CN102634461B - Verticillium dahliae 171 (Vd171) for preventing and treating cotton verticillium wilt and application of Vd171 - Google Patents
Verticillium dahliae 171 (Vd171) for preventing and treating cotton verticillium wilt and application of Vd171 Download PDFInfo
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Abstract
The invention relates to plant pathology and particularly relates to verticillium dahliae 171 (Vd171) and an application of the Vd171 in prevention and treatment on cotton verticillium wilt. On the basis of an attenuated strain of the cotton verticillium wilt, the Vd171 is obtained through separation, screening, identification and biological assay in a greenhouse. The Vd171 has a remarkable control function on the cotton verticillium wilt, and the preservation number of the Vd171 is CGMCC (China General Microbiological Culture Collection Center) N0.5903. The Vd171 can be cultured on a synthetic medium and is easy to be obtained and cultured, thus being beneficial to industrialization production as well as promotion and application. The Vd171 has a good control effect on the verticillium, and after the Vd171 is inoculated for four days, and Vd991 is inoculated, so that the cotton verticillium wilt control efficiency can reach 85.0%. The Vd171 has the remarkable control function on the cotton verticillium wilt, and compared with bacillus subtilis usually applied in a market, the control efficiency of the Vd171 is improved by about 50%, and the Vd171 does no harm to people, livestock, insects and crops and is environment-friendly.
Description
Technical field
The present invention relates to plant pathology, be specifically related to prevent and treat cotton verticillium wilt verticillium dahliae less-virulent strain Vd171 and application thereof.
Background technology
Cotton verticillium wilt is a kind of soil-borne vascular bundle disease that is caused by verticillium dahliae (Verticillium dahliae), is distributed widely in each cotton growing area, the world.Since the nineties in 20th century, this disease is caused disaster in each main product cotton region outburst of China, about 3,000,000 hectares of year generation areas, and year about 1,200,000,000 Renminbi of financial loss become one of critical limitation factor of DEVELOPMENT OF COTTON production.Because the crop rotation difficulty, lack disease-resistant variety, lack the agricultural chemicals with better prevention effect, cause that the control to verticillium almost is blank in the present production.
Owing to the reasons such as heterokaryosis of the coevolution of doing mutually with the host, ecotope, germ, verticillium dahliae is easy to variation, Physiological Differentiation usually occurs, produces new bion, comprises the differentiation of virulence, the totally different bion of virulence occurs.National cotton in 1977 is withered, verticillium integrated control research cooperative groups is organized " the verticillium dahliae bion is identified Combined Trials ", with the Cotton in China verticillium wilt pathogen be divided into strong, weak, in three bions, after the nineties, occur in succession again at the report of various places appearance about virulence differentiation and defoliation bacterial strain.The pathogenic bacteria variation is frequent, causes pathogenesis complexity, breeding for disease resistance hysteresis, varietal resistance forfeiture soon, and it is big to become this disease difficulty of prevention and cure, the major cause that outburst is caused disaster.Yet we can attempt utilizing the less-virulent strain of verticillium dahliae to develop " plant vaccine ", and inducing cotton produces immune response to verticillium, to reach the purpose of preventing and treating verticillium.
" plant vaccine " refers to and can produce resistance by inducing plant, thereby opposing pathogenic bacteria invasion, suppress the pathogenic micro-organism growth, make plant exempt from disease or alleviate the biological or abiotic material of a class of disease, mainly contain weak poison, avirulent strain is vaccine, live bacterial vaccines, the protein vaccine, the oligosaccharides vaccine, small molecules and other meta-bolites vaccines, transgenic plant vaccine.Plant vaccine easily by soil microorganisms degrade rapidly, noresidue, the resistance composition that inducing plant produces is the koinomatter of plant metabolism, to the person poultry safety, and plant vaccine wide, the disease-resistant longer duration of plant disease-resistant spectrum of inducing, repeatedly induce pathogenic bacteria is developed immunity to drugs for a long time, have concurrently and promote plant-growth and improve quality function, be the new class biological pesticide environmentally friendly, pollution-free, that development prospect is wide, become the research focus of crop disease control gradually.
