases; (4) Analysis of active components of baumannin. The antibacterial active ingredient of Bowman's bacteria liquid or baumannin is mainly Iturin A, which has the characteristics of wide bactericidal spectrum, long-lasting effect, less resistance to diseases, no phytotoxicity of plants, biosafety and environmental friendliness, etc. Leaf fungi, especially Anthracnose, Phytophthora, Botrytis cinerea, Fusarium and Rhizoctonia, have significant control effects; in addition, Bowman's liquid or Baumannin can promote the germination of rice seeds to a certain extent.

Description

Biocontrol bacteria for preventing and treating plant fungal diseases and preparation method thereof

1. Technical field

The invention belongs to the technical field of biological pesticides.

2. Background technology

Human beings are facing many challenges in the 21st century, and one of the main problems is the problem of population and food. my country's population accounts for a quarter of the world's total population, but the arable land is only 6.8% of the world's arable land. This problem is particularly prominent in our country. Therefore, increasing food production is a strategic task related to people's livelihood in our country. But according to investigations, the world's main food crops and economic crops lose 20% (Oerke et al, 1994. Crop Protection and Crop Production. Elsevier, Amsterdam, 808pp) due to diseases every year. my country's grain crops lose at least 10-15% due to diseases. If fungicides are not used at all, the losses of cucumbers in normal years are about 55%-75%, radishes are 40%, sweet baskets are 25%, apples are 30%. 24% [Chen Wanyi, 1998. Plant Protection 24(5): 33-36]. This shows the important position of fungicides in food crops and economic crops. In fact, without the use of fungicides, half of the world's crops would not be grown under current conditions.

Most of the fungicides used in agricultural production are chemical compounds. Although chemically synthesized fungicides have the advantages of high control efficiency and convenient application, they have the following disadvantages: ①The problem of resistance is serious: especially the resistance of plant pathogenic fungi to systemic fungicides develops quickly and has a high level of resistance . It is generally applied for about 3 years, and plant pathogenic fungi can develop resistance to it. It has been reported that 46 pathogenic fungi have developed high resistance to benomyl (Gullino et al, 2000. Crop Protection 19: 1-11). ②The cost of developing new fungicides is expensive: As the requirements for fungicides are becoming more and more stringent, the cost of developing new fungicides is also increasing. In the United States, more than 20,000 compounds need to be researched for every successful research and development of a new type of fungicide, costing about 0.8-100 million US dollars (Leroux, 1996, Pestic. Sci. 47: 191-197). ③Environmental pollution: Although chemically synthesized fungicides are less harmful to humans than chemically synthesized insecticides, chemically synthesized fungicides also have carcinogenic effects, are toxic to wild and non-target organisms, and have poor environmental compatibility. Therefore, in the promotion of pollution-free pesticides and sustainable agricultural production today, it has become an inevitable trend in the development of pesticides to develop and develop environmentally friendly biocides that are less likely to produce resistance and cause less pollution.

Compared with chemical synthetic fungicides, the use of biological control and natural products has more attractive prospects, especially microbial fungicides. On the one hand, microorganisms are the largest biological group on the earth. They not only have a wide range of biodiversity, but also The production of target substances can be greatly increased through strain selection, metabolic regulation, large-scale fermentation and other modern bioengineering technologies, and it is easy to realize industrial production; Safer, easy to degrade, and lower synthesis cost.

Endophytes refer to a class of special microorganisms that grow in the host without causing disease. A large number of studies have proved that endophytes regulate and enhance the environmental adaptability of host plants by producing secondary metabolites, such as nitrogen fixation, drought resistance, and Insect and disease resistance, etc. (Chanway, 1996. Can. J. Bot. 75: 331-332; Wilson et al., 1994. Mycologia 86: 635-647). Fisher et al. (Fisher et al., 1984.Bot.Helv.94: 249-253) found that 30% of endophytes can produce antifungal and/or antibacterial substances when testing the biological activity of endophytes; especially When Stierle et al. (Stierle et al., 1993.Science 260:214-216) in the United States found two endophytic fungi capable of producing paclitaxel and its analogues from Pacific berry taxus, the secondary products from the endophytic bacteria were It is more evidence-based to isolate new antibacterial or bactericidal lead compounds [Petrini et al.1992, Sydowia, 44:282-293]. The present invention will report the discovery of an endophytic bacterium—Acinetobacter baumannii LCH0606 strain and its disease prevention effect.

