CN105524841A - Broad-spectrum chaetomium strain applied to biological control of plant diseases and applications of broad-spectrum chaetomium strain - Google Patents
Broad-spectrum chaetomium strain applied to biological control of plant diseases and applications of broad-spectrum chaetomium strain Download PDFInfo
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- CN105524841A CN105524841A CN201510751523.4A CN201510751523A CN105524841A CN 105524841 A CN105524841 A CN 105524841A CN 201510751523 A CN201510751523 A CN 201510751523A CN 105524841 A CN105524841 A CN 105524841A
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Abstract
The invention provides a broad-spectrum chaetomium strain applied to biological control of plant diseases. The broad-spectrum chaetomium strain is separated and screened from scale-leaf branches of sabina chinensis; through the morphological observation and the ITS sequence analysis, the broad-spectrum chaetomium strain is identified to be C.subaffine in the Chaetomium spp fungus, the broad-spectrum chaetomium strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on Oct 19, 2015, and the preservation number of the broad-spectrum chaetomium strain is CGMCC No.11400. The broad-spectrum chaetomium strain can be applied to the biological control of plant diseases, has good inhibiting effects for various common phytopathogens (wilt fusarium oxysporum, botrytis cinerea and root rot fungus) in production, and particularly has obvious inhibiting effects for intractable soil-borne plant pathogens (verticillium dahliae and fusarium oxysporum), the successive cropping obstacle caused by root diseases under the planting mode of the facility can be overcome, and the high-quality and pollution-free development of the agricultural industry is promoted.
Description
Technical field
The present invention relates to a strain chaetomium, belong to the field of biological control in plant protection, the Plant diseases comprehensive regulation.
Background technology
Biological control refers to utilize beneficial organism and product thereof to suppress existence and the activity of pathogen, thus alleviates or suppress the control of plant disease technology of Occurrence and development of disease.Compared to chemical prevention, biological control has person poultry safety, and pathogenic bacteria can not be induced to develop immunity to drugs, and can play the advantages such as actively long-acting regulating and controlling effect to ecotope.Therefore, along with people are to the enhancing of environmental protection consciousness, research and development and applying biological agricultural chemicals have become with progressively substituting chemical pesticide the new trend that modern agriculture industryization develops energetically.
Biocontrol microorganisms plays a part very important in biocontrol of plant disease.The good protection effect showed after using biocontrol microorganisms in control of plant disease, to the security of the ecosystem and the accreditation advantages such as the toxicological harmless of the mankind and various biology having been obtained to people.Therefore, from nature find beneficial microorganism to Plant diseases carry out sustainable control and Unpolluting management become one very active and show the field of applications well prospect.
The biocontrol microorganisms being applied to biocontrol of plant disease mainly utilizes the antagonistic action between microorganism, Competition, hyperparasitism, cross-protection etc. to alleviate the generation of disease.At present, in production, the biocontrol microorganisms of Application and Development relates to fungi, bacterium, actinomycetes, virus etc.Widely used in fungi have Trichoderma, yeast, Paecilomyces lilacinus and mycorrhizal fungi etc.; Biocontrol bacteria mainly contains bacillus, Pseudomonas alba, radioactivity soil Agrobacterium etc.Wherein, edaphic bacillus, subtilis widespread use all; Applying maximum in actinomycetes is streptomycete and mutation thereof, as jinggangmeisu, pesticide corrosion 120 etc.; Virus mainly utilizes the cross-protection of low virulent strain system to carry out the control of virus disease.
Chaetomium, in the fungi of Ascomycota, gang pyrenomycetes, excrement shell order, chaetomium section, Chaetomium, was found in the ight soil of animal and plant body, soil, grit and some animals once.Research in recent years finds that chaetomium is the fungi monoid that class has Biocontrol Potential, its Biocontrol Effect is more with the research of chaetomium globosum, has reported that chaetomium globosum all has stronger restraining effect to apple black star bacteria, Colletotrichum capsici, dry thread Pyrenomycetes, wheat rust bacterium, rice blast fungus etc.Chaetomium to the restraining effect of phytopathogen with nutrient competition and produce antibacterial substance for Main Function approach, wherein with antibacterial substance most study, the antibacterial substance produced with the chaetomium found at present mainly contains chaetoglobosin, chaetoglobosin, chaetocin and chaetoaurin etc.
At present, the biocontrol microorganisms research report for biocontrol of plant disease is more, and biocontrol microorganisms also screens, preserve certain quantity, but in actual production, the biocontrol microorganisms that can apply is less.Trace it to its cause, except the microorganism active composition of biocontrol microorganisms is subject to except the impact of environmental factor in production, preservation and application, the biocontrol microorganisms of current report only acts on certain or a certain class Plant diseases mostly, antimicrobial spectrum is narrow, and to the Main Biological of the biocontrol microorganisms of separation screening and main resistance research delayed.
