CN103173385B - Streptomyces and applications thereof to prevention and control of clubroots of crucifers - Google Patents
Streptomyces and applications thereof to prevention and control of clubroots of crucifers Download PDFInfo
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Abstract
The invention discloses streptomyces and applications thereof to prevention and control of clubroots of crucifers. The streptomyces comprise streptomyces scabiei WS-24926 CCTCC NO: M 2012393 and/or streptomyces cuspidosporus WS-29246 CCTCC NO: M 2012413. The streptomyces can be fermented to produce a fermentation broth with a strong bacteriostasis, and the fermentation broth can be used for preparing a biocontrol suspension agent and a biocontrol slow-release capsule for resisting the clubroots of crucifers. The streptomyces are used for biologically preventing and controlling the clubroots of crucifers.
Description
Technical field
The present invention relates to biological pesticide technical field, specifically refer to a kind of streptomycete and the application in control Cruciferae club root thereof.
Background technology
Cruciferae club root (plasmodiophoa brassicae Woron) is a kind of worldwide soil-borne disease, and pathogenic bacterium are plasmodiophora brassica bacteria, belongs to the low mastigomycetes (also having scholar to be classified to Acarasiales) that waits of a kind of obligatory parasitism, there is no at present method artificial culture.Main harm brassicaceous vegetable is as Chinese cabbage, wild cabbage, radish etc.Its germ survives the winter the summer in soil or the barnyard manure that do not become thoroughly decomposed with sporocyst, strong to poor environment resistibility, generally can survive more than 10 years.Carry disease germs from soil and the barnyard manure that do not become thoroughly decomposed in earliest infection source, field.Germ can be carried and propagate into without lesion with dish seedling, earth again.Germ is invaded from young root or the root hair of dish seedling, and stimulates root cell to accelerate division, causes the enlargement of root deformity, causes plant transfusion tissue to be obstructed, overground part poor growth, and blade is wilted, severe one butt rot, plant death.In recent years, club root in Hubei, the ground high mountain brassicaceous vegetable growing area such as Yunnan, Sichuan, Guizhou, Zhejiang, Fujian occurs serious, become one of principal element of restriction China high mountain Chinese cabbage, wild cabbage, the production of radish anti-season, wherein especially be injured the heaviest with Chinese cabbage, wild cabbage, serious plot even has no harvest, and every mu of loss is more than 3000 yuan.
At present, control Cruciferae club root common method mainly comprises liming and regulates soil pH, crop rotation, plantation disease-resistant variety etc., but owing to having soil compaction, deficiency in economic performance, the shortcoming such as resistance to accumulating not, all spreads not of above-mentioned measure.Conventional sterilization agent has derosal, thiram, zinc manganese ethylenebisdithiocarbamate etc., but preventive effect is all not obvious, preventive effect performance is preferably 50% fluazinam SC and the 10% cyazofamid SC combination of Japanese Ishihara Sangyo Kaisha Ltd., but owing to suppressing the reasons such as seed germination and use cost high (every mu nearly 400 yuan), also not yet obtain large scale application.About Cruciferae club root biological pesticide fungi, actinomycetes, all there is certain research report the aspects such as genus bacillus, and obtain certain achievement, as Japanology person by Chinese cabbage sowing from wheatland, Rapeseed Field, in the soil that adopt back on Chinese cabbage ground and meadow, from Chinese cabbage root, separate and obtain a large amount of endogenetic fungus, screen and obtain 2 strain endogenetic fungus (Heteroconium chaetospira) H4007 by pot experiment, M4006, disease refers to be respectively 23, 15, be significantly higher than other and belong to kind of processing and a blank, show growth-promoting functions by seed treatment, there is investigator to utilize (Phoma glomerata) JCM9972 effectively to control the generation of club root, also the person that has foreign study obtains suppressing 3 trichoderma strains (TC32, TC45 and TC63) and 1 streptomycete bacterial strain (S99) of pathogen plasmodiophora by greenhouse and field test, Korea S researchist separates and obtains raw actinomycetes in 81 strains from Chinese cabbage root, by Chinese cabbage club root pot experiment, obtain the interior raw actinomycetes that 3 strains have potentiality to be exploited, wherein 2 strains (Microbispora rosea) and (subsp.rosea) preventive effect be respectively 58% and 33%, 1 strain (Streptomyces olivochromogenes) preventive effect reach 42%, the researchist of Sichuan agricultural university of China isolates 1 strain ash red streptomyces (Streptomyces griseoruber) from the Rhizosphere Soil of Sichuan, and it reaches 73.69% to Chinese cabbage club root potted plant experiment preventive effect, the researchists such as He Yueqiu are separated to 1 bacillus subtilis (Bacillus subtilis) XF-1 from Chinese cabbage rhizosphere soil, and its control efficacy to club root reaches 85%, and field efficacy reaches 68.6～84.8%, by subtilis (Bacillus subtilis), for club root integrated control, preventive effect reaches 85% with fixed nitrogen series bacillus (Paenibacillus azotofixans) in Changjiang University.
Streptomycete has antibiosis, also has advantages of can field surely to grow etc. to be developed as biological pesticide.Environmental Protection Agency EPA has announced the biological pesticide research project of 1 shot hole streptomycete (Streptomyces acidiscabies) RL-110 for weeding for 2012.The registered streptomycete agricultural chemicals on the regular payroll in the whole world has 6 kinds, registration countries and regions are respectively Korea S, European Union, Ukraine, Canada, the U.S., be specially (Streptomyces colombiensis) (Korea S), (Streptomyces kasugaensis) (Korea S), (Streptomyces griseoviridis) K61(European Union, Canada, the U.S.), (Streptomyces albus) (Ukraine), (Streptomyces avermitilis) (Ukraine), (Streptomyces lydicus) WYEC108(Canada, the U.S.), controlling object is mainly fungal disease and comprises leaf diseases and soil-borne disease (wherein S.avermitilis is used for preventing and treating beetle and tetranychid).But there is no so far, the streptomycete agriculture chemical registration of control Cruciferae club root.
