CN102660468A - Pestalotiopsis crassipes - Google Patents
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Abstract
A pestalotiopsis crassipes WH010 bacterial strain (with the storage number of CGMCCNO.4418) is obtained from water hyacinth natural disease plant leaves by means of separation, and is quite easy to produce spores under conventional culture conditions, the optimum culture medium is potato sucrose agar (PDA), the optimum temperature is 28 DEG C, the optimum pH (potential of hydrogen) is 6.5, and the best germ infection pathogenic conditions include that the inoculation concentration is larger than 105/ml, moisture preservation is performed for 24 hours after inoculation, and the temperature is kept in a range from 25 DEG C to 28 DEG C. A bacterium preparation and toxin liquid have quite high pathopoiesis and weed killing effects for water hyacinth which is worst weed, the pestalotiopsis crassipes can be used for producing environment-friendly mycoherbicide by fermentation bacterium or toxin biomimetic synthesis technology, and has a potential business development and application value. Fungal endophyte culture supernatants are extracted by ethyl acetate and evaporated by a rotary evaporator in a vacuum manner to obtain pathogenic crude toxin. A control effect of water aqua and powder of the bacterial strain to the water hyacinth in field natural conditions can be higher than 76%. Suspension and toxin liquid of the bacterial strain are safe for 21 types of important crops such as rice, and do not have adverse effects.
Description
Technical field
The invention belongs to weeds biological control technical field, particularly relate to a kind of water hyacinth fungal biocontrol agent plan dish stey (
Pestalotiopsis crassipes) bacterial strain.
Background technology
Herba Eichhorniae (
Eichhornia crassipes) claim Herba Eichhorniae again, be one of the world's ten big evil grass, also be one of important exotic invasive plant of China.Be distributed widely in a lot of countries and regions in Asia, North America, Oceania and Africa.Herba Eichhorniae imports China into the thirties in 20th century, extensively is distributed in areas such as south China, Central China and East China at present, especially with provinces and cities such as Yunnan, Sichuan, Hunan, Hubei, Jiangxi, Jiangsu, Zhejiang, Fujian and Taiwan.This grass flexibility is extremely strong, the speed of growth is very fast, rate of propagation is surprising, is difficult to effective control behind the excessive multiplication, brings serious water surrounding ecological damage and biological pollution.
Herba Eichhorniae is mainly through artificial mechanism salvaging, chemical weed control and biocontrol control at present.But artificial salvaging can only be in the short period of time, work among a small circle, and wastes time and energy.Machinery stirs and goes out, and limited by the region, and cost is high, be not suitable for promoting, and if the Herba Eichhorniae of salvaging is untimely processing, and mosquito grows, and also can have a strong impact on environment and human and livestock health.Chemical herbicide not only expense is high, and possibly directly cause environmental pollution.Therefore in recent years the biological control research of " appliable plant germ control Herba Eichhorniae " receives showing great attention to of people; Report more than 70 kind of Herba Eichhorniae pathomycete abroad, wherein had 7 kinds of strong pathogenic strainss to have the biological and ecological methods to prevent plant disease, pests, and erosion application potential that is developed to the Herba Eichhorniae mycoherbicide.Sieve get Manny tail spore (
Cercospora rodmanii) prevent and kill off Herba Eichhorniae and obtained patent protection abroad.China reported ten surplus kind of Herba Eichhorniae pathomycete, wherein a kind of false chain lattice spore of Herba Eichhorniae (
Nimbya alternantheraeThe SF193 bacterial strain) obtained China's patent protection (patent No.: 200710019832), also do not have so far about the further research of other several kinds of germs and the play-by-play of application.
Summary of the invention
The purpose of this invention is to provide a kind of careless fungi that kills that Herba Eichhorniae is had very strong pathogenic effects and control effect.According to morphological feature and molecular biology method with its be accredited as intend pestalotia bacteria (
Pestalotiopsis photiniae), confirmed its growth, produced spore and infect morbific optimum condition through test determination, discover that it makes the dead pathogenesis of weeds plant morbidity through producing toxin, has analyzed host's specialization of germ and toxin thereof.