" plant vaccine " can imitate virus, bacterium, fungi instruction plant cell, produces resistance of wide spectrum thereby inducing plant produces antibody materials, suppresses the pathogen growth.Be used at present grain, really, vegetables, the control of 600 multiple diseases of 80 various crop such as raise.Using more in the production is small molecules metabolite amino-oligosaccharide, by 3-5 monose form the oligose element.Its main raw material source is crab shell, shrimp shell, decomposes by fermentation, technology such as apple is got, purification and making.Domestic cotton register to be used for this type of preparation of control cotton verticillium wilt be the 0.5% amino-oligosaccharide aqua that Beihai Guofa Ocean Biological Pesticide Co., Ltd. produces, calendar year 2001 and 2002, bear in the national agriculture chemical registration test (task derives from Ministry of Agriculture's agricultural chemicals and identifies institute) in The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute, prevention effect to cotton verticillium wilt is 29.96%～40.64%, and prevention effect is 19.23% in big area test in 2003.Another kind is widely used to be " plant activation protein ", be a kind of novel texture albumen that utilizes biotechnology separation and Extraction from microorganism, can by with the mutual work of plant surface receptor protein, the signal conduction of inducing plant, cause a series of metabolic reactions in the plant materials, induce and activated plant self-defence system and growing system, thereby generation is to the resistance of disease and pest, promote plant-growth, improve crop yield.Domestic by with 3% the plant activation protein wettable powder of listening that green source (Beijing) biotech inc produces, obtain the registration authentication at tobacco, grape, oranges and tangerines, paddy rice, Chinese cabbage, rape, solanaceous crops etc.2008～2009 years, we were through field test, were 34.9%～38.5% to the prevention effect of cotton verticillium wilt.Also do not utilize the verticillium dahliae less-virulent strain to develop plant vaccine at present both at home and abroad, the report of control cotton verticillium wilt.
The present invention is through years of researches, and screening obtains a strain less-virulent strain from a large amount of verticillium dahliae bacterial strains, through greenhouse test in seedling stage the prevention effect of cotton verticillium wilt reached 85.0%, and the control of cotton verticillium wilt is had huge potential.
Summary of the invention
Therefore, the present inventor is for solving existing plant vaccine for the control cotton verticillium wilt, and the formulation of existence is single, and the relatively poor problem of prevention effect proposes and finish the present invention.
The purpose of this invention is to provide a kind of cotton verticillium wilt verticillium dahliae less-virulent strain Vd171 that prevents and treats.
A further object of the present invention provides a kind of cotton verticillium wilt vaccine.
A further object of the present invention provides a kind of method of preventing and treating cotton verticillium wilt.
The present inventor was in 2007～2010, gather 387 cotton verticillium wilt diseased plant samples from 12 84 counties of province (city) such as Xinjiang of China, Henan, Hebei, Shandong, separate and obtain 308 cotton verticillium wilt wild strains, biological assay by screening, evaluation, greenhouse, obtain a strain extremely weak verticillium dahliae bacterial strain Vd171 (Verticillium dahliae) of virulence on cotton, its deposit number is: CGMCC No.5903.This bacterial strain can be used as vaccine, and inducing cotton produces immunization to cotton verticillium wilt, the prevention effect of cotton verticillium wilt is reached 85.0%, and can promote growing of cotton, to people and animals' toxicological harmless effect, environmentally safe.
The verticillium dahliae verticillium dahliae is on common PDA substratum, exist mycelia type, sclerotium type, osculant etc. dissimilar, it cultivates the influence that proterties also is subjected to culture condition easily, and traditional classification based on morphology is identified and sometimes can be restricted.Development of molecular biology is identified to the classification of microorganism new effective measure is provided, and this invention is accredited as the basis with traditional morphology, and the biological means of binding molecule are identified the bacterial strain that screens, and obtain the purpose bacterial strain at last.