Summary of the invention
One of object of the present invention is that a kind of streptomycete with control Cruciferae club root purposes will be provided, and this streptomycete comprises shot hole streptomycete (Streptomyces scabiei) WS-24926CCTCC NO:M2012393 and/or sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246CCTCC NO:M2012413.Shot hole streptomycete provided by the present invention (Streptomyces scabiei) WS-24926 has been preserved in Chinese Typical Representative culture collection center (being called for short CCTCC) on October 10th, 2012; Preservation registration number is CCTCC M2012393; Preservation place is: China, Wuhan, Wuhan University.Point spore streptomycete (Streptomyces cuspidosporus) WS-29246 has been preserved in Chinese Typical Representative culture collection center (being called for short CCTCC) on October 18th, 2012; Preservation registration number is CCTCC M2012413, and preservation place is: China, Wuhan, Wuhan University.
Described shot hole streptomycete (Streptomyces scabiei) WS-24926 separates and obtains from the plant rhizosphere soil of liuyang hunan outer suburbs.
The result of morphological feature, physio-biochemical characteristics and the 16S rDNA sequential analysis of comprehensive shot hole streptomycete (Streptomyces scabiei) WS-24926, is accredited as shot hole streptomycete.Concrete qualification result is as follows:
(1) morphological specificity of thalline
Shot hole streptomycete (Streptomyces scabiei) WS-24926 bacterial strain is on ISP-2 solid medium, and bacterium colony is dry, rough, has projection, light gray; Spore is many, chain, bat shape, size (long 1.5 μ m-2 μ m, wide 0.6 μ m-0.8 μ m), grey, gas silk oyster white or grey, have spiral fibrillae of spores to produce; Pigment is not obvious; On Cha Shi solid medium, non-pigment produces, gas silk and sorus white, and base silk is colourless.Substratum silk yellow on asparagine solid medium, gas silk and sorus canescence, non-pigment produces.
(2) physio-biochemical characteristics
Do not produce melanochrome, can utilize dextrin, polysorbate40, L-arabinose, D-wood sugar, monomethyl succinate, pyruvic acid, glycerol, D-ribose, D-L-alpha-phosphate glycerine.Can not utilize: tween 80, alpha-cylodextrin, glycogen, mannosans, D-R, D-Fructose, D-cellobiose, D-semi-lactosi, D-galacturonic acid, PEARLITOL 25C, alpha-D-glucose, maltonic acid, maltose, lactulose, D-MANNOSE, ALANINE, D-alanine, succsinic acid, L-alanyl-glycine, ALANINE amine, Pidolidone, adenosine, inosine, thymidine, L-rhamnosyl, uridine, sucrose, saligenin, 6-phosphoric acid-D-Fructose, 6-phosphoric acid-D-Glucose.
(3) 16SrDNA sequential analysis
Shot hole streptomycete (Streptomyces scabiei) WS-24926 bacterial strain is through round pcr, DNA sequencer analysis, and the 16SrDNA sequence of this bacterial strain is by 1391 based compositions.In 16SrDNA sequence with blast program to shot hole streptomycete (Streptomyces scabiei) WS-24926 and GenBank, listed 16SrDNA sequence is carried out nucleotide homology comparison, result and shot hole streptomycete (S.scabiei) ATCC49173(T having reported) the 16SrDNA sequence homology of (accession number AB026199) reaches 100%, identifies that it is shot hole streptomycete.
Described sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 separates and obtains from the plant rhizosphere soil of liuyang hunan outer suburbs.
The result of morphological feature, physio-biochemical characteristics and the 16SrDNA sequential analysis of comprehensive sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246, is accredited as sharp spore streptomycete.Concrete qualification result is as follows:
(1) morphological specificity of thalline
Point spore streptomycete (Streptomyces cuspidosporus) WS-29246 is on ISP-2 solid medium, and bacterium colony is dry, rough, has projection, has fold, and oyster white or micro-yellow, have spirrillum fibrillae of spores, and 2-5 encloses not etc.; Base silk tawny, without separating, does not rupture, and is undaform, multi-branched, diameter 0.4 μ m-0.6 μ m.On Cha Shi solid medium, non-pigment produces, and bacterium colony smooth surface, does not produce spore.On PDA substratum, produce lemon yellow pigment.
(2) physio-biochemical characteristics
Do not produce melanochrome, utilize D-Glucose, L-arabinose, D-wood sugar, D-Fructose, sucrose, rhamnosyl, raffinose, inositol, PEARLITOL 25C and D-semi-lactosi, lactose, seminose, melibiose, trehalose, D-Maltose, Trisodium Citrate, sodium succinate.
(3) 16SrDNA sequential analysis
Point spore streptomycete (Streptomyces cuspidosporus) WS-29246 is through round pcr, DNA sequencer is analyzed, obtain the 16S rDNA sequence of this bacterial strain, then with blast program, in the 16SrDNA sequence to sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 and GenBank, listed 16S rDNA sequence is carried out nucleotide homology comparison, result reaches 100% with the 16S rDNA sequence homology of the sharp spore streptomycete (Streptomyces cuspidosporus) of reporting, identify that it is sharp spore streptomycete (Streptomyces cuspidosporus).