The strong pathogenic strains separation and purification of Herba Eichhorniae: repeatedly gather Herba Eichhorniae natural occurrence plant in the Chongqing region Various Seasonal; Adopt potato sucrose nutrient agar (PDA); Obtain multiple fungi through indoor tissue culture and purifying; Demonstrate,prove the disease method with Ke He Shi and confirm pathogenic bacterium wherein, relatively screen through pathogenic test at last, obtained thisly to kill careless pathogenic fungi (WH010) what Herba Eichhorniae had a very strong pathogenic effects.
Herba Eichhorniae of the present invention is intended pestalotia bacteria strain (WH010) and has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number: CGMCC NO.4418 on December 8th, 2010.
The morphological specificity of bacterial strain (Fig. 1) is identified: can reach more than 64 mm at 25 ℃ of cultivation 5 d bacterium colony (Figure 1A) diameters on the PDA substratum, illumination is produced spore with dark to the germ growth not to be had significantly to influence.The germ bacterium colony is circular, and surperficial aerial hyphae is flourishing, and white is short cotton-shaped, expansion radially; (Fig. 1 C) is colourless for mycelia, separates the about 1.3-2.7 μ of diameter m.The bacterium colony surface grows black oil spherical shape or irregular, tool gloss and moistening conidium heap radially distributes behind the 14-15 d; Prolong with incubation time, conidium is piled a large amount of the generation, and what have is in blocks in the associating of bacterium colony surface.
Conidium (Fig. 1 D) fusiform or the club-like that falls, size is 14.2-28.5 * 4.1-7.8 μ m, straight or slight curvature, 5 cells, separation is true barrier film between each cell, and the two ends cell walls is thin, and is colourless, and middle 3 cells are the olive brown, and color and luster is consistent; The top cell is colourless, and taper shape obviously diminishes than inferior top cell dia, top appendage 2-3 bar, and the colourless turbination of base portion cell, 1 of middle living formula dodds is about 1/4 of appendage length.
Conidiophore is shorter, and is born in the acervulus base portion, and is colourless or light brown, separates irregular branch.
To the diseased tissues visible acervulus cup-shaped of cutting into slices, scattered or collection is given birth to, and just buries to expose after giving birth to, and black or dun bottom are by heavy wall, horn shape, dun cellularity, and it is thin that panel surface is produced spore district cell walls, and look light.
These morphological features according to germ identify, its imperfect stage belong to intend the Pestalotia fungi (
PestalotiopsisSp.), do not find its perfect stage in the experiment.
The bacterial strain molecular engineering is identified: the genomic dna that extracts bacterial strain; Adopt rDNA ITS universal primer and β-tubulin primer: ITS5 (5 '-GGAAGTAAA AGT CGTAACAAGG-3 '), ITS4 (5 '-TCCTCCGCTTATTGATA TGC-3 '), Bt2a (5 '-GGAAGTAAAAGTCGTAACAAGG-3 '), Bt2b (5 '-ACCCTCAGTGTAGTGACC CTTGGC-3 ') to carry out the pcr amplification of rDNA ITS and β-tubulin sequence; Acquisition length is that (gene fragment (seeing sequence table for details) of β-tubulin) is respectively JF502635 and JQ694097 in the accession number of Genbank for 550bp (rDNA ITS) and 453 bp.Institute's calling sequence is logged on the www.ncbi.nlm.nih.gov website, carries out BLAST comparison, can know by comparison result, the sequence of the rDNA ITS of WH010, β-tubulin section with
Pestalotiopsis photiniaeSequence have 99% similarity, thereby confirm
WH010With Chinese photinia plan dish stey (
Pestalotiopsis photiniae) be same kind.
The best of bacterial strain is cultivated and the inoculation infection condition: this fungal colony is grown and produced conidial optimum medium is potato dextrose agar (PDA); 28 ℃ of optimum temperutures, Optimum pH are pH6.5, under these conditions; Colony growth is fast, produces macroconidium in a large number; Illumination is produced spore to fungal growth does not have obvious influence.The morbific top condition of infection process is that inoculum density is 10
5Individual/the mL order of magnitude or higher, preserve moisture after the inoculation and half-light 24 hours, and keep 25-28 ℃ of temperature in the whole phase of infecting.
