CN102071145A - Trichoderma viride fungi and preparation and application of fungicide thereof - Google Patents
Trichoderma viride fungi and preparation and application of fungicide thereof Download PDFInfo
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Abstract
The invention discloses a trichoderma viride fungi strain and a method for preparing a bio-control fungicide thereof and application. The strain is named as trichoderma viride strain ZBS6 and collected in the CGMCC, and the collection number is CGMCC No.3538. The microbial fungicide is liquid or solid fungicide, and is prepared from the trichoderma viride ZBS6 strain. The ZBS6 strain and the fungicide thereof have wide bacteriostatic spectrum and good bacteriostatic effect, and have strong inhibiting effect on botrytis cirerea, rhizoctonia solani, rhizocto-nia cerealis, gaeumannomyces grami, marssonina coronaria, fusarium oxysporum and the like; and the fungicide can be used for control of botrytis cirerea, cucumber downy mildew, rhizocto-nia cerealis, wheat soil-borne disease and wheat powdery mildew, is efficient, non-toxic and simple to use, and does not cause environmental pollution.
Description
Technical field
The present invention relates to the biocontrol of plant disease field, relate in particular to a kind of viride bacterial strain that can prevent and treat plurality of plant diseases, and utilize this bacterial strain to prepare the method for biocontrol fungicide and the application of this biocontrol fungicide.
Technical background
Plant diseases is the formidable enemy of agriculture production, is 10%～15% of ultimate production according to the annual underproduction average loss that caused by disease because of plant of Food and Argriculture OrganizationFAO (FAO) statistics.For centuries, chemical pesticide is being brought into play positive effect in the preventing and controlling of plant disease pest and weed, for the stable yields of agricultural, increase production and retrieve a loss etc. and to have made important contribution.But excessive using with pesticide abuse already caused many negative impacts, as soil fertility decline, environmental pollution, develop immunity to drugs etc.Especially in recent years, pesticide residue exceed standard and cause person poultry poisoning's incident constantly to take place in the agricultural-food, serious threat people's quality of life, limited the foreign export of agricultural products in China.Progress along with social civilization, people improve day by day to pollution-free, pollution-free green food cry, biological control has become the research focus of control of plant disease, and it all has good blending with human development in future direction on the mode of production, food safety, nutrient health, the eubiosis and species diversity.
Biocontrol microorganisms is the focus of biocontrol of plant disease research as an important ring of biological control always.Biocontrol microorganisms of a great variety, widespread use has fungi, bacterium, actinomycetes etc. in the production.Wherein the biological and ecological methods to prevent plant disease, pests, and erosion microorganism that is applied in the production is early studied and be developed to Trichoderma (Trichoderma.spp) by people.It extensively is present in the ecotopes such as soil, plant rhizosphere seed-coat, is easy to separate and cultivate, and many pathogenic fungies and pathogenetic bacteria are had antagonistic action, has good biological and ecological methods to prevent plant disease, pests, and erosion application prospect.Advantages such as this genus fungi has fast growth, and adaptability is strong, and sporulation quantity is big also have chlamydospore in the mycelium that has, and are easy to suitability for industrialized production, and can produce multiple antibiotics and enzyme, suppress or weaken pathogenic bacteria and develop immunity to drugs.Therefore, in development sustainable agriculture cry more and more higher today, it has very important effect as the resourceful antagonistic microbe of a class in biocontrol of plant disease.Add up according to incomplete data, wood is mould to have antagonistic action to 29 kinds of pathogenic fungies of 18 genus at least in external or body, be widely used in the control of various plants fungal disease, particularly the disease that rhizoctonia, sickle-like bacteria, Sclerotium rolfsii, phytophthora, pythium spp, alternaric bacteria etc. are caused has the better prevention effect.The Trichoderma that utilizes that domestic and international many scientific research personnel are also successful has has prevented and treated many important Plant diseasess, as seedling blight of red pepper, and cowpea blight, cotton seedling blight and samping off etc.In recent years, the research of adopting Trichoderma to prevent and treat banded sclerotial blight also appears in the newspapers repeatly.
Along with the needs of the mankind to the further attention and the agricultural sustainability development of ecotope, the biological control technology certainly will will further develop, and particularly the application of modern biotechnology will inevitably be opened up the frontier of biological and ecological methods to prevent plant disease, pests, and erosion science and technology.The research of microbial pesticide and be applied in the beginning of new century and welcome unprecedented historic opportunities and technological challenge, the industry development prospect is very wide.Therefore, microbial pesticide will develop rapidly in 10～20 years of future, in protection environment, assurance human health, and the more and more important effect of performance in promoting the agricultural sustainable development process.
Summary of the invention
The present invention is intended to provide a kind of efficient, active viride bacterial strain of broad-spectrum sterilization of having that can be used for biological control for plurality of plant diseases, and utilize this bacterial strain to prepare the method for biocontrol fungicide, in addition, also provide the application of this viride on control of plant disease.
The objective of the invention is to realize by following scheme.