Described shot hole streptomycete (Streptomyces scabiei) WS-24926 and described sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 adopt liquid fermentation and culture, and the substratum of its use is as follows:
(1) slant medium: culture medium prescription is as follows: take malt extract: 1.0～2.0g/L, Zulkovsky starch: 0～5g/L, yeast extract: 0.4～1.0g/L, glucose: 0～2.0g/L, sucrose: 0～2.0g/L, agar: 1.5～2.0g/L, pH value 7.0～8.0, is settled to 1000mL.
(2) seed culture medium: N.F,USP MANNITOL: 10～20g/L, soy peptone: 5～20g/L, fish meal: 0～5.0g/L, soya-bean oil: 0.5～5.0g/L, K
2hPO
4: 0.15～0.5g/L, CaCO
3: 0.1～0.4g/L, pH value 7.0～8.0, is settled to 1000mL.
(3) fermention medium: glucose: 2～8g/L, glucose: 2～8g/L, Zulkovsky starch: 5～10g/L, soyflour: 0～10g/L, cotton seed meal: 0～25g/L, yeast extract: 1～3g/L, CaCO
3: 2～5g/L, NaCl:2～5g/L, pH value 7.0～8.0, is settled to 1000mL.
Cultivate flow process:
(1) described shot hole streptomycete (Streptomyces scabiei) WS-24926 and described sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 bacterial classification are inoculated respectively on test tube slant substratum, under 28 ± 1 DEG C of conditions of temperature, activation culture 5d;
(2) bacterial classification of activation is seeded in seed culture medium, in the shaking table of 28 ± 1 DEG C of temperature, cultivates 3～4d;
(3) by step 2) in the bacterial classification cultivated be inoculated in fermention medium, cultivate 3～4d at the condition bottom fermentation of 28 ± 1 DEG C of temperature.
The invention provides the application of described streptomycete in control Cruciferae club root.
The present invention also provides biological and ecological methods to prevent plant disease, pests, and erosion suspension agent of the control Cruciferae club root that described streptomycete is prepared from and preparation method thereof.Described biological and ecological methods to prevent plant disease, pests, and erosion suspension agent is made up of 10～80% zymocyte liquid, 0.4～10% dispersion agent, 0.5～5% emulsifying agent, 0.4～10% thickening material, 0.4～10% sanitas, 0～3.0% defoamer, 0～10.0% frostproofer and the water of surplus by weight percentage, wherein, described zymocyte liquid is the bacterium liquid being obtained by described streptomycete fermentation.
Further, described dispersion agent select in sodium lignosulfonate, calcium lignin sulphonate, naphthalenesulfonic acid-formaldehyde condensate, fatty amide-N methyl taurine salt, alkyl sulfo-succinic acid sodium salt, sodium dibutyl naphthalene sulfonate, sodium lauryl sulphate, polyaryl phenol polyethenoxy ether and edetate choose any one kind of them or several; Described emulsifying agent is in alkylbenzene sulfonate, alkyl alcohol Soxylat A 25-7, alkylphenol polyoxyethylene, styroyl phenol polyethenoxy ether, sorbic ester and polyhydric alcohol fatty acid ester, to choose any one kind of them or several; Described thickening material selects xanthan gum, gelatin, gum arabic, melon glue, sodium alginate, Zulkovsky starch, mixed polysaccharide, Xylo-Mucine, sodium hydroxyethlcellulose, polyvinyl alcohol, sodium polyacrylate, poly amic acid, urea, neusilin, silicon-dioxide, diatomite, wilkinite and Attapulgite to choose any one kind of them or several; Described sanitas selects Sodium Benzoate, potassium sorbate and ethyl p-hydroxybenzoate to choose any one kind of them or several; Described frostproofer be in ethylene glycol, propylene glycol, glycerol, glycol ether, triglycol, polyoxyethylene glycol choose any one kind of them or several; Described defoamer is in isooctyl alcohol, primary isoamyl alcohol, stearic acid, lauric acid, polyglycol ether and organosilicon, to choose any one kind of them or several.
The preparation method of described biological and ecological methods to prevent plant disease, pests, and erosion suspension agent, comprises the following steps:
1) fermented liquid described streptomycete fermentation being obtained, under 4000～10000r/min condition, centrifugal 5～20min, obtains zymocyte liquid;
2) according to described weight percent, the water of described zymocyte liquid, dispersion agent, emulsifying agent, thickening material, sanitas, defoamer, frostproofer and surplus is mixed, obtain mixture;
3) after mixture shearing is uniformly dispersed, is ground to 90% above raw meal particle size degree and reaches below 5 μ m, stir, regulate pH to 4.0～5.0, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
Experiment shows: biological and ecological methods to prevent plant disease, pests, and erosion suspension agent of the present invention reaches 55～80% to Cruciferae club root field test prevention effect, and its pot experiment prevention effect is 75～90%, contrasts medicament higher than fluazinam with cyazofamid etc.
Biological and ecological methods to prevent plant disease, pests, and erosion suspension agent using method of the present invention is: spray method: before transplanting, adopt spray method to carry out soil treatment, 50～200 times of sprayings of described suspension agent dilution are used 1～2 time by the usage quantity of described suspension agent 1000～2000mL by mu.Root-pouring method: adopt root-pouring method processing when transplanting, by 50～200 times of suspension agent dilutions, by the suspension 50～100mL after dilution described in every strain filling root, mu is 1000～3000mL with described suspension agent dosage, fills with root whole vegetative period 1～3 time.
The present invention also provides biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule of the control Cruciferae club root that a kind of described streptomycete is prepared from and preparation method thereof.Described biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule comprises gelatin or the Xylo-Mucine of the gum arabic of the zymocyte liquid of 1～3 part of weight, 1～3 part of weight or seaweed gel, 1～3 part of weight, wherein, described zymocyte liquid is the bacterium liquid being obtained by described streptomycete fermentation.