Acinetobacter baumannii is a common endophytic bacterium in plants, but there are few studies on its biological function. It has been reported that the bacterium can degrade herbicides and petroleum pollution, and has a certain environmental protection effect (Roque et al., 2000.Scientia Agricola 57 (4): 723-728; Sarma et al., 2004.Can.J.Microbiol. 50(6):405-414). In recent years, it has been found that this fungus can be used for the prevention and treatment of citrus canker (Tan Xiaoyan et al., 2006, Acta Microbiology 46(2): 292-296). The invention isolates a strain of Acinetobacter baumannii from the stem of camphor tree, which has broad-spectrum antifungal activity. These research results show that Acinetobacter baumannii has rich intraspecific diversity and a wide range of biological activities, and is a biopesticide with great development potential for the control of plant diseases.

3. Contents of the invention

The purpose of the present invention is to search for a new source of biopesticides, and to develop a broad-spectrum, high-efficiency, and industrially-producible biocontrol bacterial strain that can be used for plant fungal disease control by using plant endophytic bacteria resources. Provide the strain of Acinetobacter baumannii LCH0606 and the screening and identification methods of the strain; provide the products of the strain Baumannii liquid and baumannin and its preparation process; A method of use for promoting germination of rice seeds.

Concrete technical scheme of the present invention is as follows:

The bacterium that the present invention adopts is Acinetobacter baumannii LCH0606 bacterial strain (Acinetobacterbaumannii LCH0606) (hereinafter abbreviated as LCH0606 bacterial strain), is preserved in China Microorganism Strain Preservation Management Committee Common Microorganism Center, preservation date is July 6th, 2006, deposits The number is CGMCCNo.1752.

The production strain LCH0606 bacterial strain of the present invention is separated from the camphor tree stem in Zijinshan area, Jiangsu Province, China. Through microscopic observation of colony and morphology, staining reaction, routine physiological and biochemical characteristic experiments and 16s rDNA sequence analysis, the bacterial strain is identified as Bowman's A new strain of Acinetobacter (A.baumannii) (LCH0606) has the following characteristics: ①The colony is regular and round, with smooth edges, protruding in the middle, opaque off-white, and the colony is dry; ②The bacterial body is short rod-shaped or club-shaped, Gram staining is negative, no spores and flagella; ③ Physiological and biochemical reactions: contact enzyme test, arginine dihydrolase test, malonate utilization test, nitrate and nitrite reduction test are all positive; oxidation Enzyme test, gelatin liquefaction test, urease test, and phenylalanine deaminase test were all negative; glucose, lactose, galactose, mannose, xylose, and rhamnose could be utilized, but sucrose, maltose, and mannose could not be utilized Alcohol and fructose; ④The 16s rDNA sequence analysis results were compared with the maximum homology in GenBank, and it was found that the 16s rDNA sequencing results had 100% homology with the 16s rDNA sequence of Acinetobacter.

The biocontrol agent of the present invention is baumannii liquid and baumannin, which have significant control effects on various important plant fungal diseases, such as soybean anthracnose, capsicum phytophthora, tomato gray mold, wheat head blight and rice sheath blight, etc., and have the basic traits of industrial production.

The present invention is prepared according to conventional methods. That is, isolate and screen the antibacterial active strain LCH0606 from camphor trees; prepare the biological fungicide——Bowman's bacteria liquid through liquid fermentation and centrifugation; the Bowman's bacteria liquid is absorbed by macroporous resin, ethanol eluted and concentrated under reduced pressure to prepare high-efficiency Biological fungicide - baumannycin; the inhibitory effect of baumannycin liquid or baumannin on soybean anthracnose, capsicum phytophthora, tomato gray mold, wheat head blight and rice sheath blight Determination by plate experiment; the activity of Bowman's bacteria liquid or baumannycin in preventing and treating rice sheath blight was proved by pot experiment; The determination of the antibacterial components of baumin or baumannin was obtained through repeated chromatography on silica gel column and Sephadex LH-20, and was identified as lipopeptide substances IturinA2 and A3 by modern spectral techniques such as NMR, HSQC, DEPT, and LC/MS. , A6.

Advantages and effects of the present invention: the present invention has a stable source of natural raw materials, the strains are derived from camphor tree stems, and the screening method is simple; the raw materials used in the fermentation process mainly include potatoes and sucrose, and the cost is relatively low; Bowman's bacteria liquid and Bowman's The preparation of the bacteriocin is simple, the baowmanicin is obtained by processing the macroporous resin, the treatment capacity is large, the operation is convenient, and the method is suitable for large-scale industrial production. The product quality is stable, and it has good stability to light, heat, acid and alkali. This product is low-toxic, non-toxic to humans and animals, has no phytotoxicity to crops, is green and environmentally friendly, and has a significant effect in preventing and controlling plant diseases.