Green trichodermin bacterial strain called after viride of the present invention (Trichoderma viride) bacterial strain ZBS6, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, culture presevation number is CGMCC No.3538, and preservation date is: on December 23rd, 2009.Its bacterium colony is circle or subcircular on the PDA culture plate, and the mycelial growth initial stage is that white is velvet-like, and the later stage presents the dark green product spore district of concentric wheel stripe shape.Microscopically is observed, and mycelia is very thin colourless, and tool is separated, multi-branched; Conidiophore bears from the side shoot of mycelia, arranges in the form of a ring, and tool repeats branch several times, sporogenic stigma doleiform estranged or taper; It is spherical that conidium mostly is, green or blue-greenish colour.
Biocontrol fungicide provided by the present invention, bacterial strain ZBS6 is prepared from by viride (Trichoderma viride), is liquid or solid-state microbial inoculum.
The preparation method of above-mentioned biocontrol fungicide is:
(1). liquid bacterial preparation process
The viride bacterial strain ZBS6 that preserves is moved on the PDA culture medium flat plate, and 26 ℃ of alternation of light and darkness are cultivated 4d, scrape under the aseptic condition and get conidium, with sterilized water preparation 1 * 10
7Conidial suspension, inoculate by 1ml spore suspension/100ml PDB substratum, place 26 ℃, shaking culture 3d under the 150rpm condition, electric mixer stirs 2min with the 2000rpm rotating speed, mix (including 0.1% auxiliary agent tween 100 and 0.2% preservative benzoic acid) with the 1.9L phosphoric acid buffer, promptly obtain liquid biocontrol fungicide.
(2). solid-state bacterial preparation process
The viride bacterial strain ZBS6 that preserves is moved on the PDA culture medium flat plate, and 26 ℃ of alternation of light and darkness are cultivated 4d, scrape under the aseptic condition and get conidium, with sterilized water preparation 1 * 10
7Conidial suspension, inoculate by 2ml spore suspension/100g matrix substratum, after 26 ℃ of alternation of light and darkness were cultivated 8-10d, mixing promptly got solid-state biocontrol fungicide.
Described matrix substratum material is made into and is wheat bran, Semen Maydis powder, and rice chaff, water, mass ratio is: 2: 1: 2: 5, promptly contain 20g wheat bran in the 100g matrix substratum, the 10g Semen Maydis powder, 20g rice chaff and 50g water, the high pressure moist heat sterilization is standby behind the mixing.
The application process of biocontrol fungicide of the present invention is: method such as the filling root of liquid microbial inoculum by routine, seed soaking, spraying is used.Solid-state microbial inoculum mainly uses by root method such as execute, spread manuer in holes.Can use separately, also can mix use with other sterilant, plant modifying agent that this biocontrol strain is had no side effect.Generally use in crop sowing time or the disease phase of starting.Before the sowing, adopt the seed dressing of liquid microbial inoculum and seed, or directly root is executed, is spreaded manuer in holes; Or start the phase in disease, by irritating that root applies crop rhizosphere or by foliar spray, bud and fruit surface spray application.
Green trichodermin bacterial strain antimicrobial spectrum provided by the present invention is wide, to comprising fusarium graminearum, withered germ of water-melon, botrytis cinerea pers, pear cucumerinum, gaeumannomyces graminis, cucumber parasitica, marssonina coronaria, the sesame wilt, Chinese yam anthrax bacteria, rhizoctonia cerealis, verticillium dahliae, the capsicum wilt, phytopathogen such as Rhizoctonia solani Kuhn and Valsa mali all has bacteriostatic action preferably, and except that the bacteriostasis rate to the Chinese yam anthrax bacteria is lower than 50%, all the other are all more than 70%, show competition, hyperparasitism also shows antagonistic action to the part germ.
The invention provides the biocontrol fungicide that is prepared from by viride bacterial strain ZBS6, indoor and field test proves to the various crop disease to have remarkable prevention effect as gray mold of cucumber, cucumber downy mildew, rice sheath blight disease, wheat hypochnus, take-all, wheat powdery mildew etc.
The invention has the advantages that: viride ZBS6 bacterial strain antimicrobial spectrum is extensive, and fungistatic effect is obvious, and the biocontrol fungicide kind is many, and the application process scope is wide, and multiple diseases is all had fungistatic effect preferably in indoor and field.Simultaneously,, environment is not polluted,, have good blending species diversity and not influence of the eubiosis to the person poultry safety.
The present invention will be further described below in conjunction with specific embodiment.
Embodiment
Method therefor among the following embodiment if no special instructions, is ordinary method.
Embodiment 1: the separation screening of viride ZBS6 bacterial strain
(1) the mould separation and purification of wood will be adopted after the soil sample of coming dries in the shade, grinds, and take by weighing 5g, put into the triangular flask that 150ml fills 45ml physiological saline.Adopt the soil isolation by dilution method, the diluent of drawing 1mL joins in the test tube that contains 9mL water, and the vibration mixing makes 10
-2The soil diluent.Get 0.1ml soil diluent and coat in the PDA substratum that contains Sodium Propionate and Vetstrep, be upside down in 25 ℃ the constant incubator and cultivate 3d.The Trichoderma that picking is separated to falls, carry out the monospore separation and purification after, change the PDA slant medium over to, treat that mycelia and spore cover with the inclined-plane after, add the sterilization paraffin oil and seal up for safekeeping standby.
