CN104164382A - Biocontrol strain G58 capable of controlling plant fungal disease, and microbial agent preparation method and application thereof - Google Patents
Biocontrol strain G58 capable of controlling plant fungal disease, and microbial agent preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a biocontrol strain G58 capable of controlling plant fungal diseases, and a microbial agent preparation method and an application thereof. The biocontrol strain is named as bacillus amyloliquefaciens G58 and has a preservation number of CGMCCNO.8977. The preparation method of the biocontrol microbial agent comprises the steps of inoculating an activated strain G58 in an NA culture solution; culturing for 22-26 days to obtain a seed solution; inoculating the seed solution in a fermentation culture medium; and culturing for 48-72 h to obtain the biocontrol microbial agent. The biocontrol microbial agent containing the strain G58 presents a significant control effect for rhizoctonia solani; and a control rate can reach 73.6%. The strain G58 has the characteristics of broad spectrum, high planting capacity, stable effect, low preparation cost, simple application method, environment protection and no toxicity, and has very good application prospects in control of the plant fungal diseases.
Description
Technical field
The invention belongs to agricultural biological technical field, be specifically related to prevent and treat biocontrol strain G58 and bacterial preparation process and the application of fungal diseases of plants.
Background technology
According to Food and Argriculture OrganizationFAO (FAO) statistics, the Crop damage causing due to the crop pest such as grain, vegetables problem every year has 10% ~ 15% at least, and wherein 70% ~ 80% Plant diseases is all by fungus-caused.On a kind of crop, can find several even tens kinds of fungal diseases.Many pathogenic fungies form the variation that special tissue or spore can tenable environments, experience severe cold and heat.Fungi is mainly propagated by air-flow, current in field; In addition, wind, rain, insect also can be propagated fungal disease.Therefore, the more difficult control of pathogenic fungi, easily wide-scale distribution.Due to feature and the route of transmission of fungal disease, the general chemical agent that adopts carries out protective control more at present, or is prevented and treated by improving cultivation management measure.
Chemical prevention and control method is simple, and effect is remarkable.But the life-time service of chemical pesticide band a series of serious negative impacts.A large amount of pesticide residue in soil and agricultural-food are there are, agricultural product security Quality Down; The problems such as in soil, a large amount of beneficial microorganisms are killed, and have produced resistance pathogenic micro-organism, and disease control is more difficult.Sustainable development and the agricultural product security problem of agricultural receive much concern at present, and this will the producer will protect and improve environment in utilizing Land Environment resource; When ensureing agricultural output, more to pay close attention to quality and the safety of agricultural-food.The biological control of Plant diseases is proposed in nineteen sixty-five by Britain Plant diseases scholar Garrett.Biological control is taking ecology as basic controlling plant diseases.The problem such as a series of plant protection, environment that can avoid chemical prevention to bring.Reduce the residual of chemical pesticide, improved agricultural product security, promoted agriculture Sustainable development.Genus bacillus is the microorganism being most widely used in current biological control.Genus bacillus can pass through competition mechanism, secretion antimicrobial substance, and the mechanism such as bacteriolysis and induction of resistance produces resistant effect.What in genus bacillus, research was the most thorough is subtilis, and the research of preparing biological prevention and control agent controlling plant diseases with subtilis is the focus of studying both at home and abroad all the time.And bacillus amyloliquefaciens is also reported for work and had the effects such as disease-resistant, growth-promoting in recent years, therefore bacillus amyloliquefaciens also receives increasing concern aspect biological control.
Rice sheath blight disease be by fungi dry thread Pyrenomycetes (
rhizoctonia solaniKuhnone of) the topmost disease of paddy rice that causes.Rice sheath blight disease more and more comes into one's own with its normal massive losses of sending out property and rice yield is caused, and on the south the Changjiang river, many provinces have been classified as the first disease in Rice Production.The general underproduction 10% ~ 30% of this disease, can reach 50% when serious.Consider health and environmental factors, European Union banned use of jingganmycin in 2002.Therefore, combine anti-benefit, the control strategy for rice sheath blight disease that necessary exploration is new in order to improve.Utilizing biocontrol microorganisms is the important channel of rice sheath blight disease controlling, is with a wide range of applications.