Summary of the invention
Technical problem to be solved by this invention is: provide the Plant diseases biocontrol strain that a strain has broad spectrum bacteriostatic action, illustrate its Main Biological and resistance, and provides its application scheme aborning.
Biocontrol strain LB-1 provided by the invention is separated from cypress scale leaf sprig, screening, by morphological observation and ITS sequence analysis, identify its be Chaetomium (
chaetomiumnearly echinid shell spp) in fungi (
chaetomiumsubaffine), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 19th, 2015, culture presevation registration number is CGMCCNo.11400.Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
The nearly edge chaetomium of biological and ecological methods to prevent plant disease, pests, and erosion provided by the invention may be used for the biological control of Plant diseases (especially intractable soil-borne disease).
Beneficial effect of the present invention: biocontrol strain LB-1 of the present invention all has good restraining effect to the pathogenic bacteria (as botrytis cinerea, Exserohilum turcicum, Rhizoctonia solani Kuhn, pathogenic Alternariaspp etc.) of common intractable soil-borne plant pathogen (as Fusarium oxysporum, Cochliobolus sativus) and frequently seen plants leaf diseases in production, the problem of environmental pollutions such as the pesticide residue that the continuous cropping obstacle caused for root disease in effective solution agriculture production and common leaf diseases chemical prevention cause, promote the high-quality of agricultural industry, nuisanceless development.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is biocontrol strain morphological specificity of the present invention.
Fig. 2 is the systematic evolution tree that the ITS sequence of bacterial strain of the present invention is analyzed.
Fig. 3 is bacterial strain of the present invention to the inhibition of pathogenic Fusarium oxysporum and Alternariaspp.
Fig. 4 is bacterial strain fermentation liquor of the present invention to the inhibition of pathogenic Fusarium oxysporum and Alternariaspp.
Embodiment
The screening of embodiment 1 bacterial strain
Adopt tissue isolation, be separated from cypress scale leaf sprig, screen this biocontrol strain.First remove the dustiness on healthy cypress scale leaf sprig with tap water, 75% ethanol, 0.1% mercuric chloride disinfect rear aseptic water washing 5 times successively.Cut the sterilization cypress scale leaf sprig tissue that 2-3mm is long, in 25 DEG C, cultivate on PDA flat board under 12h illumination condition, the dominant strain purifying on PDA flat board again of acquisition, observes and records colony morphology characteristic.
Strain identification: 1. morphologic observation: the bacterium colony of separation and purification is made bacteria suspension Temporary slide by the glucose solution with 1%, by means of the morphological specificity of digital micrograph sem observation pathogenic bacteria; 2. ITS sequence analysis: CTAB method extracts bacterial classification genomic dna, fungi universal primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') carries out pcr amplification to the DNA extracted.Amplified production detects through the agarose gel electrophoresis of 1%, TaKaRa sepharose reclaims test kit and reclaims, checks order.The rDNA-ITS sequence having logged in fungi in sequencing result and GenBank database carries out tetraploid rice, the adjacent method phylogenetic tree construction in MEGA4.0 software.
The results are shown in Fig. 1-2.
Biological characteristics: adopt pure culture biscuits involvng inoculation PDA dull and stereotyped, right-angled intersection method measures colony diameter to pass judgment on the method for growth situation, measures the temperature of biocontrol strain of the present invention, pH, best carbon and nitrogen sources and to Main Biologicals such as the tolerances of ultraviolet, water stress and salt stress.
Biological property analysis result shows, the temperature range that the biocontrol strain LB-1 of this invention grows is 4-38 DEG C, and optimum temperuture is 25-30 DEG C, and fatal temperature is 65 DEG C; PH value range is 4-13, all can grow very well in the scope of pH7-10; Dark and the growth of natural lighting to germ does not have a significant effect, but continuous light can obviously suppress its mycelia to expand; This bacterial strain Absorbable rod utilizes multiple common monosaccharides, disaccharides and polysaccharide and organic and inorganic nitrogen-sourced, but optimum carbon source of its growth is sweet dew alcohol and glucose, and optimum nitrogen source is urea.The biocontrol strain of this invention all has fine tolerance to uv irradiating, salt stress, water stress, and continue uv irradiating less than 120 minutes, in growth matrix, NaCl content PEG content in 0.75M, growth matrix all has no significant effect its growth below 25%.
Cultural method: adopt traditional PDA solid culture can obtain a large amount of LB-1 hyphostroma; With glucose or N.F,USP MANNITOL for carbon source, shaking culture 3-4 days under the condition of 23-25 DEG C, can obtain the thalline fermented liquid with fine bacteriostatic action.