Pathogenic and the specialization of bacterial strain: pathogenic effects and growth inhibitory effect that Herba Eichhorniae plan dish stey is good to Herba Eichhorniae.Mainly infect plant leaf.Behind the direct spray inoculation of the conidial suspension of indoor germ, 4 d can fall ill, and typical case's morbidity scab appears in plant leaf; The sick plant rate of 12 d reaches 100%, and plant leaf begins the dehydration yellow; The later on whole plant of 18 d is wilted dead, and its control effect to Herba Eichhorniae restrains track with the enemy, Glyphosate 62 IPA Salt is suitable.Inoculation back effect manifests slightly and postpones the 8th whole yellows in d plant top, the death of wilting behind 18 d in the field.
Use high density conidial suspension (10 under optimum conditions
5Individual/mL) inoculation test result shows; This fungi is all not pathogenic at interior 21 kinds of water of 7 sections, Dry crop to important crops such as paddy rice, wheat, corn, pea, rape, capsicum, tomato, cottons; Do not influence their seed germination and plant strain growth yet; This explanation Herba Eichhorniae plan dish stey is the obligate pathogenic bacterium of Herba Eichhorniae, if be used to develop the Herba Eichhorniae weedicide, can guarantee the safety of other plant in the habitat.
The thick toxin of bacterial strain: after cultivating 7 d with potato glucose (hereinafter to be referred as PD) nutrient solution; Four layers of filtered through gauze 1 time; Gained filtrating is filtered with double-deck chromatography filter paper again; Centrifugal 20 min that filtrate then, rotating speed is 4000 r/min, the gained supernatant obtains the thick toxin that causes a disease through ethyl acetate extraction, rotatory evaporator vacuum-evaporation.Promptly show signs of toxicity, 5 d intra vane yellows in 2 d behind the steep water cucurbit blade respectively with the water liquid of this toxin.Do not observe signs of toxicity handle seed and the plant of aforementioned 20 kind of plant with this thick toxin soiutions after, explain that this toxin also is highly specialized.
7.
WH010The preparation of bacterial strain biological prevention and control agent and application: the preservation bacterial classification moved to grow in test tube PDA slant medium carry out activation; After cultivating 5 d on the immigration of the plan dish stey WH010 after the activation PDA plate culture medium; Get the bacterium cake and be inoculated in the PD liquid nutrient medium, the shaking culture after-filtration obtains the mixing liquid of spore and culturing filtrate, can gained filtrating directly be processed into suspension agent; Or with bacterial classification inoculation the sterilization wheat bran on, collect spore behind the solid fermentation and be processed into pulvis.Be sprayed at the Herba Eichhorniae blade face equably after the biological prevention and control agent dilution with gained, every square metre of spraying consumption is bacterium liquid 50 mL after diluting.Herba Eichhorniae began morbidity, 15 d rear section plant withered deaths (Fig. 5) after 3 d were handled in spraying; Measure after 1 month, the growth inhibition ratio of Herba Eichhorniae can reach 76.3%.Its advantage is: the biological prevention and control agent of plan dish stey WH010 bacterial strain preparation all has good preventive effect to Herba Eichhorniae; To people, animal safety, environment is not polluted.
Advantage of the present invention is: the Herba Eichhorniae plan dish stey WH010 bacterium that separation obtains from physical environment (
Pestalotiopsis crassipes), its inoculum to the Herba Eichhorniae of one of the world's ten big malignant weeds (
Eichhornia crassipes) have very strong pathogenic control action kou, its meta-bolites
(Toxin
)This weeds also had very strong toxicity lethal effect; Biological prevention and control agent through strain liquid fermentation and solid fermentation preparation has good preventive effect to Herba Eichhorniae; Microbial inoculum of germ and toxin all do not have detrimentally affect to other plant in important crop and the environment; Show to the height of Herba Eichhorniae and specially change removing activity; Have potential business development and using value, in the biological control practice of Herba Eichhorniae, have important application prospects.
Description of drawings
Fig. 1 is the morphological specificity of Herba Eichhorniae plan dish stey WH010: the last 5d bacterium colony of A. PDA; B. the last 5d bacterium colony of the PDA back side; C. mycelia; D. conidium
Fig. 2 is Herba Eichhorniae plan dish stey WH010 thick toxin indoor bioassay result (5 d): A. plan dish stey WH010 toxin is handled; B. clear water contrast
Fig. 3 is symptom (scab) characteristic of Herba Eichhorniae plan dish stey WH010 bacterium piece inoculation back 5 d
Fig. 4 is 12 d plant incidences behind the WH010 spore suspension live body indoor inoculation: A. clear water contrast B. plan dish stey WH010 handles
Fig. 5 is field morbidity and the control action kou of WH010 preparation to Herba Eichhorniae.