The plant vaccine that is used for control of plant disease, should be easy to by plant absorb, characteristics such as environmentally friendly, pollution-free.Cotton verticillium wilt is a kind of tracheomycosis evil, and pathogenic bacteria is infected from the tip of a root of cotton plant, spreads the harm cotton by upwards transportation of vascular bundle, expansion.The vaccine strains that the present invention obtains and cause that the pathogenic bacterium of cotton verticillium wilt belong to verticillium dahliae has identical ecological habit, infects the mode of cotton and the expansion approach in cotton.Therefore, utilize this bacterial strain to control cotton verticillium wilt as vaccine, can infect cotton smoothly and grow in that cotton plants is decided at the higher level but not officially announced.
The vaccine strains Vd171 that the present invention obtains by dipping in root inoculation conidial suspension, is easy to grow at plant Gen Biaoding, successfully infects cotton, and expands in the plant body, induces plant to produce immune response, the harm of opposing verticillium.
Utilize verticillium dahliae less-virulent strain Vd171 to control cotton verticillium wilt as vaccine and aspect following three, produce beneficial effect.
1.Vd171 can cultivate at artificial medium, obtain easily and cultivate, the vaccine strains that is used for disease control must be able to be cultivated at artificial medium, is beneficial to batch production production and applies.VD171 be a strain from the verticillium dahliae less-virulent strain of cotton, well-grown on PDA, Cha Shi and cottonseed substratum is conducive to breed in a large number and apply.
2.Vd171 effective to cotton verticillium wilt control, inoculation Vd171 inoculated verticillium dahliae after 4 days earlier, prevention effect to cotton verticillium wilt reaches 97.2%, verticillium is had significant control action kou, and the subtilis more with producing upward application compared, and prevention effect improves about 50%.
3.Vd171 be environmentally friendly vaccine strains, VD171 separates from cotton, exist naturally in the soil, to the cotton virulence extremely a little less than, to people and animals, insect and crop safety, environmentally safe.
Description of drawings
Fig. 1 is Vd171 growing state on PDA, A: colonial morphology, B: conidium;
Fig. 2 be Vd171 through the amplification of verticillium dahliae special primer, A:P1/P2 primer amplification result, B:VDS1/VDS2 primer amplification result; Swimming lane 1 is Vd991, and swimming lane 2 is Vd171, and swimming lane 3 is Gn111 (Gibellulopsis nigrescens).
The control action kou of the cotton verticillium wilt of Fig. 3 Vd171,1-1, Ji cotton 11 inoculation in after planting 18 days Vd171, inoculation verticillium dahliae strong virus force bacterial strain Vd991 after 4 days; 1-2, the Shandong cotton is ground 21 after planting 18 days inoculation Vd171, inoculation verticillium dahliae strong virus force bacterial strain Vd991 after 4 days; 2-1, Ji cotton 11 inoculation in after planting 22 days verticillium dahliae strong virus force bacterial strain Vd991; 2-2, the Shandong cotton is ground 21 after planting 22 days inoculation verticillium dahliae strong virus force bacterial strain Vd991;
Verticillium dahliae Vd171 (Virticillium dahliae), be stored in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on March 15th, 2012, Institute of Microorganism, Academia Sinica, 100101), its deposit number is: CGMCC No.5903.
Embodiment
The separation of embodiment 1 bacterial strain
(1) sample collecting
2007～2009, gather 387 cotton verticillium wilt diseased plant samples in 12 84 counties of province (city) such as Xinjiang of China, Henan, Hebei, Shandong, separate obtaining 308 cotton verticillium wilt wild strains.