(2) comprise fusarium graminearum (Fusarium graminearum) for the examination pathogenic bacteria, withered germ of water-melon (Fusarium oxysporium), botrytis cinerea pers (Botrytis cirerea), pear cucumerinum (Venturia nashicola), gaeumannomyces graminis (Gaeumannomyces grami), cucumber parasitica (Phytophthora melonis), marssonina coronaria (Marssonina coronaria), sesame wilt (Fusarium oxysporum), Chinese yam anthrax bacteria (Colletotrid umgloeosporiodes), rhizoctonia cerealis (Rhizocto-nia cerealis), verticillium dahliae (Verticillium dahliae), capsicum wilt (Fusarium oxysporum), Rhizoctonia solani Kuhn (Rhizoctonia solani) and Valsa mali (Valsmali Miyabe et Yamada).
(3) the mould screening of antagonism wood trichoderma strain that separation and purification is arrived is a target for the examination pathogenic fungi, adopts dull and stereotyped face-off culture method to carry out preliminary screening.Concrete grammar is: total eclipse germ and the Trichoderma cake (d=0.8cm) of face-off inoculation activation 3d on 9cm PDA flat board, both are respectively apart from culture dish edge 1cm, 25 ℃ of light and shades are alternately cultivated, with the flat board of independent inoculation pathogenic bacteria in contrast, every processing repeats 3 times, cultivates 4d, regularly observes, record colony radius and parasitic rate, and the mould inhibiting rate of wood when calculating growth velocity and the dull and stereotyped 4d of cultivation to pathogenic bacteria.
Inhibiting rate (%)=[(colony radius is cultivated in contrast colony radius-face-off)/contrast colony radius] * 100
(4) result is divided into from obtaining 12 strain wood mouldly from wheat paddock soil, is numbered ZBS1-ZBS12 respectively.The trichoderma strain bacterium colony of growing on the PDA substratum is subcircular, and the initial stage is that white is velvet-like, and the later stage presents green to deep green, the mould closely knit product spore district of concentric wheel stripe shape that presents of part wood.Fast growth can cover with full ware and big volume production spore about 4d.
In the present embodiment, be the target bacterium with 12 pathogenic bacterias such as gaeumannomyces graminis, by the face-off screening, having obtained a strain all has the trichoderma strain ZBS6 of fungistatic effect by force to various tested diseases, and concrete fungistatic effect sees Table 1.
Table 1 trichoderma strain ZBS6 is to the fungistatic effect of each pathogenic bacteria
Annotate: " +++": bacteriostasis rate is more than 80%; " ++ ": bacteriostasis rate 50-80%; "+": bacteriostasis rate 20-50; "-": bacteriostasis rate＜30%
The evaluation of embodiment 2:ZBS6 bacterial strain
(1) morphological classification is identified and is adopted colonial morphology observation and microscopic observation method to identify that wood is mould, and searching document is determined its kind.
(2) the mould extracting genome DNA of Molecular Identification antagonism wood is extracted with reference to fungal DNA extracting method such as Wu Fahong, and uses ddH
2The O dissolving DNA is to 500ng/ μ l.The upstream and downstream primer of wooden mould ITS sequence of being used to increase is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, is respectively ITS4:5 '-GGAAGTAAAAGTCGTAACAAGG-3 ' and ITS6:5 '-TCCTCCGCTTATTGATATGC-3 '.With the mould genomic dna of antagonism wood is template, utilizes primer I TS4 and ITS6 to carry out pcr amplification.The PCR reaction system is 25 μ l:10 * PCR Buffer 2.5 μ l, template 1 μ l, and dNTP (10mM each) 0.5 μ l, primer I TS4 (10mM) 1 μ l, ITS6 (10mM) 1 μ l, archaeal dna polymerase (5U/ μ l) 0.25 μ l adds deionized water to 25 μ l.Pcr amplification condition: 94 ℃ of 5min; 94 ℃ of 1min; 55 ℃ of 30sec; 72 ℃ of 1min; 33 circulations; 72 ℃ of 10min.The PCR reaction product adopts 1% agarose gel electrophoresis to detect.After 600bp size purpose band adopts DNA to reclaim test kit (AXYGEN company) recovery purifying, (Takara PMD-19T) is connected with the T-carrier, the transformed competence colibacillus cell, on the flat board that contains Amp, IPTG, X-Gal, carry out the screening of blue hickie then, and carry out bacterium colony PCR and filter out recombinant plasmid.Empirical tests is that the segmental positive colony of purpose is delivered to Shanghai and given birth to the biological company limited of worker and check order.Sequencing result adopts BLAST to carry out sequence identity relatively by retrieval Genbank (http://www.nebi.nlm.nih.gov/Blast/).Simultaneously, download the type strain rDNA ITS sequence of other kind of Trichoderma and other genus fungi, and use CLUSTAL X (Version 1.8) software to carry out contraposition and arrange, being aided with artificial check and correction, the sequence that obtains is used MEGA3.1 molecular evolution genetic analysis software analysis.Calculate in Kimura 2-parameter method on the basis of genetic distance, adopt adjacent method to make up the NJ phylogenetic tree, each ramose degree of confidence of genealogical tree is carried out 1000 circulations altogether with bootstrapping method of inspection (bootstrap) check.