Embodiment
Below in conjunction with embodiment the present invention is further described
Embodiment 1:Kill separation and purification and the screening of careless fungi
1. Herba Eichhorniae pathogenic bacteria separation, purifying
1.1. the separation of bacterial strain: choose the blade or the cane that have typical scab respectively; Clean with flushing with clean water, in the tissue block of strong intersection clip 2 mm of disease * 2 mm size, at Bechtop successively at 70% alcohol; Sterilize in 0.1% the mercuric chloride; Continuously rinsing 3 times in aqua sterilisa is then taken out the back and is inhaled with the thieving paper of sterilization and go to be placed in the PDA flat board after the redundant moisture, in 25 ℃ of constant incubators, cultivates.
Treat that bacterium colony on the PDA plate culture medium is long during to a certain size, carry out separation and purification and after moving into the PDA test tube slant pathogenic mensurations after under 4 ℃ in refrigerator preservation subsequent use.
1.2. host plant source and cultivating: gather the relative healthy plant of not obvious disease symptom, remove be transplanted in the tank in the greenhouse behind the old and feeble blade subsequent use.Chose the healthy water cucurbit plant that grows fine and be of moderate size in preceding 2 days in inoculation test, implant respectively in the polypots, the polypots bottom is affixed by a small amount of nutrition soil and fills tap water.Basin alms bowl plant is placed on growth under the greenhouse natural temperature.
1.3. Herba Eichhorniae disease screening and tieback: the pathogenic bacteria bacterial classification of separation and purification gained is got about 1 * 1 cm mycelia piece, be inoculated in potato sucrose (PS) liquid nutrient medium, in 25 ℃ of constant incubators, cultivate 7 d.Weighing mycelia weight in wet base behind the suction filtration.With mortar mycelia is ground into the mycelia segment, is mixed with mycelia suspension-s in the ratio of mycelia weight in wet base and sterilized water 1:100.The inoculation of employing streak method is applied in mycelia on the Herba Eichhorniae blade with aseptic hairbrush equably, begins till following until mycelia liquid on the blade, and the Herba Eichhorniae of smearing with sterilized water compares.Processing of each blade, co-processing 5 strain Herba Eichhorniaes, every strain 5-6 blade.Inoculation back with plastic bag cover on Herba Eichhorniae with the maintenance high humidity.Throw off plastics bag behind 48 h, temperature is 25-28 ℃ between seed stage, and the inoculation back is observed incidence in good time and write down the disease symptom.
After symptom appears in the blade of waiting to inoculate plant, pathogenic bacteria is separated again, morphological specificitys such as cultivation proterties, conidium and conidiophore of separate front and back pathogenic bacteria etc. are compared with tissue isolation.
The screening of Herba Eichhorniae biocontrol microorganisms
Under 25 ℃ of conditions, each separating obtained bacterial strain is mixed with mycelia suspension-s in the ratio of mycelia weight in wet base and sterilized water 1:100 respectively.Before the inoculation, the Herba Eichhorniae blade is rinsed well with sterilized water.The inoculation of employing streak method is applied in mycelia on the Herba Eichhorniae blade with aseptic hairbrush equably, begins till following until mycelia liquid on the blade.The Herba Eichhorniae of smearing with sterilized water compares.Processing of each blade, co-processing 5 strain Herba Eichhorniaes, every strain 5-6 blade.After the inoculation, on Herba Eichhorniae, keep high humidity with plastic bag cover.Behind 48 h, remove plastics bag.Behind 14 d, susceptible rate of observed and recorded plant and disease severity (grade scale of Charudattan).
The Herba Eichhorniae Taxonomic Status of Pathogenic Fungus is identified
3.1. the morphology of pathogenic bacteria is identified: cultivate pathogenic bacteria on the potato sucrose plate culture medium; The various morphological specificitys of in good time observed and recorded pathogenic bacteria: bacterium colony is directly taken with digital camera; Under opticmicroscope, observe after the mycelia of fungi, conidium and the conidiophore film-making, and take with micro imaging system.The main characteristics such as CF according to conidial shape, color, size, omphalion and barrier film number and conidiophore of the evaluation of pathogenic bacteria are made.