(2) separation of bacterial strain
Earlier the sample (cotton cane) that collects is cleaned with clear water, peeling, placing diameter is the culture dish of the sterilization of 9cm, the alcohol disinfecting 1min with 75%.With 5.0% the clorox 60s that carries out disinfection, with sterilization washing 3 times, each time is no less than 3min again.Be stained with the moisture of doing the surface with the thieving paper of sterilization then, be cut into the long segment of 0.4cm with the scissors of sterilizing, place the culture dish of 9cm, 25 ℃ of cultivations.To the bacterium colony of doubtful verticillium dahliae, in time be transferred in the culture dish that diameter is 6cm and cultivate, observe its bacterium colony size, color, surface characteristic, quality then.
(3) feature of Vd171
1. morphological specificity
On the PDA flat board, the bacterium colony of Vd171 is white, neat in edge, and surface compact has the nebenkern literary composition, does not produce black germ nuclear.Microscopically is observed, and wide 1.94 ± 0.39 μ m that are about of mycelia produce the dendritic conidium of wheel, whenever takes turns 2-3 branch, and conidium is oval, size be 4.33 ± 0.55 * 2.20 ± 0.48 (growing * wide) (Fig. 1).
2. physiological characteristic
Vd171 all can grow under 5-35 ℃ of condition, and optimum growth temperature is 30 ℃.Colony diameter under 5,10,15,20,25,30,35 ℃ of conditions is respectively 0.0,3.0, and 6.8,32.8,47.2,52.5 and 27.5mm.Under 30 ℃, the colony diameter maximum.
3. characterization of molecules
Adopt verticillium dahliae rrna ITS section special primer (P1,5 '-CATCAGTCTCTCTGTTTATACCAACG-3 '/P2,5 '-CGATGCGAGCTGTAAC TACTACGCAA-3 ') and (VDS 15 '-CACATTCAGTTCAGGAGACGGA-3 '/VDS25 '-CCTTCTACTGGAGTATTTCGG-3 ') carry out pcr amplification, the result shows, all obtains special purpose band.
According to Vd171 morphological specificity, physiological characteristic and Molecular Identification result, the Vd171 ownership is Verticillium dahliae.
(4) substratum and culture condition
1.PDA substratum
Potato 200 grams, glucose 20 grams, agar 15-20 gram, 1 liter in water, natural pH.Take by weighing the 200g potato of peeling, being cut into small pieces places pot to add poach mashed (boiling 20-30 minute), after filtered through gauze, adding glucose and agar continuation heating stirs evenly in the filtrate, moisturizing is to 1L again after the cooling slightly, and branch installs to Erlenmeyer flask, the back 120 ℃ of sterilizations of the wrapping of jumping a queue 20 minutes.
2. look into that (Czapek) liquid nutrient medium
KNO
3, 2.00g; K
2HPO
4, 1.00g; KCI, 0.50g; MgSO
4.7H
2O, 0.50g; FeSO
4.7H
2O, 0.01g; Sucrose, 30.00g; Add water to 1L, divide to install to Erlenmeyer flask, the back 120 ℃ of sterilizations of the wrapping of jumping a queue 20 minutes.
3. cottonseed substratum
Made cottonseed suction moisture in 6 hours with being with the cottonseed of short flannel to boil 5 minutes, soaking, pull out and drench the solid carbon dioxide branch, put into Kolle flask, sterilized 30 minutes for 120 ℃.
28 ℃, dark culturing 15d.Liquid culture need be put shaking table 150rpm, and concussion is cultivated.
The control action kou of the cotton verticillium wilt of embodiment 2VD171
1. test method
With disease tolerant variety Shandong cotton grind 21 and susceptible variety Ji cotton 11 be experimental cultivar, adopt the vermiculite sandy soil quantitatively to dip in bacterium liquid method.Vermiculite after the sterilization and sand were mixed by 6: 4, make the no base stock alms bowl of diameter 6cm, place in the vinyl disc of 45cm * 35cm, 8 in every alms bowl sowing cotton seed stays seedling 5 strains after emerging, 15 alms bowls of every kind, and per 5 alms bowls are a repetition, 3 repetitions.Behind the sowing cotton seed, vinyl disc is put in the greenhouse, treated the long a slice true leaf of cotton seedling, according to test inoculating spores suspension 10ml is set, spore concentration is 1 * 10
7Individual spore/ml.Inoculation method is, the 10ml spore suspension poured in the culture dish of diameter 10cm, paper pot taken out from vinyl disc be placed on 40s in the culture dish, treat that spore suspension is blotted after, paper pot is put back in the vinyl disc.Cotton seedling is put in 18 ℃-28 ℃ the greenhouse, carries out the classification investigation of disease by 5 grades of grade scales.Calculate diseased plant rate, disease index and prevention effect.