(3) the morphological observation result shows as a result, trichoderma strain ZBS6 bacterium colony circle or subcircular on the PDA culture plate after the separation and purification, and the mycelial growth initial stage is that white is velvet-like, the later stage presents the dark green product spore district of concentric wheel stripe shape.Microscopically is observed, and mycelia is very thin colourless, and tool is separated, multi-branched; Conidiophore bears from the side shoot of mycelia, arranges in the form of a ring, and tool repeats branch several times, sporogenic stigma doleiform estranged or taper; It is spherical that conidium mostly is, green or blue-greenish colour, and searching document judges that tentatively trichoderma strain ZBS6 is viride (Trichoderma viride).For further determining the mould ZBS6 strain classification of antagonism wood status, we are template with this strain gene group DNA, amplifying 1 length with ITS sequence universal primer is expection fragment about 600bp, meets ITS report of high conservative and consistent length all in all fungies.Give birth to worker Bioisystech Co., Ltd through Shanghai and carry out dna sequencing, record the ITS gene complete sequence of ZBS6, comprise ITS1 district, 5.8S district and ITS2 district, readable sequences length total length is 526bp.Submit readable 526bp sequence to GenBank, use the search of BLAST homology, carry out similarity analysis, reach 100% with the bacterial strain consistence of Trichoderma virens with each bacterial strain sequence in the database.By the systematic evolution tree analysis of bacterial strain ZBS6 and other type strains rDNA ITS sequence construct as can be seen, the sequence of bacterial strain ZBS6 is gathered into a class with Trichoderma viride, constitute a branch separately, genetic distance is the shortest, a tree reliability reaches 81%, in conjunction with its biological characteristics and morphological feature, identify that this bacterial strain is a viride.
ZBS6-ITS DNA complete sequence:
1 CCGAGTTTAC?AACTCCCAAA?CCCAATGTGA?ACGTTACCAA?ACTGTTGCCT?CGGCGGGATC
61 TCTGCCCCGG?GTGCGTCGCA?GCCCCGGACC?AAGGCGCCCG?CCGGAGGACC?AACCAAAACT
121?CTTATTGTAT?ACCCCCTCGC?GGGTTTTTTA?CTATCTGAGC?CATCTCGGCG?CCCCTCGTGG
181?GCGTTTCGAA?AATGAATCAA?AACTTTCAAC?AACGGATCTC?TTGGTTCTGG?CATCGATGAA
241?GAACGCAGCG?AAATGCGATA?AGTAATGTGA?ATTGCAGAAT?TCAGTGAATC?ATCGAATCTT
301?TGAACGCACA?TTGCGCCCGC?CAGTATTCTG?GCGGGCATGC?CTGTCCGAGC?GTCATTTCAA
361?CCCTCGAACC?CCTCCGGGGG?GTCGGCGTTG?GGGATCGGCC?CTTTACGGGG?CCGGCCCCGA
421?AATACAGTGG?CGGTCTCGCC?GCAGCCTCTC?CTGCGCAGTA?GTTTGCACAC?TCGCATCGGG
481?AGCGCGGCGC?GTCCACAGCC?GTTAAACACC?CCAAACTTCT?GAAATG
Embodiment 3: a kind of preparation of microbiobacterial agent
(1) preparation of liquid microbial inoculum moves into the viride bacterial strain ZBS6 that preserves on the PDA culture medium flat plate, and 25 ℃ of alternation of light and darkness are cultivated 4d, scrapes under the aseptic condition and gets conidium, with sterilized water configuration 1 * 10
7Conidial suspension, inoculate by 1ml spore suspension/100ml PDB substratum, place 25 ℃, shaking culture 3d under the 150rpm condition, electric mixer stirs 2min with the 2000rpm rotating speed, mix (including 0.1% auxiliary agent tween 100 and 0.2% preservative benzoic acid) with the 1.9L phosphoric acid buffer, promptly obtain liquid biocontrol fungicide.
(2) preparation of solid-state microbial inoculum moves into the viride bacterial strain ZBS6 that preserves on the PDA culture medium flat plate, and 25 ℃ of alternation of light and darkness are cultivated 4d, scrapes under the aseptic condition and gets conidium, with sterilized water configuration 1 * 10
7Conidial suspension, inoculate by 2ml spore suspension/100g matrix substratum, after 26 ℃ of alternation of light and darkness were cultivated 8-10d, mixing promptly gets solid-state biocontrol fungicide, and (the matrix substratum was: wheat bran, Semen Maydis powder, rice chaff, water, its mass ratio is: 2: 1: 2: 5, be to contain 20g wheat bran in the 100g matrix substratum, the 10g Semen Maydis powder, 20g rice chaff and 50g water, the high pressure moist heat sterilization is standby behind the mixing).