3.2. the Molecular Identification of pathogenic bacteria: chloroform/isoamyl alcohol extracting method is adopted in the extraction of total DNA.Employing rDNA ITS universal primer and β-tubulin primer: ITS5 (5 '-GGAAGTAAA AGT CGTAACAAGG-3 '); ITS4 (5 '-TCCTCCGCTTA TTGATATGC-3 '); Bt2a (5 '-GGAAGTAAAAGTCGTAACAAGG-3 '); Bt2b (5 '-ACCCTCAGT GTAGTGACCCTTGGC-3 ') carries out the pcr amplification of rDNA ITS and β-tubulin sequence; The PCR product that obtains is checked order, the bacterial strain rDNA ITS sequence of acquisition and β-tubulin sequence are carried out the BLAST comparison in the www.ncbi.nlm.nlh.gov DB, and then the classification position of definite pathogenic bacteria again.
Embodiment 2: Herba Eichhorniae plan dish stey biological characteristics
1. substratum is to the influence of bacterial strain colony growth and sporulation quantity
On the bacterium colony of 25 ℃ of constant temperature culture 5 d, using punch tool to beat cut-off along colony edge directly is that the bacterium cake of 5.0 mm is connected to PDA, PSA, CAA, WA, WLA, OSA, WS and WD culture medium flat plate (9 cm) central authorities.25 ℃ of constant temperature culture are observed continuously.Day by day measure the record colony diameter with vertical cross method behind the 5th d; Measure sporulation quantity behind the 18th d; Add sterile purified water, scrape the bacterium colony surface gently, wash-out, filtration then with the sterilization slide; 0.1% tween-80 that adds certain volume is made into suspension-s; Draw spore suspension with liquid-transfering gun and drip on Neubauer (Neubauer) blood counting chamber, the 10x20 power microscope is counting down, calculates spore concentration.More different substratum are to the influence of colony growth and sporulation quantity, and each handles repetition 3 times.
Temperature is to the influence of bacterial strain colony growth and sporulation quantity
On the bacterium colony of 25 ℃ of constant temperature culture 5 d; Use punch tool to buy the bacterium cake that cut-off directly is 5.0 mm along colony edge; Place PDA culture medium flat plate (9 cm) central authorities; Respectively in 10 ℃, 19 ℃, 16 ℃, 22 ℃, 25 ℃, 28 ℃, 31 ℃ and 34 ± 0.5 ℃ of 8 illumination boxs, measure colony diameter and sporulation quantity as stated above, each Temperature Treatment repeats 3 times.
Potential of hydrogen is to the influence of bacterial strain colony growth and sporulation quantity
Behind the PDA medium sterilization; In the aseptic technique platform, the pH value of substratum is adjusted to 4.0,5.0,6.0,6.5,7.0,7.5,8.0,9.0,10.0,11.0 and 12.0 (measuring with the electronics pH meter) respectively, pours 9 cm petridish (about 5 mL/ wares) into and process plate culture medium with the NaOH of 1.0 mol/L and HCl solution.Inoculate pathogenic bacteria as stated above, measure colony diameter and sporulation quantity, 25 ℃ of constant temperature culture, every processing repetition 3 times.
Illumination is to the influence of bacterial strain colony growth and sporulation quantity
Adopt the PDA substratum, illumination every day 0h, 6h, 12h, 18h, 5 kinds of processing of 24h are set, inoculate 25 ℃ of constant temperature culture behind the pathogenic bacteria as stated above, measure colony growth amount and sporulation quantity, every processing repetition 3 times.
Carbon source is to the influence of bacterial strain colony growth and sporulation quantity
To look into those (Czapek) substratum is basic medium.Sucrose in the glucose, sucrose, D-fructose, lactose, SANMALT-S, wood sugar, D-N.F,USP MANNITOL, Zulkovsky starch replacing base substratum of mole carbon atom such as get as carbon source, and establish not sugaring basic medium as contrast (CK).Connecing bacterium as stated above is placed on 25 ℃ of constant temperature culture, measures colony growth amount and sporulation quantity, every processing repetition 3 times.