Summary of the invention
In order to address the above problem, Plant diseases is multiple, control difficulty, cost is higher, and chemical pesticide is residual serious, and soil microflora is destroyed, the problems such as Crop securify, the invention provides effect stability, colonization ability is strong, has a broad antifungal spectrum, the biocontrol strain G58 of the controlling plant diseases of non-environmental-pollution.
The object of the present invention is to provide a strain novel bacterial: bacillus amyloliquefaciens (
bacillus amyloliquefaciens) G58.
Another object of the present invention is to provide the application of above-mentioned bacterial strains in control of plant disease process.
A further object of the present invention is to provide one to contain
bacillus amyloliquefaciensthe plant disease controlling agent of G58.
A further object of the present invention is to provide biocontrol strain
bacillus amyloliquefaciensthe preparation method of G58 microbial inoculum.
The technical solution used in the present invention is:
A kind of fungal diseases of plants biocontrol strain G58, through being accredited as bacillus amyloliquefaciens
(Bacillus amyloliquefaciens)be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 28th, 2014, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC NO.8977, and the Classification And Nomenclature of suggestion is
bacillus amyloliquefaciensg58, survives in the bacterial strain of qualification preservation on March 28th, 2014.
Above-mentioned biocontrol strain
bacillus amyloliquefaciensthe application of G58 in control of plant disease process.
Further, above-mentioned Plant diseases is fungal diseases of plants.
A kind of plant disease controlling agent, it contains above-mentioned fungal diseases of plants biocontrol strain
bacillus amyloliquefaciensg58.
Biocontrol strain
bacillus amyloliquefaciensthe preparation method of G58 microbial inoculum, comprise the following steps: biocontrol strain G58 is inoculated in to NA flat board and under 29～31 DEG C of conditions, activates, the biocontrol strain G58 after activation is inoculated in to NA nutrient solution, 29～31 DEG C, under 105～115 rpm conditions, cultivate 22～26h, obtain seed liquor; Seed liquor is inoculated in to fermention medium by 1% ~ 3% inoculum size, 30～37 DEG C, under 150 ~ 180rpm condition, cultivate 48～72 h, obtain fermented liquid, i.e. biocontrol strain
bacillus amyloliquefaciensg58 microbial inoculum.
Further, above-mentioned fermentative medium formula contains 0.8 ~ 1.2g/L sucrose, 8 ~ 12 g/L yeast powders, 0.6 ~ 1 g/L KCl, and pH is 5 ~ 6.
The invention has the beneficial effects as follows:
1) Bacillus amyloliquefaciens strain G58 microbial inoculum preparation process of the present invention is simple, and cost is lower, toxicological harmless, non-environmental-pollution, the alternative or application of minimizing to chemical pesticide, reduces pesticide residue, colonization ability is strong, improves soil microorganisms environment, improves agricultural product security.
2) biocontrol strain G58 of the present invention shows good competition inhibition in the dull and stereotyped antagonistic effect of testing laboratory, greatly the inhibition of degree plant epiphyte cause of disease bacteria growing.Competition suppresses can avoid bringing because of the decomposition of antibacterial substance the problem of fungistatic effect decline or disappearance.Meanwhile, this bacterium also can produce the growth that antibacterial substance suppresses plant epiphyte cause of disease bacterium.
3) biocontrol strain G58 of the present invention shows in greenhouse test result: effectively suppressed the growth of fungal pathogens containing the biocontrol fungicide of biocontrol strain G58 of the present invention, shown significant prevention effect for rice sheath blight disease, preventive effect can reach 73.6%.
Brief description of the drawings
Fig. 1 be biocontrol strain G58 suppress rice sheath blight disease (
rhizoctonia solani) dull and stereotyped antagonistic effect.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited to this.
embodiment 1
one, the separation of biocontrol strain G58, screening and identification
A kind of fungal diseases of plants biocontrol strain G58, called after
bacillus amyloliquefaciensg58, is separated and is obtained by near water paddy soil Zengcheng City of Guangdong Province.It specifically separates, screening, authentication method are as follows:
(1) separation of biocontrol strain G58
Take the pedotheque that 10.0 g mix, add 90 ml sterilized waters, in 30 ° of C, under 180 rpm, vibration 30 min, make 10
-1bacteria suspension, it is 10 that redilution becomes gradient
-2～10
-6bacteria suspension, the bacteria suspension 100 μ l that draw suitable gradient are uniformly coated on nutrient agar medium (10
-5, 10
-6) on flat board, each extent of dilution in triplicate, is cultivated 1～2 d in 30 ° of C constant incubators.According to bacterium shape, size, the difference of the physiological characters such as color and luster, picking list bacterium colony is coated with flat board again until obtain the bacterial strain of single form, finally, by microscopy, further observes its form, is really purifying for single form, and after purifying, 4 ° of C refrigerations are for subsequent use.