(3) methods such as the filling root of the liquid microbial inoculum of application process by routine, seed soaking, spraying are used.Solid-state microbial inoculum is mainly used by root method such as execute, spread manuer in holes.Can use separately, also can mix use with other sterilant, plant modifying agent that this biocontrol strain is had no side effect.Generally use in crop sowing time or the disease phase of starting.Before the sowing, adopt the seed dressing of liquid microbial inoculum and seed, or directly root is executed, is spreaded manuer in holes; Or start the phase in disease, by irritating that root applies crop rhizosphere or by foliar spray, bud and fruit surface spray application.
The application of embodiment 4 bacterial strain ZBS6 on the control gray mold of cucumber
(1) indoor face-off
The face-off cultivation results shows that ZBS6 has extremely strong fungistatic effect to the botrytis cinerea pers bacterium.On the PDA substratum, the per day growth velocity of ZBS6 is more than 26.7mm, and during face-off was cultivated, its per day speed of growth also about 19.5mm, had the obvious growth advantage than ash arrhizus bacteria, shows intensive competition effect; Simultaneously, botrytis cinerea mycelial growth rate (6.2mm/d) during face-off is cultivated is starkly lower than the pathogenic bacteria mycelial growth rate (12.8mm/d) of independent inoculation, shows that antagonism wood is mould to secrete the normal growth that secondary metabolite etc. has suppressed pathogenic bacteria in process of growth.Face-off was cultivated after 3 days, and trichoderma strain ZBS6 begins parasitic germ bacterium colony, and to the 4th day, its bacteriostasis rate reached 72.7%, and parasitic rate reaches 100%.
(2) warmhouse booth diseases prevention
Experiment place: the Da Ran village, Liu market town, Zhongmou County, Kaifeng (green cucumber booth 1)
Cucumber variety: excellent No. 1 of Tianjin
Experimental period: the 9-12 month in 2009
Experiment is handled: 1. clear water contrast
2. liquid microbial inoculum stoste spraying control (every 10d spraying is once handled 3 times continuously after mid-October)
3. 10 times of liquid sprayings of liquid microbial inoculum control (every 10d spraying is once handled 3 times continuously after mid-October)
4. 500 times of liquid of 50% derosal wettable powder
Investigation method: investigation on (1) November 15 disease index
(2) 5 samplings are adopted in every sub-district, investigate 2 strains at every, investigate whole blade gray mold of cucumber grade scales of every strain:
0 grade: no scab;
1 grade: single blade has 3 of scabs
3 grades: single blade has scab 4-6
5 grades: single blade has scab 7-10
7 grades: single blade has scab 11-20, and part is intensive in flakes
9 grades: single blade has the intensive leaf area that accounts for of scab more than 1/4th
By control experiment, find that the ZBS6 microbial inoculum has the better prevention effect to the tomato in plastic greenhouse gray mold, the results are shown in Table 2 to the warmhouse booth cucumber.After the control of ZBS6 microbial inoculum stoste, its sickness rate is 15% only, is starkly lower than 52% of contrast, and prevention effect also reaches 70.97%, is higher than 70.28% of derosal preventive effect.
Table 2:ZBS6 microbial inoculum is prevented and treated experimental result to the tomato in plastic greenhouse gray mold
The application (warmhouse booth diseases prevention) of embodiment 5 bacterial strain ZBS6 on the control cucumber downy mildew
Experiment place: the Da Ran village, Liu market town, Zhongmou County, Kaifeng (green cucumber booth 2)
Cucumber variety: excellent No. 1 of Tianjin
Experimental period: the 9-12 month in 2009
Experiment is handled: 1. clear water contrast
2. liquid microbial inoculum stoste spraying control (every 10d spraying is once handled 3 times continuously after mid-October)
3. 10 times of liquid sprayings of liquid microbial inoculum control (every 10d spraying is once handled 3 times continuously after mid-October)
4. 200 times of liquid of 40% phosethyl Al wettable powder
Investigation method: investigation on (1) November 15 disease index
(2) every sub-district is got 4 investigation at random, every some investigation two strains, and whole blade cucumber downy mildew grade scales are investigated in every strain:
0 grade: no scab;
1 grade: lesion area accounts for below 5% of whole leaf area;
3 grades: lesion area accounts for the 6%-15% of whole leaf area;
5 grades: lesion area accounts for the 16%-25% of whole leaf area;
7 grades: lesion area accounts for the 26%-50% of whole leaf area;
9 grades: lesion area accounts for more than 50% of whole area.
Shown in table 3 test-results, ZBS6 microbial inoculum stoste and ten times of diluents have fabulous prevention effect to plastic tent cucumber oidium, and after treatment, its sickness rate is respectively 41.67% and 33.33%, is lower than 75.00% of contrast; Its preventive effect reaches 73.87% and 70.11% respectively simultaneously, all is higher than the preventive effect of 40% phosethyl Al.