The preparation method of described biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule: comprise the following steps:
1) fermented liquid first described streptomycete fermentation being obtained, under 4000～10000r/min condition, centrifugal 5～20min, obtains zymocyte liquid, and the zymocyte liquid of getting 1～3 part of weight adds reaction vessel;
2) secondly, get the gelatin of 1～3 part of weight or Xylo-Mucine and add water that to be configured to weight percent be, after 3～8% the aqueous solution, to add reaction vessel, and regulate pH value to 3～5 of reactant with acetum;
3) again, get the gum arabic of 1～3 part of weight or seaweed gel and add water that to be mixed with weight percent be, after 2～8% the aqueous solution, to add reaction vessel, continue to stir 10～30min;
4) then, in reaction vessel, add formalin to solidify cyst wall, continue to stir 10～30min, obtain the microcapsule aqueous solution;
5) last, spraying will be obtained after microcapsule aqueous solution centrifugal treating and biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule can be obtained after dry.
Experimental results show that: biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule of the present invention is 65～80% to Chinese cabbage club root field efficacy, when field operation technique is Chinese cabbage or wild cabbage transplanting, 2g biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule is spread fertilizer over the fields in every cave.
Brief description of the drawings
Fig. 1 is shot hole streptomycete (Streptomyces scabiei) WS-24926 colonial morphology on ISP-2 solid medium.
Fig. 2 is the colonial morphology of sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 on ISP-2.
Embodiment
In order to explain better the present invention, below in conjunction with specific embodiment and Figure of description, streptomycete of the present invention and the application in control Cruciferae club root thereof are described in further detail.
Separation, the screening of embodiment 1 shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 bacterial strain
Soil sampling, adopts that conventional method separates, purifying and preservation.We isolate altogether 30000 strain actinomycetes from soil, adopt the active way of following the trail of of lead compound, in conjunction with HPLC-MS natural product compound database, select relative low toxicity, easy cultivation to there are 109 strain actinomycetes of Commercialization Trends, by Chinese cabbage club root pot experiment and field test, experimental result is in table 1.Final definite WS-24926 and WS-29246 optimum, its disease index minimum, proofreaies and correct preventive effect and is respectively 95% and 79%.
Pot experiment implementation step: by after each strains tested actication of culture, adopt secondary shake flask culture, elementary shake flask fermentation adopts seed culture medium, and culture temperature is 28 DEG C, 150rpm, second order fermentation adopts fermention medium, inoculum size is 5～10%, 28 DEG C of culture condition, rotating speed 150rpm, incubation time 4～6d, packs nutrient solution 100mL in the every 500mL triangular flask of liquid amount.5 bottles of every strain culturing, every bottled 100mL fermention medium.
Adopt cave dish to cultivate the healthy seedling of Chinese cabbage, grow to 2～3 leaf periods for experiment.
Pot experiment adopts the inoculation of bacterium local method, and the Chinese cabbage knee of collecting is shredded with potting soil by 1:100(v/v) mix, pack flowerpot (ф into
on=15cm, ф
under=10cm, h=13cm) middle for experiment.When transplanting, pour into each bacterial strain shake flask fermentation liquid, every alms bowl 100mL, fills with root 1 time every 10d afterwards, and each 100mL, fills with root whole vegetative period 4 times altogether.Result demonstration WS-24926 and WS-29246 are best to the prevention effect of Chinese cabbage club root, proofread and correct preventive effect and are respectively 95% and 79%.
Club root grade scale:
0 grade: do not adhere to knee;
1 grade: knee is attached on lateral root, and quantity accounts for 1～25%;
2 grades: on main root, have knee to adhere to, on lateral root, knee quantity accounts for more than 25%;
3 grades: knee quantity accounts for 50～75%; On main root, there is knee to adhere to;
4 grades: on main root, have knee to adhere to, knee quantity accounts for more than 75% root system.
Calculation formula:
The results from pot experiment test of table 1109 strain actinomycetes to Cruciferae club root
Note: economize " WS-" before strain number.
Embodiment 2 shot hole streptomycetes (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 strain identification
The bacterial strain screening is through morphological observation, and Physiology and biochemistry experiment and molecular biology identification, finally determine that WS-24926 is streptomyces shot hole streptomycete, and WS-29246 is streptomyces point spore streptomycete.
Shot hole streptomycete of the present invention (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 bacterial strain colonial morphology on ISP-2 flat board are shown in respectively accompanying drawing 1, Fig. 2.
16S rDNA sequential analysis: extract shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 genomic dna, carry out pcr amplification in 50 μ L systems.Adopt primer:
PrimerA：5’-AGAGTTTGATCCTGGCTCAG-3’；
PrimerB：5’-TACGGCTACCTTGTTACGACTT-3’。
Reaction conditions: 94 DEG C of denaturation 5min, 94 DEG C of 1min, 55.5 DEG C of 1min, 72 DEG C of 3min, 35 circulations, 72 DEG C are extended 10min.16SrDNA sequencing result utilizes BLAST software to carry out homology search and sequence alignment analysis in GenBank.WS-29246 bacterial strain 16SrRNA gene order GenBank accession number is JQ712974.
By pot experiment and field test, obtain shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246.By shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) the WS-29246 fermented liquid obtaining that ferments, centrifugal 5～20min under 4000～10000r/min condition respectively, obtains respectively the zymocyte liquid of the zymocyte liquid of shot hole streptomycete (Streptomyces scabiei) WS-24926, sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246; Two kinds of zymocyte liquids are mixed to get to the zymocyte liquid of shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246.Three kinds of zymocyte liquids are sampled respectively for subsequent use.