(2) screening of biocontrol strain G58
Adopt dull and stereotyped antagonistic effect method to carry out the screening of biocontrol strain.
Drawing concentration is 1 × 10
5the plant epiphyte cause of disease bacterium bacterium liquid 100 μ l of CFU/ml put into the coating of PDA flat board evenly, utilize aseptic punch tool on flat board, to make a call to 3 holes, in every hole, be infused in respectively the nutrient solution 50 μ l that 30 ° of C cultivate the different isolated strains of 24h, each processing in triplicate.In 30 ° of C constant incubators, cultivate after 3 ~ 5 d, observe and have or not inhibition zone, calculate bacteriostatic activity.
Bacteriostatic activity represents with antibacterial circle diameter: antibacterial circle diameter=transparent circle diameter-aperture (8 mm).
The bacterial strain with bacteriostatic activity is transferred and cultivated 10 times (every 7 d once) continuously on NA flat board, measure the antagonistic effect of each succeeding transfer culture bacterial strain.
The stability of bacteriostatic activity according to the bacteriostatic activity to rice sheath blight disease, citrus anthracnose, pepper anthracnose, anthracnose of maize, leaf muld of tomato, wheat scab, wax gourd blight, sugarcane Black Rotten, cotton verticillium wilt, sclerotinia rot of colza etc. and after going down to posterity, filter out the wide spectrum plant fungal disease resistance bacterial strain of a strain effect stability, by after bacterial strain purifying, be stored in-70 DEG C of Ultralow Temperature Freezers with 50% glycerine.
(3) qualification of biocontrol strain G58
Through form, dyeing, biochemical reactions and molecular biology identification be
bacillus amyloliquefaciens, called after G58.
A. prevent and treat the morphological specificity of the bacillus amyloliquefaciens G58 of fungal diseases of plants
Observe the single colony morphology characteristic forming known after NA plate loop method 24h, GB58 colonial morphology is less, edge is irregular, surface nothing projection, dry, opaque, is creamy white.When Static submerge-culture, there is mycoderm to form.By gramstaining, gemma and flagella staining, to examine under a microscope, cell is rod-short, gram-positive microorganism, central spore, peritrichous.
B. the bacillus amyloliquefaciens G58 physiological and biochemical property (in table 1) of control fungal diseases of plants of the present invention
Aerobism, glucose fermentation type, catalase, Starch Hydrolysis, nitrate reduction, casein hydrolysis, gelatin hydrolysis, glycitols fermentation, M.R. and V-P test-results are positive; Citrate trianion hydrolysis, cellulose decomposition and tyrosine hydrolysis test result are negative.
The physiological and biochemical property of table 1 biocontrol strain G58
Note: "+" positive, "-" feminine gender.
C. the molecular biology identification of biocontrol strain G58
Adopt Fast DNA kit to extract bacterial genomes DNA, utilize the universal primer of 16srRNA to check order.The primer sequence is: 8F (5'-AGAGTTTGA TCATGG CTCAG-3') and 907R (5'-CCGTCAATTCCTTTGAGTTT-3') amplify 16S rDNA gene fragment from genomic dna.PCR product entrusts Cornell university order-checking center to measure, and sequencing result is submitted to GenBank database and obtains accession number KJ801304, utilizes Blast program search homologous sequence, the NC009725 that result and accession number are
bacillus amyloliquefaciensfZB42 homology is 99%, and therefore, in conjunction with colonial morphology and physiological and biochemical property, biocontrol strain G58 is accredited as
bacillus amyloliquefaciens.
two, the preparation of biocontrol strain G58 microbial inoculum
To be stored in the described biocontrol strain G58 of NA slant medium, be inoculated in the dull and stereotyped activation of NA, the biocontrol strain G58 after activation is inoculated in to NA nutrient solution, 30 DEG C, under 110 rpm conditions, cultivate 24 h, obtain seed liquor.Seed liquor is inoculated in to fermention medium by 1% inoculum size, 30 DEG C, under 150rpm condition, cultivate 48h, obtain fermented liquid (biocontrol microorganisms liquid), i.e. biocontrol strain G58 microbial inoculum, wherein biocontrol strain G58 viable count is 10
8~ 10
9cFU/ml.