Table 3:ZBS6 microbial inoculum is prevented and treated experimental result to plastic tent cucumber oidium
Embodiment 6 bacterial strain ZBS6 are in the application (field diseases prevention) that prevents and treats on the rice sheath blight disease
The experiment place: Yuanyang, the Henan Province institute of agricultural sciences experimental field
Wheat breed: former rice No. 1
Experimental period: in July, 2009
Application method: liquid microbial inoculum stoste spraying control
Experiment is handled: 1. clear water contrast
2. liquid microbial inoculum stoste spraying control
3. 5% jingganmycin aqua spraying control
Investigation method: investigated in 10 days after (1) last dispenser
(2) 5 samplings of every sub-district diagonal lines, every continuous 5 strains of investigation, totally 25 strains, record diseased plant number and sick progression
The rice sheath blight disease grade scale:
0 grade: complete stool is anosis;
1 grade: quaterfoil and following each leaf sheath thereof, blade morbidity (is first leaf with sword-like leave);
3 grades: the 3rd leaf and following each leaf sheath thereof, blade morbidity;
5 grades: second leaf and following each leaf sheath thereof, blade morbidity;
7 grades: sword-like leave blade and following each leaf sheath thereof, blade morbidity;
9 grades: the complete stool morbidity, withered ahead of time.
The field control experimental result of rice sheath blight disease is as shown in table 4.After the liquid microbial inoculum stoste spraying of ZBS6 was handled, its sickness rate was 33.33% only, is starkly lower than 60.00 of contrast, but was higher than 26.67% of contrast chemicals treatment; Its prevention effect to rice sheath blight disease reaches 76.68%, and is suitable with the contrast pharmacy control efficacy.
Table 4ZBS6 microbial inoculum is to the rice sheath blight disease field control effect
The application of embodiment 7 bacterial strain ZBS6 on the control wheat hypochnus
(1) indoor face-off
The face-off cultivation results shows that ZBS6 has extremely strong fungistatic effect to rhizoctonia cerealis.On the PDA substratum, the per day growth velocity of ZBS6 is more than 26.7mm, and during face-off was cultivated, its per day speed of growth also about 21.5mm, had than the obvious growth advantage than sheath blight fungus, shows intensive competition effect; Simultaneously, the withered bacterium mycelial growth rate of line (7.2mm/d) during face-off is cultivated is starkly lower than the pathogenic bacteria mycelial growth rate (13.4mm/d) of independent inoculation, shows that antagonism wood is mould to secrete the normal growth that secondary metabolite etc. has suppressed pathogenic bacteria in process of growth.Face-off was cultivated after 3 days, and trichoderma strain ZBS6 begins parasitic germ bacterium colony, and the growth of rhizoctonia cerealis is subjected to obvious suppression, and to the 4th day, its bacteriostasis rate reached 75.5%, and parasitic rate also reaches 100%, and after this inhibiting rate keeps this level always.
(2) meta-bolites antagonistic experiment
Glassine paper is cut into the disk of diameter 9cm, behind the high-temperature sterilization, is attached to the PDA media surface under the aseptic condition.ZBS6 bacterium cake is inoculated in the PDA media surface of posting glassine paper, establishes cover glass paper but the substratum that do not connect bacterium is contrast.Under 25 ℃ of conditions, light and shade is alternately cultivated, treat that bacterial strain ZBS6 covers with ware after, the glassine paper that will contain bacterium colony moves, rhizoctonia cerealis bacterium cake is inserted contain in the culture dish of wooden mould metabolite, dark culturing under 25 ℃ of conditions, every processing repeats 3 times, observations behind the 7d.Experimental result shows that in containing the culture dish of wooden mould metabolite, rhizoctonia cerealis is suppressed fully, can not grow fresh mycelia, and in the contrast, the wheat hypochnus bacteria growing is normal, and 7d can cover with full ware.Illustrate that ZBS6 can produce the growth that certain or multiple metabolite suppress rhizoctonia cerealis.
(3) indoor pot
This experiment is carried out in the greenhouse of Henan Academy of Agricultural Sciences, for the examination wheat breed is 9023, for the examination pathogenic bacteria is rhizoctonia cerealis, carry disease germs mode that the wheat substratum mixes soil of employing is carried out pathogenic bacteria inoculation (the wheat substratum is carried disease germs: soil=1: 15), the reagent agent contrast is 50% derosal WP (Fuda, Jiangyin City farmingization company limited) and 5% jingganmycin aqua, 3 repetitions of every processing whenever repeat to use native 500g, sow 20 seeds.
7 processing are established in test altogether.