4. Description of drawings:

Figure 1: The inhibitory effect of Bowman's bacteria liquid on the growth of plant pathogenic fungi. The test strains were: 1. C. parasitica; 2. G. glycines; 3. P. capsici; 4. F. oxysporum; 5. B. cinerea; 6. F. graminearum; 7. R. solani. In the figure, A represents Baumannii solution (concentration: 0.1ml/ml), B represents baumannin (concentration: 0.1mg/ml), C represents negative control, treated with sterile water, and D represents 75% dacryline wettable powder (concentration: 0.1mg/ml), E represents triadimefon wettable powder containing 15% triazolone (concentration: 1mg/ml), culture temperature: 30°C, observation time: 40h.

Figure 2: The control effect of baumannycin on rice sheath blight. The rice seeds are germinated at 30°C until the root length is 1cm, inoculated with a rhizoctonia mycelium cake with a diameter of 6mm near the base of the root, and sprayed 2ml of different solutions on the inoculation site, kept it moist at 30°C for 24 hours, then removed the cake, and continued to cultivate for 24 hours. Observe the length of brown or black roots of different treatments to evaluate the control effect on rice sheath blight. Among the figure, A is the commercial bactericide dactomin (positive control), the concentration is 1mg/ml, B is the sterilized water (negative control), C is the Baumannii liquid, D is baumannin, the concentration is 1mg/ml.

5. Specific implementation

The present invention is further described in detail below with examples, but the content of the present invention is not limited thereto.

Example 1: Isolation and identification of LCH0606 strain

Fresh camphor tree stems are collected from Zijin Mountain in Nanjing, Jiangsu Province, washed with tap water, dried and cut into small pieces of about 1cm. Sequentially immerse in 75% ethanol and 40% formaldehyde solution to disinfect the surface for 3 minutes each, then wash 3 times with sterile water to remove surface contamination bacteria, then cut the stem longitudinally with sterile scissors, put the cut side down on the potato sucrose On the agar plate, 5 stem segments per plate, 5 plates in total. Cultivate at 30°C, observe the appearance of new colonies every day for a total of 7 days, pick a single colony, and purify by streaking. In addition, put 2 to 3 uncut stems or leaves in the same potato sucrose medium plate as a control to test whether the surface sterilization is thorough. The obtained strains were preserved in a -80°C refrigerator by glycerin preservation method for future use.

Inoculate 10 endophytic bacteria from camphor tree stems into 15mL test tubes containing 4mL potato sucrose medium, and culture them on a shaker at 37°C and 100rpm for 48 hours; after the culture solution was filtered and sterilized, take 1mL and 9mL of dissolved potato sucrose for culture Mix the base, pour it into a petri dish with a diameter of 9 cm, and make a potato sucrose medium plate containing 10% of the fermentation broth. After cooling, put it into a fresh phytopathogenic fungus cake with a diameter of 7 mm, with the bacterial face down, at 30 ° C After culturing in the incubator for 40 hours, measure the diameter of the colony, and use the same volume of medium instead of the fermentation broth as a blank control, calculate the inhibition rate of the fermentation broth to the growth of the test fungus, and repeat three times. Wherein, inhibition rate (%)=[(control group average diameter of colonies-sample group average diameter of colonies)/(control group average diameter of colonies-original bacterial sheet diameter)] * 100%, colony diameter unit: centimeter (cm).

According to the inhibitory activity of the fermentation broth against plant pathogenic fungi Anthracnose, Phytophthora, Botrytis cinerea, Fusarium and Rhizoctonia, a broad-spectrum, high-efficiency antifungal active strain was screened, and the morphology, staining, physiology and biochemistry and 16S rDNA sequence Analysis, identified as Acinetobacter baumannii LCH0606 strain.

Embodiment two: the preparation method of Bowman's bacteria liquid

The seed medium was potato sucrose medium, and a single colony of activated LCH0606 was picked and inoculated into 4 mL potato sucrose medium (15 mL test tube) for culture, and cultured on a shaker at 37° C. and 100 rpm for 12 hours. Inoculate 1% seed solution in 400mL potato sucrose medium shake flask (1L), culture on a shaker at 37°C and 100rpm for 48-60h, and the obtained bacterial solution is centrifuged at 5000-6000rpm to remove bacteria, and then it becomes Baumann's bacterial solution.

Embodiment three: the preparation method of bowmancin

The Bowmansin solution described in Example 2 was adsorbed by macroporous resin X-5, eluted with 80% ethanol, and dried by rotary evaporation under reduced pressure to obtain baumannin. The amount of macroporous resin used was 1% of the Baumannii solution, and the flow rate of sample loading and elution was 4 mL/min. The yield of baowmanicin was 568mg/L.