Embodiment 3
Getting THE ADIABATIC SHEAR IN in the water that 4kg xanthan gum adds 112kg mixes well, add afterwards zymocyte liquid for subsequent use described in 800kg (described three kinds of zymocyte liquids for subsequent use respectively to be got once, the present embodiment can prepare three kinds of streptomycete biological and ecological methods to prevent plant disease, pests, and erosion suspension agents), 4kg sodium lignosulfonate, 20kg alkylbenzene sulfonate, 30kg sorbic ester, 20kg potassium sorbate, 10kg propylene glycol, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
For high mountain anti-season Chinese cabbage club root, adopt root-pouring method, in the time transplanting, fill with root 100mL, after fill with root 1 time every 10d, fill with root whole vegetative period 3 times.Three kinds of streptomycete biological and ecological methods to prevent plant disease, pests, and erosion suspension agents of the present embodiment and the transnational agricultural chemicals Japan chemical pesticide 50% fluazinam SC of Ishihara Sangyo Kaisha Ltd. of company and 10% cyazofamid SC combination prevent and treat high mountain Chinese cabbage club root field test results and show, streptomycete biological and ecological methods to prevent plant disease, pests, and erosion suspension agent disease index of the present invention is lower than contrast medicament.Prevention effect is shown in Table 2 in detail.
Table 2 streptomycete biological and ecological methods to prevent plant disease, pests, and erosion suspension agent control Chinese cabbage club root disease index and prevention effect
Note: in table 2, " 50% fluazinam SC+50% cyazofamid SC " processes and adopt 150 grams/acre of (50% fluazinam SC300mL) even spraying soil of fluazinam, fills with 2 cyazofamids of root (1500 times of liquid of 10% cyazofamid SC dilution) vegetative period.Contrast medicament A is 1,000,000,000 living spores/gram bacillus amyloliquefaciens wettable powder, and contrast medicament B is 50% carbendazol wettable powder, and contrast medicament C is the wettable powder of good fortune more than 50%.Remember that with column data acceptance of the bid the mean number of different lowercases has significant difference (P<0.05, Duncan multiple comparisons method (Duncan ' S multiple range test, DMRT)).
Embodiment 4
Get 15kg xanthan gum, 25kg gelatin, 30kg gum arabic, 20kg melon glue and 10kg urea add THE ADIABATIC SHEAR IN in the water of 611kg to mix well, add afterwards 100kg shot hole streptomycete for subsequent use (Streptomyces scabiei) WS-24926 zymocyte liquid, 15kg sodium dibutyl naphthalene sulfonate, 20kg sodium lauryl sulphate, 25kg polyaryl phenol polyethenoxy ether, 25kg alkyl alcohol Soxylat A 25-7, 4kg ethyl p-hydroxybenzoate, 30kg glycerol, 20kg glycol ether, 40kg triglycol and 10kg polyoxyethylene glycol, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
At Lichuan, Hubei Province high mountain wild cabbage growing area, select club root that relatively serious plot occurs, by 100 times of gained suspension agent dilutions, adopt healthy seedling, in the time that wild cabbage is transplanted, root 50～100mL is filled with in every strain, every mu of ground usage quantity is 3kg, and the field test prevention effect of club-root is reached to 66.67%, the heavy 0.89kg of strain.
Embodiment 5
Getting 25kg silicon-dioxide and 25kg diatomite adds THE ADIABATIC SHEAR IN in 225kg water to mix well, add afterwards the zymocyte liquid of 500kg point spore streptomycete (Streptomyces cuspidosporus) WS-29246, 50kg sodium lignosulfonate, 40kg calcium lignin sulphonate, 10kg naphthalenesulfonic acid-formaldehyde condensate, 5kg sorbic ester, 20kg Sodium Benzoate, 10kg potassium sorbate, 12kg isooctyl alcohol, 18kg primary isoamyl alcohol, 50kg ethylene glycol, 10kg propylene glycol, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
In experimental plot, Hubei Prov. Acdemy of Agricutural Sciences Wuhan, by knee by 1 ︰ 100(v/v) to mix rear dress alms bowl for subsequent use with potting soil, by 50～100 times of shot hole streptomycete (Streptomyces scabiei) WS-24926 suspension agent dilutions, in the time that purple flowering stalk is transplanted, root 50～100mL is filled with in every strain, after 10d, fill with again root 1 time, 60d " Invest, Then Investigate " preventive effect, streptomycete reaches 90% to the pot experiment prevention effect of purple flowering stalk club root, to plant without poisoning.
Embodiment 6
Getting 10kg and 55kg urea xanthan gum adds the water THE ADIABATIC SHEAR IN of 300kg to mix well, add afterwards the zymocyte liquid mixed solution of 395kg shot hole streptomycete for subsequent use (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246, 30kg polyaryl phenol polyethenoxy ether, 50kg edetate, 15kg alkylphenol polyoxyethylene, 25kg styroyl phenol polyethenoxy ether, 100kg Sodium Benzoate, 20kg stearic acid, , obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
At Lichuan, Hubei Province high mountain wild cabbage growing area, by gained biological and ecological methods to prevent plant disease, pests, and erosion suspension agent dilution 5 barrels of water of 200 times every mu even spraying soil (every barrel of 15～20kg water), mu suspension agent 1000mL.Transplant healthy seedling, 2～3 barrels of water of again spraying in the time having been punched in cave in wild cabbage transplanting, every mu of ground usage quantity is 500mL, and the field test preventive effect of club-root is greater than to 77.14%, the heavy 1.09kg of strain.