2. test-results
Test-results in the greenhouse showed in 2009 and 2010, inoculate Vd171 earlier, inoculate the verticillium dahliae bacterial strain Vd991 of strong virulence after 4 days, in the Ji cotton 11 and the Shandong cotton average disease of grinding on 21 refer to be respectively 8.9 and 8.4, extremely significantly be lower than the strong virulence bacterial strain of direct inoculation verticillium dahliae Vd991 (70.9 and 48.0); Prevention effect in 11 last two years of Ji cotton is respectively 90.3% and 84.5%, and the prevention effect of grinding for 21 last two years in the Shandong cotton is respectively 78.9% and 86.3%, and average prevention effect reaches 85.0%.Only inoculate the cotton seedling that Vd171 handles, the average diseased plant rate on Ji cotton 11 and average disease refer to be 9.6 and 2.8, grind in the Shandong cotton and do not show the disease symptom on 21, show this bacterial strain to the virulence of cotton extremely a little less than.The cotton seedling of vaccination bacterial strain Vd171, plant height are also significantly high than direct inoculation Vd991 and clear water contrast.Show that Vd171 not only has good prevention effect to cotton verticillium wilt, and the effect (Fig. 4, table 1-1, table 1-2) that promotes cotton growth to grow is arranged.
The control action kou of the cotton verticillium wilt of table 1-1:Vd171
The control action kou of the cotton verticillium wilt of table 1-2:Vd171
A:4 different processing.1, after planting 18 days inoculation Vd171, inoculation verticillium dahliae strong virus force bacterial strain Vd991 after 4 days; 2, after planting 22 days inoculation verticillium dahliae strong virus force bacterial strain Vd991; 3, after planting 22 days inoculation Vd171; 4, after planting 22 days inoculation clear water.
B, c: cotton variety.
D: the different letters of disease index and plant height back, difference reaches utmost point conspicuous level (LSD, P＜0.01) between the expression different treatment.
Claims (5)
1. verticillium dahliae ((Verticillium dahliae)) less-virulent strain Vd171, its deposit number is: CGMCCNo.5903.
2. the application of the described verticillium dahliae of claim 1 ((Verticillium dahliae)) less-virulent strain Vd171 in the prevention cotton verticillium wilt.
3. the cultural method of the described verticillium dahliae of claim 1 ((Verticillium dahliae)) less-virulent strain Vd171 is characterized in that, at the PDA substratum, looked into those liquid nutrient mediums or 28 ℃ of dark culturing of cottonseed substratum 15 days.
4. a cotton verticillium wilt vaccine is characterized in that, comprises the described verticillium dahliae of claim 1 ((Verticillium dahliae)) less-virulent strain Vd171.
5. a method of preventing cotton verticillium wilt is characterized in that, is included as the step of the cotton inoculation described verticillium dahliae of claim 1 ((Verticillium dahliae)) less-virulent strain Vd171.
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| CN104480085B (en) * | 2014-12-11 | 2017-02-22 | 石河子大学 | VdUDG gene and application thereof in reducing pathogenicity of verticillium dahliae |
| CN108018214A (en) * | 2017-12-06 | 2018-05-11 | 中国农业科学院棉花研究所 | The method for separating verticillium dahliae in soil |
| CN116987161A (en) * | 2023-08-01 | 2023-11-03 | 内蒙古农业大学 | Protein exciton for external secretion of attenuated verticillium melanophore and application thereof |
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