The bacteriostatic experiment of embodiment 2 bacterial strain
(1) inhibition of bacterial strain of the present invention to pathogenic Fusarium oxysporum and Alternariaspp is tested.
Adopt dull and stereotyped opposite culture method to screen biocontrol microorganisms, and detect bacteriostasis rate: the LB-1 bacterial strain of the separation and purification of inoculation diameter 6mm respectively and indicator bacterium cake, on the relative position at PDA substratum two ends, are positioned in illumination box and cultivate (25 DEG C, 8h illumination).If the indicator of single culture is contrast.After 3 days, employing right-angled intersection method measures the trend radius (Rp) in the colony radius (Rc) and opposite culture flat board contrasting single culture in flat board, according to following formulae discovery bacteriostasis rate, the biocontrol microorganisms that Analysis and Screening goes out is to the restraining effect (Deng Zhenshan etc., 2009) of indicator: bacteriostasis rate (%)=[(Rc-Rp)]/Rc × 100.The results are shown in
Fig. 3. in figure, the left side is Fusarium oxysporum, and the right is Alternariaspp.
(2) inhibition of bacterial strain fermentation liquor of the present invention to pathogenic Fusarium oxysporum and Alternariaspp is tested.
The acquisition of fermented liquid: biocontrol strain LB-1 is activated on PDA flat board, scraping mycelia, with in the inoculum size access liquid PDA substratum of 0.1%, shaking culture 4d(25 DEG C, rotating speed is 120r/min).The fermentation liquor double gauze cultivated is crossed and is filtered mycelia, and centrifugal 15 minutes of filtrate 10000r/min, the supernatant liquor obtained is fermented liquid.
Fermented liquid bacteriostatic action measures: fermented liquid is divided into two parts, a through high-pressure sterilizing pot sterilizing, the non-sterilizing of portion, then by two portions of fermented liquids with mix with the volume ratio of 1:2 with PDA substratum respectively, the PDA made containing LB-1 fermented liquid is dull and stereotyped, indicator (Alternariaspp and Fusarium oxysporum) the bacterium cake of inoculation diameter 6mm, in dull and stereotyped central authorities, is placed in illumination box and cultivates 3d(25 DEG C, 12h illumination).Contrast is the PDA flat board not containing fermented liquid.According to the colony diameter size of indicator in contrast and process PDA flat board, calculate the bacteriostasis rate of sterilization fermentation liquid.The results are shown in Figure 4.In figure, the left side is Fusarium oxysporum, and the right is Alternariaspp.
Biocontrol strain of the present invention all has good restraining effect to pathogenic Fusarium oxysporum, Alternariaspp, verticillium dahliae etc., wherein 75.3% is reached to the maximum bacteriostasis rate of Fusarium oxysporum, and its cultivation and fermentation liquid can play obvious solvency action to the mycelia of pathogenic bacterium.Meanwhile, because biocontrol microorganisms of the present invention has good resistance to ultraviolet, salt stress and water stress, therefore, be the excellent biocontrol strain that a strain has fine environmental compatibility.Use by modes such as root inoculation, foliage-sprays in production application.
Claims (2)
1. a strain chaetomium, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 19th, 2015, culture presevation registration number is CGMCCNo.11400.
2. the application of chaetomium described in claim 1 on biocontrol of plant disease.
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| CN106754413A (en) * | 2016-12-15 | 2017-05-31 | 河南省农业科学院 | A kind of Chinese toon endogenetic fungus TS4 and its secondary metabolite, preparation method and application |
| CN107318891A (en) * | 2017-01-18 | 2017-11-07 | 东北林业大学 | A kind of wettable powder for preventing and treating root rot |
| CN108504361A (en) * | 2017-02-24 | 2018-09-07 | 深圳市昶裕隆实业有限公司 | A kind of soil reformation method and its transformation agent |
| CN112725188A (en) * | 2019-10-28 | 2021-04-30 | 潍坊学院 | Plant disease biocontrol strain LB-1 and method for obtaining bacteriostatic crude extract thereof |
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| CN107318891A (en) * | 2017-01-18 | 2017-11-07 | 东北林业大学 | A kind of wettable powder for preventing and treating root rot |
| CN108504361A (en) * | 2017-02-24 | 2018-09-07 | 深圳市昶裕隆实业有限公司 | A kind of soil reformation method and its transformation agent |
| CN112725188A (en) * | 2019-10-28 | 2021-04-30 | 潍坊学院 | Plant disease biocontrol strain LB-1 and method for obtaining bacteriostatic crude extract thereof |
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