Embodiment 7
Getting 34kg Zulkovsky starch and 52kg mixed polysaccharide adds THE ADIABATIC SHEAR IN in 246kg water to mix well, add afterwards 435kg shot hole streptomycete for subsequent use (Streptomyces scabiei) WS-24926 zymocyte liquid, 48kg polyaryl phenol polyethenoxy ether, 36kg alkylphenol polyoxyethylene, 75kg sodium lignosulfonate, 5kg polyglycol ether, 69kg polyoxyethylene glycol, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
In experimental plot, Hubei Prov. Acdemy of Agricutural Sciences Wuhan, by knee by 1 ︰ 100(v/v) to mix rear dress alms bowl for subsequent use with potting soil, by 90 times of the present embodiment streptomycete suspension agent dilutions, in the time that purple flowering stalk is transplanted, root 60～100mL is filled with in every strain, after 10d, fill with again root 1 time, 60d " Invest, Then Investigate " preventive effect, streptomycete reaches 92% to the pot experiment prevention effect of purple flowering stalk club root, to plant without poisoning.
Embodiment 8
Get 25kg poly amic acid add with 144kg water in THE ADIABATIC SHEAR IN mix well, add afterwards the zymocyte liquid of 680kg point spore streptomycete (Streptomyces cuspidosporus) WS-29246, 20kg sodium lauryl sulphate, 30kg alkylphenol polyoxyethylene, 50kg potassium sorbate, 15kg organosilicon, 36kg triglycol, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
The high mountain wild cabbage growing area of bestowing favour in Hubei Province, by gained biological and ecological methods to prevent plant disease, pests, and erosion suspension agent dilution 5 barrels of water of 150 times every mu even spraying soil (every barrel of 15～20kg water), mu suspension agent 1000mL.Transplant healthy seedling, 2～3 barrels of water of again spraying in the time having been punched in cave in wild cabbage transplanting, every mu of ground usage quantity is 500mL, and the field test preventive effect of club-root is greater than to 75.22%, the heavy 1.13kg of strain.
Embodiment 9
Getting THE ADIABATIC SHEAR IN in the water that 23kg polyvinyl alcohol and 43kg sodium polyacrylate add 414kg mixes well, add afterwards the zymocyte liquid mixed solution of 336kg shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246, 11kg polyaryl phenol polyethenoxy ether, 66kg alkyl sulfo-succinic acid sodium salt, 22kg ethyl phenol Soxylat A 25-7, 48kg ethyl p-hydroxybenzoate, 12kg stearic acid, 25kg propylene glycol, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
In experimental plot, Hubei Prov. Acdemy of Agricutural Sciences Wuhan, by knee by 1 ︰ 100(v/v) to mix rear dress alms bowl for subsequent use with potting soil, by 150 times of the suspension agent dilutions of the present embodiment, in the time that purple flowering stalk is transplanted, root 60～100mL is filled with in every strain, after 10d, fill with again root 1 time, 60d " Invest, Then Investigate " preventive effect, streptomycete reaches 89.3% to the pot experiment prevention effect of purple flowering stalk club root, to plant without poisoning.
Embodiment 10
Getting THE ADIABATIC SHEAR IN in the water that 33kg polyvinyl alcohol adds 382kg mixes well, add afterwards 442kg shot hole streptomycete for subsequent use (Streptomyces scabiei) WS-24926 zymocyte liquid, 43kg sodium lignosulfonate, 15kg sorbic ester, 42kg Sodium Benzoate, 28kg isooctyl alcohol, 15kg glycol ether, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
In experimental plot, Hubei Prov. Acdemy of Agricutural Sciences Wuhan, by knee by 1 ︰ 100(v/v) to mix rear dress alms bowl for subsequent use with potting soil, by 120 times of the present embodiment streptomycete suspension agent dilutions, in the time that purple flowering stalk is transplanted, root 90～120mL is filled with in every strain, after 15d, fill with again root 1 time, 60d " Invest, Then Investigate " preventive effect, streptomycete reaches 81.08% to the pot experiment prevention effect of purple flowering stalk club root, to plant without poisoning.
Embodiment 11
Getting 86kg xanthan gum adds THE ADIABATIC SHEAR IN in 449kg water to mix well, add afterwards zymocyte liquid, 95kg sodium lignosulfonate, 45kg alkyl alcohol Soxylat A 25-7,60kg potassium sorbate, 20kg defoamer lauric acid, the 15kg ethylene glycol of 230kg point spore streptomycete (Streptomyces cuspidosporus) WS-29246, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
At Tujia Autonomous County of Changyang, Hubei Province high mountain wild cabbage growing area, by gained biological and ecological methods to prevent plant disease, pests, and erosion suspension agent dilution 5 barrels of water of 120 times every mu even spraying soil (every barrel of 15～20kg water), mu suspension agent 980mL.Transplant healthy seedling, 2～3 barrels of water of again spraying in the time having been punched in cave in wild cabbage transplanting, every mu of ground usage quantity is 600mL, and the preventive effect of club-root is greater than to 76.09%, the heavy 0.98kg of strain.
Embodiment 12
Getting 20kg poly amic acid adds THE ADIABATIC SHEAR IN in 177kg water to mix well, add afterwards the zymocyte liquid mixed solution of 723kg shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246, 12kg sodium lignosulfonate, 15kg polyhydric alcohol fatty acid ester, 35kg potassium sorbate, 12kg polyglycol ether, 6kg glycol ether, obtain mixture, dispersed with stirring is even, after grinding by horizontal sand mill firsts and seconds, through granularity Detection, more than 90% fineness of materials reaches below 5 μ m, stop grinding and stirring, be packaged as finished product, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
At Lichuan, Hubei Province high mountain wild cabbage growing area, by gained biological and ecological methods to prevent plant disease, pests, and erosion suspension agent dilution 6 barrels of water of 120 times every mu even spraying soil (every barrel of 15～20kg water), mu suspension agent 1100mL.Transplant healthy seedling, 2～3 barrels of water of again spraying in the time having been punched in cave in wild cabbage transplanting, every mu of ground usage quantity is 600mL, and the field test preventive effect of club-root is greater than to 78.02%, the heavy 1.03kg of strain.