Nitrogenous source is to the influence of bacterial strain colony growth and sporulation quantity
To look into those (Czapek) substratum is basic medium.SODIUMNITRATE, Carnis Bovis seu Bubali cream, peptone, saltpetre, an ammonium nitrate, peptone, urea, the yeast extract of the mole of nitrogen atom such as getting are nitrogenous source, and the SODIUMNITRATE in the replacing base substratum is as nitrogenous source, and establish and do not add the nitrogenous source basic medium as contrast (CK).Connecing bacterium as stated above is placed on 25 ℃ of constant temperature culture, measures colony growth amount and sporulation quantity, every processing repetition 3 times.
Embodiment 3: the thick toxin of pathogenic bacteria extracts
Herba Eichhorniae plan dish stey produces poison and cultivates
On Bechtop, take out cultured Herba Eichhorniae and intend pestalotia bacteria strain flat board, using diameter is the punch tool of 5 mm, beats and gets the bacterium cake, inserts in potato sucrose (PS) nutrient solution every bottle 4 ferfas cake respectively.Put into the shaking table shaking culture, it is 25 ℃, rotating speed 110 r/min that temperature is set, and 12 h are alternately dark, cultured continuously 7 d.
The extraction of thick toxin
With the fermented liquid of gained, with four layers of filtered through gauze 1 time, gained filtrating is with double-deck chromatography filter paper filtration, centrifugal 20 min that filtrate then, and rotating speed is 10000 r/min, the gained supernatant is the crude extract of toxin.
Extraction test
Choose three kinds of extraction agent sherwood oils, methylene dichloride, ETHYLE ACETATE and respectively thick toxin is carried out extraction experiments.The thick toxin of 100 mL of in separating funnel, packing into earlier reinstalls isopyknic extraction agent, and fully concussion makes and leaves standstill two kinds of liquid mixings to layering, from slowly shifting out organic phase down.Add isopyknic extraction agent to aqueous phase again, so continuous extraction is three times, merges organic phase, obtains extracted organic phase and aqueous phase extracted.Organic phase is rotated evaporation (pressure is 0.1 MPa, 30 ℃ of temperature), evaporates organic solvent, 4 ℃ of refrigerators of extraction product sealing are preserved subsequent use.
The toxin biological activity determination
The excised leaf needle punching: put into petridish to the filter paper of the petridish of sterilization size, one in every ware, and add 1 mL sterile purified water.The clip robust growth does not have the blade of scab, with flushing with clean water clean after, clean one time with 70% alcohol, and then dry with behind the aseptic distillation water rinse 3 times, face of blade upwards expansion is put into petridish, every ware is put 1, the cultivation of preserving moisture is subsequent use.Cause the microtrauma mouth in the about 1 cm place acupuncture of blade proximal edge, inhale liquid 40 μ L to be measured respectively, drip in the wound, preserve moisture with the preservative film sealing, repeat 3 times, do contrast with PD nutrient solution and aqua sterilisa, incubator was preserved 5 days for 25 ℃.Observed and recorded produces spot diameter.
Pickling process: put into petridish to the filter paper of the petridish of sterilization size, one in every ware, and add 1 mL sterile purified water.The clip robust growth does not have the blade of scab, with flushing with clean water clean after, with 70% alcohol scouring one time, and then dry with behind the aseptic distillation water rinse 3 times, blade is immersed in the culturing filtrate, take out the cultivation of preserving moisture behind 2 min.And compare observed and recorded incidence and degree behind 5 d with aqua sterilisa, PD nutrient solution.
Embodiment 4: safety (specialization) the property mensuration of pathogenic bacteria and toxin thereof
WH010 is to the pathogenic mensuration of crop cauline leaf
In petridish, put into filter paper; Add an amount of aqua sterilisa, will use the healthy seed of 21 kinds of crops of warm water soaking 12 h or 24 h (deciding) to be placed on the wet filter paper, cover the ware lid according to seed category; Vernalization in 25 ℃ of thermostat containers is seeded in the plastic tub (diameter 12 cm) after waiting to expose plumule.Add sandy soil in the basin, add a certain amount of aqua sterilisa and composite fertilizer then through hyperthermia drying.After planting under outdoor natural condition, grow, inoculate processing when treating plant length to the 5th leaf.Make species 3 basins for every kind.