Embodiment 4: Antibacterial activity experiment in Bauman's bacteria liquid and Bowmansin dish

After 1mL of the Bauman's bacteria liquid described in Example two is mixed with 9mL of the potato sucrose medium that has melted, pour it into a petri dish with a diameter of 9cm to make a culture plate containing 10% of the Bowman's bacteria liquid, and then put Put various fresh phytopathogenic fungal cakes with a diameter of 7mm, with the bacteria face down, measure the diameter of the colony after culturing in a 30°C incubator for 40 hours, and replace the supernatant with the same volume of medium as a blank control, and calculate the supernatant The inhibition rate of various test fungal growth was repeated three times.

Inhibition rate (%)=[(average colony diameter of control group-average colony diameter of sample group)/(average colony diameter of control group-original bacterial sheet diameter)]×100%, colony diameter unit: centimeter (cm).

The same method was used to measure the bacteriostasis of baumannycin described in Example 3, using a concentration of 0.1 mg/mL; 1 mg/mL of triadimefon (15% triazolone wettable powder) was used as a positive control. The results showed that the inhibitory effects of the Baumannii liquid and baumannin on the growth of each tested phytopathogenic fungus were equivalent to those of the positive control triadimefon, with no significant difference (Table 1, Figures 1-2).

Table 1 Inhibition of the growth of phytopathogenic fungi by Bowman's bacteria liquid and baumannin (%) sample C. parasitica P. capsici B. cinerea F. graminearum R. solani Bowman's Bacteria 90.35 66.70 78.63 60.75 76.00 Bowmansin 89.10 55.51 60.04 52.96 70.18 Triadimefon 87.53 42.85 57.11 60.04 88.58

Embodiment five: the analysis of active substance:

6.85 g of baowmanicin described in Example 3 was eluted by silica gel column chromatography and chloroform-methanol gradient, and the obtained components were subjected to an antibacterial experiment in a dish. According to the antibacterial results, the active components were combined and concentrated to dryness under reduced pressure. , to obtain 280mg brown extract. The extract was dissolved in methanol and purified repeatedly with Sephadex LH-20 to obtain 40 mg of white powder active ingredient. According to mass spectrometry and nuclear magnetic resonance detection and analysis, the antibacterial compounds are lipopeptide compounds, which are Iturin A2, A3 and A6 respectively. The mass spectrometer model is VG-ZAB-HS, the nuclear magnetic resonance instrument model is Bruker AM500FT-NMR, and the chromatography silica gel is 200-300 mesh.

Embodiment 6: Experiment of controlling rice sheath blight with Bowman's bacteria liquid and baumannin

(1) In-dish experiment: 50 rice seeds were germinated at 30°C until the root length was 1cm, and a rhizoctonia mycelium cake with a diameter of 6mm was inoculated near the base of the root. Remove the fungus cake, continue to cultivate for 24 hours, observe the length of brown roots or black roots of different treatments, and evaluate the control effect on rice sheath blight. The results showed that the control effect of Baumannii liquid or baumannin (1 mg/ml) on rice sheath blight was equivalent to that of the commercial fungicide Dactin (positive control, concentration 1 mg/ml) (Figure 3).

(2) Pot experiment: get the rice seedlings with a root length of about 2cm after germination, inoculate the soil-borne fungus disease rice sheath blight bacteria at the root, then use the Bowman's bacteria liquid described in embodiment two and the method described in embodiment three respectively Spray the roots of rice seedlings with the baumannin solution (0.1mg/mL) and treat with sterile water as a control. 50 seeds in each group were cultured at 30°C for 72 hours to investigate and record the sheath blight infestation. Classify according to 5 levels Standard (level 0: no disease symptoms; level 1: light gray lesion, length less than 0.5cm; level 2 disease: brown lesion, length 0.5cm-1cm; level 3: black lesion, length more than 1cm, severely damaged roots ), and calculate the average disease index of rice in each group, the average disease index=∑[(number of diseased plants at all levels×relative disease progression)]/total number of plants under investigation. The results show that the baumannii liquid has a good control effect on rice sheath blight, and the baumannin solution with a concentration of 0.1mg/mL also has certain control effects on rice sheath blight (table two). The control effect of baowmancin (0.1mg/mL) was 48%.