Embodiment 13
Getting 1kg shot hole streptomycete for subsequent use (Streptomyces scabiei) WS-24926 zymocyte liquid adopts sand mill grinding 1h to be added in reactor, getting 3kg gelatin is added in 100kg water after dissolving and is added in aforesaid reaction vessel, 45 DEG C of temperature controls, rotating speed is 400r/min rotation 60min, and adding weight percent is that 10% acetic acid is adjusted to 4～5 by system pH.Continue to stir 15min, adding 60kg weight percent is 5% the gum arabic aqueous solution, adjusting rotary speed, to 300r/min, stirs 30min, adds 1.8kg formaldehyde solution, stirring reaction 10min, obtain the slow-release microcapsule aqueous solution, more centrifugal rear employing water cleans 1～2 time, more centrifugal concentrated solution, after spraying is dry, sieve after shaping, obtain slow releasing capsule.
In high mountain anti-season growth of Chinese cabbage district of baked wheaten cake level ground township, Tujia Autonomous County of Changyang (annual 5～October), in the time that Chinese cabbage is transplanted punching, sustained-release granular formulation 2g is spread fertilizer over the fields in every cave, 0.5 mu of ground of co-processing, at market vegetables ripening stage investigation club root incidence, calculate by statistics, it is 69.4% to Chinese cabbage club root field control effect.
Embodiment 14
Get 3kg seaweed gel, add in the water of 97kg and dissolve.Zymocyte liquid, the employing sand mill of getting 2kg point spore streptomycete (Streptomyces cuspidosporus) WS-29246 grind 1h and are added in reactor.Get 3kg gelatin and be added in 97kg water after dissolving and be added in aforesaid reaction vessel, 45 DEG C of temperature controls, rotating speed is 300r/min rotation 60min, adding weight percent is that 10% acetic acid is adjusted to 4～5 by system pH.Continue to stir 15min, add the prepared seaweed gel aqueous solution, adjusting rotary speed, to 300r/min, stirs 30min.Add 1.8kg formaldehyde solution, stirring reaction 10min, obtains the slow-release microcapsule aqueous solution, more centrifugal rear employing water cleans 3 times, more centrifugal concentrated solution, after spraying is dry, sieves after shaping, obtains slow releasing capsule.
In high mountain anti-season growth of Chinese cabbage district, He Jiaping town, Tujia Autonomous County of Changyang (annual 5～October), in the time that Chinese cabbage is transplanted punching, sustained-release granular formulation 2g is spread fertilizer over the fields in every cave, 0.5 mu of ground of co-processing, at market vegetables ripening stage investigation club root incidence, calculate by statistics, it is 74.5% to Chinese cabbage club root field control effect.
Embodiment 15
Get 2kg seaweed gel, add in the water of 78kg and dissolve, get 3kg shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 zymocyte liquid mixed solution, adopt sand mill to grind 1h to be added in reactor.Get 3kg Xylo-Mucine and be added in 96kg water after dissolving and be added in aforesaid reaction vessel, 45 DEG C of temperature controls, rotating speed is 400r/min rotation 10min, adding weight percent is that 10% acetic acid is adjusted to 4～5 by system pH.Continue to stir 15min, add the prepared seaweed gel aqueous solution, adjusting rotary speed, to 300r/min, stirs 30min.Add 1.8kg formaldehyde solution, stirring reaction 10min, obtains the slow-release microcapsule aqueous solution, more centrifugal rear employing water cleans 1～2 time, more centrifugal concentrated solution, and slow releasing capsule sieves after spraying is dry after shaping to obtain.
High mountain anti-season growth of Chinese cabbage district (annual 5～October) bestows favour in Hubei Province, in the time that Chinese cabbage is transplanted punching, sustained-release granular formulation 2g is spread fertilizer over the fields in every cave, 0.5 mu of ground of co-processing, at market vegetables ripening stage investigation club root incidence, calculate by statistics, it is 71.6% to Chinese cabbage club root field control effect.
Embodiment 16
Get the gum arabic of 2kg, add in the water of 78kg and dissolve, get 2kg shot hole streptomycete (Streptomyces scabiei) WS-24926 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246 zymocyte liquid mixed solution, adopt sand mill to grind 1h to be added in reactor.Get 5kg gelatin and be added in 950kg water after dissolving and be added in aforesaid reaction vessel, 45 DEG C of temperature controls, rotating speed is 200r/min rotation 60min, adding weight percent content is that 10% acetic acid is adjusted to 4～5 by system pH.Continue to stir 15min, add the prepared gum arabic aqueous solution, adjusting rotary speed, to 300r/min, stirs 30min.Add 1.8kg formaldehyde solution, stirring reaction 10min, obtains the slow-release microcapsule aqueous solution, more centrifugal rear employing water cleans 1～2 time, more centrifugal concentrated solution, after spraying is dry, sieves after shaping, obtains slow releasing capsule.
In anti-season growth of Chinese cabbage district, mountain, mayor Yanggao County, Lichuan (annual 5～October), in the time that Chinese cabbage is transplanted punching, sustained-release granular formulation 2g is spread fertilizer over the fields in every cave, 0.5 mu of ground of co-processing, at market vegetables ripening stage investigation club root incidence, calculate by statistics, it is 78.2% to Chinese cabbage club root field control effect.
Claims (8)
1. a streptomycete, this streptomycete is shot hole streptomycete (Streptomyces scabiei) WS-24926CCTCC NO:M2012393.