1). mycelia is to the mensuration that infects of crop: with hand sprayer mycelia suspension-s is sprayed at and supplies to study that (ratio in mycelia weight in wet base and sterile distilled water 1:100 is mixed with mycelia suspension-s, and spray amount is about 100 mL/m on the thing plant
2), again with transparent plastic bag 24 h that preserve moisture.Compare with aqua sterilisa spraying, connect continued growth under outdoor natural condition behind the bacterium, observed and recorded result behind 18 d.
2). spore is to the mensuration that infects of crop: with manual sprayer spore suspension is sprayed at and supplies to study that (spray amount is about 100 mL/m on the thing plant
2), again with transparent plastic bag 24 h that preserve moisture.Compare with aqua sterilisa spraying, connect continued growth under outdoor natural condition behind the bacterium, observed and recorded result behind 18 d.
3). thick toxin is to the mensuration of crop cauline leaf growth: with hand sprayer spore suspension is sprayed at and supplies to study that (spray amount is about 100 mL/m on the thing plant
2), and compare with aqua sterilisa and nonvaccinated PD nutrient solution, under indoor natural condition, grow, observe behind 18 d and the record result.
Influence to the crop seed germination rate
1). mycelia suspension-s is measured the sprouting of crop seed: mycelia suspension-s is added in the petridish that is covered with filter paper make filter paper saturated; Choosing the confession of soaking 2 h studies species and is placed on the filter paper; Each petridish is put 50 seeds, and every kind of crop is placed 3 wares, and does contrast with sterile purified water; Rate of emergence is observed and calculated to dark culturing under 25 ℃ of conditions.
Son sum * 100 are planted experimentally in percentage of germination (%)=normal chitting piece number/confession
2). thick toxin is measured the sprouting of crop seed: thick toxin is added in the petridish that is covered with filter paper make filter paper saturated; Choosing the confession of soaking 2 h studies species and is placed on the filter paper; Each petridish is put 50 seeds, and every kind of crop is placed 3 wares, and with aqua sterilisa with do not connect bacterium PD nutrient solution and do contrast; Rate of emergence is observed and calculated to dark culturing under 25 ℃ of conditions.
Embodiment 5: the preparation method of plan dish stey WH010 bacterial strain biological prevention and control agent
1). aqua: the preservation bacterial classification moved to grow in test tube PDA slant medium carry out activation; Plan dish stey WH010 after the activation is moved into cultivation 5 d on the PDA plate culture medium; Using diameter then is that the punch tool of 5 mm is beaten and got the bacterium cake, inserts in the PD nutrient solution (2/100 mL).Put into the shaking table shaking culture, 25 ℃ of temperature, rotating speed 110 r/min are set, 12 h are alternately dark, and sterile gauze filters behind cultured continuously 14 d, obtain mixing filtrating (the Bao Zinongdu > of spore and culturing filtrate; 10
6Individual/mL, more than operation is all under gnotobasis).Adopt Rotary Evaporators will obtain mixed solution and evaporate, obtain liquid concentrator (Bao Zinongdu>10
10Individual/mL), i.e. the suspension formulation of WH010.
2). pulvis: get wheat bran and add an amount of water, 120 ℃ of moist heat sterilizations, naturally cooling.The preservation bacterial classification moved to grow in test tube PDA slant medium carry out activation; Plan dish stey WH010 after the activation moved into cultivate 5 d on the PDA plate culture medium, using diameter then is that the punch tool of 5 mm is beaten and got the bacterium cake, and mycelia faces down and is inoculated in the sterilization wheat bran (2/100 g); The plastic film sealing; Place 25 ℃ of static cultivations of thermostat container dark condition (above operation is all under gnotobasis), 15 d, culture is smashed with kibbler after 40 ℃ of air dried, and sieve at last (200 order) obtains spore powder; Be the wettable powder of thalline, its spore content is about 1.0262 * 10
10Individual/g.
Embodiment 6:WH010The application of biological prevention and control agent and advantage thereof
Get WH010 aqua or pulvis, being diluted with water to concentration is 10
5The soup of individual/mL is sprayed at the Herba Eichhorniae blade face at dusk equably in fine or cloudy day, and every square metre of spraying consumption is bacterium liquid 100 mL after the dilution.Herba Eichhorniae began morbidity, 15 d rear section plant withered deaths (Fig. 5) after 3d was handled in spraying; Measure after 1 month, the growth inhibition ratio of Herba Eichhorniae can reach 76.3%.