1. clear water contrast
2. connect the bacterium contrast
3. the liquid microbial inoculum stoste seed soaking of ZBS6 2h directly sows
4. irritate root, every strain 5ml microbial inoculum amount behind the liquid microbial inoculum sowing of the ZBS6 7d
5. solid-state microbial inoculum is mixed soil and is handled (microbial inoculum: soil=1: 49)
6. 50% derosal WP irritates root
7. 5% jingganmycin aqua, 50 * liquid seed soaking
Grade scale:
0 grade: do not fall ill
1 grade: leaf sheath is fallen ill but cane is not fallen ill
3 grades: the leaf sheath morbidity, and invaded stem, but cane scab ring stem less than 1/2
5 grades: cane scab ring stem surpasses 1/2, but non-lodging or fracture
7 grades: withered, lodging, withered dead ears
By the greenhouse pot culture test, the result shows that in all biological and ecological methods to prevent plant disease, pests, and erosions were handled, the preventive effect of carrying out root irrigation with the liquid microbial inoculum of ZBS6 was the highest, reaches 70.89, only a little less than 5% jingganmycin effect, but is higher than 50% derosal.And the liquid microbial inoculum seed soaking of ZBS6 treatment effect is not obvious, and preventive effect may fail fully to be adsorbed by wheat less than 50% owing to microbial inoculum.In addition, after the ZBS6 microbial inoculum was handled, the wheat seedling rate all was improved to some extent.
Table 5ZBS6 microbial inoculum is to the preventive and therapeutic effect of wheat hypochnus
Embodiment 8 bacterial strain ZBS6 are in the application that prevents and treats on the take-all
(1) indoor face-off
The face-off cultivation results shows that ZBS6 has extremely strong fungistatic effect to gaeumannomyces graminis.On the PDA substratum, the per day growth velocity of ZBS6 is more than 26.7mm, and during face-off was cultivated, its per day speed of growth also about 24.8mm, had the obvious growth advantage than the total eclipse germ, shows intensive competition effect; Simultaneously, total eclipse germ mycelial growth rate (5.1mm/d) during face-off is cultivated is starkly lower than the pathogenic bacteria mycelial growth rate (11.4mm/d) of independent inoculation, shows that antagonism wood is mould to secrete the normal growth that secondary metabolite etc. has suppressed pathogenic bacteria in process of growth.Face-off was cultivated after 3 days, and trichoderma strain ZBS6 begins parasitic germ bacterium colony, and the growth of gaeumannomyces graminis is subjected to obvious suppression, and to the 4th day, its bacteriostasis rate reached 82.5%, and parasitic rate also reaches 80%, and after this inhibiting rate keeps this level always.
(2) meta-bolites antagonistic experiment
Glassine paper is cut into the disk of diameter 9cm, behind the high-temperature sterilization, is attached to the PDA media surface under the aseptic condition.ZBS6 bacterium cake is inoculated in the PDA media surface of posting glassine paper, establishes cover glass paper but the substratum that do not connect bacterium is contrast.Under 25 ℃ of conditions, light and shade is alternately cultivated, treat that bacterial strain ZBS6 covers with ware after, the glassine paper that will contain bacterium colony moves, and gaeumannomyces graminis bacterium cake is inserted contain in the culture dish of wooden mould metabolite, dark culturing under 25 ℃ of conditions not handle and repeats observations behind the 7d 3 times.Experimental result shows that in containing the culture dish of wooden mould metabolite, gaeumannomyces graminis is suppressed fully, can not grow fresh mycelia, and in the contrast, the take-all bacteria growing is normal, and 7d can cover with full ware.Illustrate that ZBS6 can produce the growth that certain or multiple metabolite suppress gaeumannomyces graminis.
(3) field diseases prevention
The experiment place: Yuanyang, the Henan Province institute of agricultural sciences experimental field
Wheat breed: short by anti-58
Experimental period: year May in October, 2009-2010
Application method: after planting solid-state microbial inoculum is spreaded manuer in holes and is sowed preceding liquid microbial inoculum seed soaking
Experiment is handled: 1. clear water contrast
2. solid-state microbial inoculum spread manuer in holes (2g/ cave)
3. liquid microbial inoculum stoste seed soaking
4. total eclipse is dressed seed only
Investigation method: (1) jointing stage is carried out root system investigation once, and the every sub-district of dead ears rate (2) of investigating every sub-district before the results in 7-10 days is 5 sampling surveys at random, investigates every of dead ears and gets the 1m duplicate rows and investigate total spike number and dead ears number.20 strain root samples are got in every of root system investigation, will put in order strain with spade and dig out, and clean up investigation root system incidence under white background in the water.
The take-all grade scale:
0 grade: anosis
1 grade: the root system onset area accounts for 1%-5%
3 grades: the root system onset area accounts for 6%-20%
5 grades: the root system onset area accounts for 21%-40%
7 grades: the root system onset area accounts for 41%-60%
9 grades: the root system onset area accounts for more than 61%
As can be seen from Table 6, the ZBS6 microbial inoculum has certain prevention effect to gaeumannomyces graminis, adopts different treatment processs, and its sickness rate all obviously reduces; Preventive effect a little less than the contrast medicament prevention effect of gaeumannomyces graminis disease, wherein, adopts the method for spreading manuer in holes also all more than 50%, and effect is better relatively.