Fermentative medium formula is: sucrose 1g/L, yeast powder 10 g/L, KCl concentration 0.8 g/L, initial pH5.
three, the restraining effect of biocontrol strain G58 to phytopathogen
(1) dull and stereotyped antagonistic effect
Drawing concentration is 1 × 10
5pathogenic bacteria (concrete pathogenic bacteria is as shown in table 2) the bacterium liquid 100 μ l of CFU/ml put into the coating of PDA flat board evenly, utilize aseptic punch tool on flat board, to make a call to 3 holes, inject respectively the biocontrol strain G58 microbial inoculum 50 μ l of above-mentioned " two " preparation in each hole.In 30 DEG C of constant incubators, cultivate after 3 ~ 5 d, observe and have or not inhibition zone, measure inhibition zone activity, antibacterial circle diameter for bacteriostatic activity (mm) represents: antibacterial circle diameter=transparent circle diameter-aperture (8 mm).
The antibacterial circle diameter (mm) of table 2 biocontrol strain G58 to plant epiphyte cause of disease bacterium
The measurement result of inhibition zone activity is as shown in table 2, therefrom can find out that biocontrol strain G58 microbial inoculum of the present invention is to 14 kinds of common plant epiphyte cause of disease bacterium rice sheath blight disease pathogenic bacterias, anthracnose of orange pathogenic bacteria, leaf muld of tomato pathogenic bacteria, pepper anthracnose pathogenic bacteria, anthracnose of maize pathogenic bacteria, wheat scab pathogenic bacteria, wax gourd Pathogen of Fusarium Wilt, Pathogen of Bitte Gourd Wilt In Guangdong, sugarcane Pathogen of Black Rot bacterium, banana anthracnose pathogenic bacteria, Pathogen of Rice Blast Fungus, rice green smut pathogenic bacteria, sclerotinia rot of colza pathogenic bacteria and cotton wilt pathogenic bacteria all have obvious fungistatic effect.Illustrate that biocontrol strain G58 of the present invention all shows good inhibition to various plants fungal pathogens, has wider antimicrobial spectrum.
Wherein biocontrol strain G58 microbial inoculum to the antibacterial figure of rice sheath blight disease pathogenic bacteria as shown in Figure 1, therefrom can find out that biocontrol strain G58 microbial inoculum has produced obvious restraining effect to this phytopathogen.
In sum, biocontrol strain G58 of the present invention has restraining effect very to frequently seen plants fungal pathogens.
(2) prevention effect of biocontrol strain G58 microbial inoculum to rice sheath blight disease measured in greenhouse test
Pot experiment: rice varieties is No. 2, Yuejingsimiao, after presprouting of seeds, adopts seedling-cultivation plate 20d-25d, then transplants.Illumination condition 14h/10h, humidity 90% ~ 95%, 32 DEG C of temperature.Rhizoctonia solani Kuhn is cultivated after 5d on PDA flat board, is cut into the square fritter of being permitted about 5 mm, rice tillering contain the phase or latter stage Inoculated Rice banded sclerotial blight.Utilize masking foil that the rice sheath blight disease agar block of 5 mm is fixed on rice leaf, every 1 of blade inoculation (having the upper layer of mycelia to be close to the surface of blade by long in agar block), 3 ~ 4 upper blade of each tillering node inoculation.Mycelia piece is embedded to rice strain stem stalk the 3rd leaf sheath inner side reciprocal, and keep leaf sheath phimosis state constant.After there is typical cytopathic spot on cane, open masking foil.After Inoculated Rice banded sclerotial blight, 1d, 7d, 15d, 21d spray containing the microbial inoculum of biocontrol strain G58 respectively, i.e. the biocontrol strain G58 microbial inoculum of preparation in above-mentioned " two ", and biocontrol strain G58 viable count is 1 × 10
8cFU/ml.