Belong to biotechnological formulation with the WH010 microbial inoculum, have many tangible advantages.It can be numerous through a large amount of expansions of fermentation, and the microbial inoculum production technique is simpler, and the preparation that is developed into all has good biocontrol effect to Herba Eichhorniae, but to people and animals and hydrocoles safety, can not cause the pesticide residue in the environment.
Sequence table
The rDNA ITS sequence of Herba Eichhorniae plan dish stey WH010
1?agaaggcttg?ggtatctacc?tgatcgaggt?caccacaaaa?aattgggggt?ttagcggctg
61?ggagttatag?cacctaacaa?aagcgagaaa?aaaaattact?acgctcagag?gatactacaa
121?atccgccgtt?gtatttcagg?aactacaact?aataaaagaa?gtagattccc?aacactaagc
181?taggcttaag?ggttgaaatg?acgctcgaac?aggcatgccc?actagaatac?taatgggcgc
241?aatgtgcgtt?caaagattcg?atgattcact?gaattctgca?attcacatta?cttatcgcat
301?ttcgctgcgt?tcttcatcga?tgccagaacc?aagagatccg?ttgttgaaag?ttttgactta
361?ttaaaataag?acgctcagat?tacataaaat?aacaagagtt?taatggtcca?ccggcagcag
421?ctataagaag?acctataact?tctgccgagg?caacaaaagg?taagttcaca?tgggttggga
481?gtttagaaaa?ctctataatg?atccctccgc?tggttcacca?acggagacct?tgttacgatt
541?tttacttcca
β-tubulin sector sequence of Herba Eichhorniae plan dish stey WH010
1?gtgataactg?tctgctcgac?acggccttaa?tacgacgttt?ttcgtgcctg?cacgacggcc
61?ccgaacagtg?atataggtca?agatagaggg?aacatgatgc?taataggtca?ttgataggca
121?aaccatctct?ggcgagcacg?gtctcgacag?caatggagtg?tatgtactat?ttttaattcc
181?tcctgcttcc?tgttaagctt?gtaggctgac?tcgatggcca?tttagctaca?acggtacctc
241?cgagctccag?ctcgagcgta?tgagcgtcta?cttcaacgag?gcttccggca?acaagtacgt
301?tcctcgtgcc?gtcctcgtcg?atctcgagcc?cggtaccatg?gatgccgtcc?gcgccggtcc
361?cttcggccag?ctcttccgcc?ctgacaactt?cgtcttcggt?cagtccggtg?ctggcaacaa
421?ctgggccaag?ggtcactcac?ctggagggta?aaa
Claims (1)
- A water hyacinth fungal biocontrol agent plan dish stey ( Pestalotiopsis crassipes), from Herba Eichhorniae ( Eichhornia crassipes) separation and purification and obtaining on the natural occurrence plant leaf, the toxin of its thalline, spore and extraction all has very strong causing a disease or intoxicating effect and control effect to Herba Eichhorniae, it is characterized in that bacterial strain is WH010, and the preserving number of this bacterial strain is CGMCC NO.4418.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110607346A (en) * | 2019-11-01 | 2019-12-24 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Identification method and application of pathogenic bacteria of weedy rice |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101371666A (en) * | 2007-08-21 | 2009-02-25 | 董晔欣 | Application of Pestalotiopsis guepinii Stey. toxin in herbicide |
| CN101538537A (en) * | 2009-02-13 | 2009-09-23 | 中国科学院微生物研究所 | Pestalotiopsis fici and discovery and application of generated anti-tumor active compounds thereof |
| CN101608165A (en) * | 2009-07-22 | 2009-12-23 | 广西大学 | One strain blazons intends pestalotia bacteria strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101371666A (en) * | 2007-08-21 | 2009-02-25 | 董晔欣 | Application of Pestalotiopsis guepinii Stey. toxin in herbicide |
| CN101538537A (en) * | 2009-02-13 | 2009-09-23 | 中国科学院微生物研究所 | Pestalotiopsis fici and discovery and application of generated anti-tumor active compounds thereof |
| CN101608165A (en) * | 2009-07-22 | 2009-12-23 | 广西大学 | One strain blazons intends pestalotia bacteria strain and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607346A (en) * | 2019-11-01 | 2019-12-24 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Identification method and application of pathogenic bacteria of weedy rice |
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