Table 6ZBS6 microbial inoculum is to the take-all field control effect
The application (field diseases prevention) of embodiment 9 bacterial strain ZBS6 on the control wheat powdery mildew
Experiment place: two bases, academy of agricultural sciences, Henan Province (Yuanyang)
Wheat breed: general wheat No. 8
Experimental period: in May, 2009
Application method: liquid microbial inoculum stoste spraying control
Investigation method: investigation state of an illness radix before (1) dispenser, two weeks were reinvestigated disease index after the dispenser
(2) 5 samplings of every sub-district diagonal lines, every some investigation 4 strains, totally 20 strains.Record diseased plant number calculates disease index and prevention effect.
The wheat powdery mildew grade scale
0 grade: anosis;
1 grade: lesion area accounts for below 5% of one-piece blade area;
3 grades: lesion area accounts for 6～15% of one-piece blade area;
5 grades: lesion area accounts for 15～25% of one-piece blade area;
7 grades: lesion area accounts for 26～50% of one-piece blade area;
9 grades: lesion area accounts for more than 50% of one-piece blade area.
The field control experimental result of wheat powdery mildew is as shown in table 7.The ZBS6 microbial inoculum reaches 60.36% to the prevention effect of wheat powdery mildew, and is suitable with the contrast pharmacy control efficacy.
Table 7 ZBS6 microbial inoculum is to the wheat powdery mildew field control effect
Claims (10)
1. green trichodermin bacterial strain is characterized in that: this bacterial strain called after viride (Trichoderma viride) bacterial strain ZBS6, culture presevation number is CGMCC No.3538.
2. green trichodermin bacterial strain according to claim 1 is characterized in that: the bacterium colony of this bacterial strain is circle or subcircular on the PDA culture plate, and the mycelial growth initial stage is that white is velvet-like, and the later stage presents the dark green product spore district of concentric wheel stripe shape; Microscopically is observed, and mycelia is very thin colourless, and tool is separated, multi-branched; Conidiophore bears from the side shoot of mycelia, arranges in the form of a ring, and tool repeats branch several times, sporogenic stigma doleiform estranged or taper; It is spherical that conidium mostly is, green or blue-greenish colour.
3. utilize the liquid or solid-state biocontrol fungicide of the described green trichodermin bacterial strain preparation of claim 1.
4. prepare the method for the described liquid biocontrol fungicide of claim 3, it is characterized in that:
(1). liquid bacterial preparation process
The viride bacterial strain ZBS6 that preserves is moved on the PDA culture medium flat plate, and 26 ℃ of alternation of light and darkness are cultivated 4d, scrape under the aseptic condition and get conidium, with sterilized water configuration 1 * 10
7Conidial suspension, inoculate by 1ml spore suspension/100ml PDB substratum, place 26 ℃, shaking culture 3d under the 150rpm condition, electric mixer stirs 2min with the 2000rpm rotating speed, mix (including 0.1% auxiliary agent tween 100 and 0.2% preservative benzoic acid) with the 1.9L phosphoric acid buffer, promptly obtain liquid biocontrol fungicide.
(2). solid-state bacterial preparation process
The viride bacterial strain ZBS6 that preserves is moved on the PDA culture medium flat plate, and 26 ℃ of alternation of light and darkness are cultivated 4d, scrape under the aseptic condition and get conidium, with sterilized water configuration 1 * 10
7Conidial suspension, inoculate by 2ml spore suspension/100g matrix substratum, after 26 ℃ of alternation of light and darkness were cultivated 8-10d, mixing promptly got solid-state biocontrol fungicide.
5. method according to claim 4, it is characterized in that: described matrix substratum, material is made into and is wheat bran, Semen Maydis powder, rice chaff, water, mass ratio is: 2: 1: 2: 5, promptly contain 20g wheat bran in the 100g matrix substratum, the 10g Semen Maydis powder, 20g rice chaff and 50g water, the high pressure moist heat sterilization is standby behind the mixing.
6. the application process of the described biocontrol fungicide of claim 3 is characterized in that: methods such as the filling root of liquid microbial inoculum by routine, seed soaking, spraying are used, and solid-state microbial inoculum mainly uses by root method such as execute, spread manuer in holes.Can use separately, also can mix use with other sterilant, plant modifying agent that this biocontrol strain is had no side effect.
7. application process according to claim 6 is characterized in that: generally use in crop sowing time or the disease phase of starting, before the sowing, adopt the seed dressing of liquid microbial inoculum and seed, or directly root is executed, spreaded manuer in holes; Or start the phase in disease, by irritating that root applies crop rhizosphere or by foliar spray, bud and fruit surface spray application.
8. the described green trichodermin bacterial strain of claim 1 is suppressing fusarium graminearum, withered germ of water-melon, botrytis cinerea pers, pear cucumerinum, gaeumannomyces graminis, cucumber parasitica, marssonina coronaria, the sesame wilt, the Chinese yam anthrax bacteria, rhizoctonia cerealis, verticillium dahliae, the capsicum wilt, the application on Rhizoctonia solani Kuhn and the Valsa mali.
9. the application of the described green trichodermin bacterial strain of claim 1 on the control cucumber downy mildew.
10. the application of the described green trichodermin bacterial strain of claim 1 on the control wheat powdery mildew.
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