Table 2. The control effect of Bowman's bacteria liquid and baumannin on rice sheath blight infection sterile water Bowman's Bacteria Bowmansin mean illness index 2.56 0.46 1.32

Embodiment seven: the influence of Bowman's bacteria liquid on the germination of rice seeds

Select healthy rice seed grains, pre-germinate at 37°C for 24h, then soak the seeds at 30°C for 12h with the Bowmansin solution (0.1mg/mL) described in Example 2 and Example 3 respectively, Seeds soaked in sterile water were used as the blank control, with 50 seeds in each group. The treated seeds were then placed in a petri dish with sterile water-moistened filter paper on the bottom of the dish, incubated at 30°C for 3 days, and the germination rate, main root length, root hair number and stem length of each group of treated seeds were detected. The results showed that the baumannii solution and baumannin had no significant effect on rice seed germination, but had certain promoting effects on root length, root hair number and stem length (Table 3).

Table 3. Effects of Bowman's bacteria liquid and Bowmansin on rice seed germination sterile water Bowman's Bacteria Bowmansin Germination rate(%) 92.59 88.46 92.30 Main root length (cm) 4.12 4.55 5.28 Root hair number (root) 28.0 29.7 37.8 stem length(cm) 1.37 2.14 2.01

Claims (9)

1. microorganism formulation that suppresses the various plants pathomycete, it is characterized in that this biologic product is Bauman bacteria solution and Bao Man rhzomorph, producing bacterial strain was Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain (Acinetobacterbaumannii LCH0606), and this bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 6th, 2006.

2. according to the preparation method of the described Bao Man of claim 1 acinetobacter calcoaceticus LCH0606 bacterial strain, it is characterized in that from the camphor tree stem, being separated to endogenetic bacteria, concrete steps are: plant stem is cut into segment about 1cm, immerse 75% ethanol and 40% each 3min of formalin surface sterilization successively, use sterile water wash then 3 times, to remove the assorted bacterium of surface contamination, with sterile scissors stem is longitudinally cut again, tangent plane is put on the potato sucrose agar plate down, every dull and stereotyped 5 stem tuber sections, totally 5 flat boards, 30 ℃ of cultivations, by day appearance of the new bacterium colony of observation, totally 7 days, picking list bacterium colony, the line purifying, obtained strains obtains the plant pathogenic fungi anthrax-bacilus through antagonistic experiment, phytophthora, botrytis cinerea, sickle-like bacteria and rhizoctonia have the bacterial strain of remarkable inhibiting activity---Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain, and in-80 ℃ of refrigerator preservations.

3. the Bauman bacteria solution that Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain according to claim 1 is produced is characterized in that the mixed culture fermentation liquid of Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain, the aseptic supernatant of gained behind the centrifugal 30min of 5000-6000rmp.

4. the preparation method of the Bauman bacteria solution that Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain according to claim 3 is produced, it is characterized in that Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain is fermented, zymotechnique comprises the preparation and the 1L shake flask fermentation of seed liquor, being prepared as of seed liquor adds 4mL potato sucrose medium in the 15mL test tube, the inoculation back is in 37 ℃, the 100rpm shaking table is cultivated 12h, be inoculated in the potato sucrose medium of 400ml by 1% seed liquor then, 37 ℃, 100rpm shake flask fermentation 48h, zymotic fluid is behind the centrifugal 30min of 5000-6000rmp, and the aseptic supernatant of gained is Bauman bacteria solution.

5. the Bao Man rhzomorph that Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain according to claim 1 is produced is characterized in that the medicinal extract of Bauman bacteria solution gained after macroporous resin adsorption concentrates.

6. the preparation method of the Bao Man rhzomorph that Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain according to claim 5 is produced, it is characterized in that using macroporous resin adsorption to concentrate, the macroreticular resin particle size is 100-400, with the fermentating liquid volume ratio be 1: 100, flow velocity is 4mL/min; Eluting solvent is 80% ethanol, and flow velocity is 4mL/min, and eluent is evaporated to dried with Rotary Evaporators, be the Bao Man rhzomorph.

7. the antifungus active substance that Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain according to claim 1 is produced is characterized in that lipopeptid class material, through being accredited as Iturin A2, A3 and A6.

8. Bauman bacteria solution that Bao Man acinetobacter calcoaceticus LCH0606 bacterial strain according to claim 1 is produced and Bao Man rhzomorph are in the application of control soybean anthracnose, Phytophthora capsici disease, graw mold of tomato, wheat scab and rice sheath blight disease plurality of plant diseases.

9. Bauman bacteria solution that Bao Man acinetobacter calcoaceticus according to claim 1 is produced and Bao Man rhzomorph promote that rice paddy seed is taken root, application in the rudiment.

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