2. a combination bacterium that comprises streptomycete described in claim 1, is characterized in that: described combination bacterium is combined by shot hole streptomycete (Streptomyces scabiei) WS-24926CCTCC NO:M2012393 and sharp spore streptomycete (Streptomyces cuspidosporus) WS-29246CCTCC NO:M2012413.
3. described in a claim 1, described in streptomycete or claim 2, combine the application of bacterium in control Cruciferae club root.
4. one kind is utilized the biological and ecological methods to prevent plant disease, pests, and erosion suspension agent that combines the control Cruciferae club root that bacterium is prepared from described in streptomycete described in claim 1 or claim 2, it is characterized in that: described biological and ecological methods to prevent plant disease, pests, and erosion suspension agent is made up of 10～80% zymocyte liquid, 0.4～10% dispersion agent, 0.5～5% emulsifying agent, 0.4～10% thickening material, 0.4～10% sanitas, 0～3.0% defoamer, 0～10.0% frostproofer and the water of surplus by weight percentage, wherein, described zymocyte liquid is by described streptomycete or the combination bacterium bacterium liquid obtaining that ferments.
5. one kind is utilized the biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule that combines the control cress club root that bacterium is prepared from described in streptomycete described in claim 1 or claim 2, it is characterized in that: described biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule comprises gelatin or the Xylo-Mucine of the gum arabic of the zymocyte liquid of 1～3 part of weight, 1～3 part of weight or seaweed gel, 1～3 part of weight, wherein, described zymocyte liquid is by described streptomycete or the combination bacterium bacterium liquid obtaining that ferments.
6. biological and ecological methods to prevent plant disease, pests, and erosion suspension agent according to claim 4, is characterized in that: described dispersion agent be in sodium lignosulfonate, calcium lignin sulphonate, naphthalenesulfonic acid-formaldehyde condensate, fatty amide-N methyl taurine salt, alkyl sulfo-succinic acid sodium salt, sodium dibutyl naphthalene sulfonate, sodium lauryl sulphate, polyaryl phenol polyethenoxy ether and edetate choose any one kind of them or several; Described emulsifying agent is in alkylbenzene sulfonate, alkyl alcohol Soxylat A 25-7, alkylphenol polyoxyethylene, styroyl phenol polyethenoxy ether, sorbic ester and polyhydric alcohol fatty acid ester, to choose any one kind of them or several; Described thickening material is that xanthan gum, gelatin, gum arabic, melon glue, sodium alginate, Zulkovsky starch, mixed polysaccharide, Xylo-Mucine, sodium hydroxyethlcellulose, polyvinyl alcohol, sodium polyacrylate, poly amic acid, urea, neusilin, silicon-dioxide, diatomite, wilkinite and Attapulgite are chosen any one kind of them or several; Described sanitas is that Sodium Benzoate, potassium sorbate and ethyl p-hydroxybenzoate are chosen any one kind of them or several; Described defoamer is in isooctyl alcohol, primary isoamyl alcohol, stearic acid, lauric acid, polyglycol ether and organosilicon, to choose any one kind of them or several; Described frostproofer be in ethylene glycol, propylene glycol, glycerol, glycol ether, triglycol, polyoxyethylene glycol choose any one kind of them or several.
7. a preparation method for biological and ecological methods to prevent plant disease, pests, and erosion suspension agent described in claim 4, is characterized in that, it comprises the following steps:
1) by described streptomycete or the combination bacterium fermented liquid obtaining that ferments, under 4000～10000r/min condition, centrifugal 5～20min, obtains zymocyte liquid;
2) according to described weight percent, the water of described zymocyte liquid, dispersion agent, emulsifying agent, thickening material, sanitas, defoamer, frostproofer and surplus is mixed, obtain mixture;
3) after mixture shearing is uniformly dispersed, is ground to 90% above raw meal particle size and reaches below 5 μ m, stir, regulate pH to 4.0～5.0, obtain biological and ecological methods to prevent plant disease, pests, and erosion suspension agent.
8. a preparation method for biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule described in claim 5, is characterized in that, it comprises the following steps:
1) first by described streptomycete or the combination bacterium fermented liquid obtaining that ferments, under 4000～10000r/min condition, centrifugal 5～20min, obtains zymocyte liquid, and the zymocyte liquid of getting 1～3 part of weight adds reaction vessel;
2) secondly, get the gelatin of 1～3 part of weight or Xylo-Mucine and add water that to be configured to weight percent be, after 3～8% the aqueous solution, to add reaction vessel, and regulate pH value to 3～5 of reactant with acetum;
3) again, get the gum arabic of 1～3 part of weight or seaweed gel and add water that to be mixed with weight percent be, after 2～8% the aqueous solution, to add reaction vessel, continue to stir 10～30min;
4) then, in reaction vessel, add formalin to solidify cyst wall, continue to stir 10～30min, obtain the microcapsule aqueous solution;
5) last, will obtain spray drying treatment after microcapsule aqueous solution centrifugal treating, can obtain biological and ecological methods to prevent plant disease, pests, and erosion slow releasing capsule.
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| CN201410128658.0A CN103911330B (en) | 2013-02-07 | 2013-02-07 | Point spore streptomycete and the application in preventing and treating Cruciferae clubroot thereof |
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| CN104663403B (en) * | 2015-03-06 | 2017-03-01 | 四川省农业科学院植物保护研究所 | A kind of float seedling method of preventing and treating crop in cruciferae clubroot |
| CN114176097A (en) * | 2021-11-15 | 2022-03-15 | 贵州省植物保护研究所 | Pesticide composition of seed treatment suspending agent for preventing and treating clubroot of cruciferous vegetables |
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