Pot experiment arranges 2 processing altogether, each processing 5 basins, and every basin is containing 2 strain paddy rice.
The conventional group for the treatment of group 1(): general planting, do not inoculate pathogenic bacteria and biocontrol microorganisms;
Treatment group 2: Inoculated Rice banded sclerotial blight cause of disease+spray G58 microbial inoculum (15ml/ basin);
Treatment group 3: Inoculated Rice banded sclerotial blight pathogenic bacteria+spray jinggangmeisu 60A (40 μ g/ml, 15ml/ basin);
Treatment group 4(control group): Inoculated Rice banded sclerotial blight cause of disease+spray sterilized water;
After 40 d, measure the preventive effect of biocontrol strain G58 microbial inoculum to rice sheath blight disease, measuring result is as shown in table 3.
The prevent and treat situation of table 3 G58 microbial inoculum to rice sheath blight disease
Biocontrol strain G58 microbial inoculum reaches 73.6% to the preventive effect of rice sheath blight disease as can be seen from Table 3, is significantly higher than the preventive effect (49.6%) of traditional jinggangmeisu 60A.Illustrate, biocontrol strain G58 of the present invention has good fungal disease preventive and therapeutic effect to plant.
(3) the field planting situation of biocontrol microorganisms G58 microbial inoculum is measured in greenhouse test
By the rice transplanting for the treatment of group 2 in above-mentioned (2), after one month, from every basin, the random each 1g of clip blade (2 left and right), is cut into 1cm long, weighs, and puts into the triangular flask containing 100 mL PBS.After 25 DEG C, 150rpm shaking culture 2h, use PBS dilute sample.Drawing 100ul puts into and contains 100ug/ml Rifampin flat board (G58 has rifampicin resistance because of biocontrol strain through the sample of suitably dilution, therefore utilize containing the flat board of Rifampin and carry out enumeration), each diluent is established 3 repetitions, at 30 DEG C, cultivate after 24 h, counting to get G58 colony number in conjunction with colonial morphology is 9.7 × 10
6cFU/g, illustrates that bacterial strain G58 has good colonization ability.
Claims (6)
1. prevent and treat the biocontrol strain of fungal diseases of plants for one kind
bacillus amyloliquefaciensg58, is deposited in Chinese common micro-organisms culture presevation administrative center, and its preserving number is
cGMCC NO.8977.
2. biocontrol strain claimed in claim 1
bacillus amyloliquefaciensthe application of G58 in control of plant disease process.
3. application according to claim 2, is characterized in that: described Plant diseases is fungal diseases of plants.
4. a plant disease controlling agent, is characterized in that: in described control agent, contain fungal diseases of plants biocontrol strain claimed in claim 1
bacillus amyloliquefaciensg58.
5. biocontrol strain claimed in claim 1
bacillus amyloliquefaciensthe preparation method of G58 microbial inoculum, it is characterized in that: comprise the following steps: biocontrol strain G58 claimed in claim 1 is inoculated in to NA flat board and under 29～31 DEG C of conditions, activates, biocontrol strain G58 after activation is inoculated in to NA nutrient solution, 29～31 DEG C, under 105～115 rpm conditions, cultivate 22～26h, obtain seed liquor; Seed liquor is inoculated in to fermention medium by 1 ~ 3% inoculum size, 30～37 DEG C, under 150 ~ 180rpm condition, cultivate 48～72 h, obtain fermented liquid, i.e. biocontrol strain
bacillus amyloliquefaciensg58 microbial inoculum.
6. biocontrol strain according to claim 5
bacillus amyloliquefaciensthe preparation method of G58 microbial inoculum, is characterized in that: described fermentative medium formula contains 0.8 ~ 1.2g/L sucrose, 8 ~ 12 g/L yeast powders, 0.6 ~ 1 g/L KCl, and pH is 5 ~ 6.
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Inventor after: Gu Wenjie Inventor after: Lu Yusheng Inventor after: Yang Shaohai Inventor after: Jie Kaizhi Inventor after: Li Wenying Inventor after: Jiang Ruiping Inventor after: Sun Lili Inventor before: Gu Wenjie |
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Free format text: CORRECT: INVENTOR; FROM: GU WENJIE TO: GU WENJIE LU YUSHENG YANG SHAOHAI JIE KAIZHI LI WENYING JIANG RUIPING SUN LILI |
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