CN104694397A - Chaetomium globosum and application thereof - Google Patents
Chaetomium globosum and application thereof Download PDFInfo
- Publication number
- CN104694397A CN104694397A CN201410796936.XA CN201410796936A CN104694397A CN 104694397 A CN104694397 A CN 104694397A CN 201410796936 A CN201410796936 A CN 201410796936A CN 104694397 A CN104694397 A CN 104694397A
- Authority
- CN
- China
- Prior art keywords
- fsr
- ginseng
- chaetomium globosum
- brown
- spore
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a chaetomium globosum FSR-74. The collection unit of the chaetomium globosum FSR-74 is China General Microbiological Culture Collection Center, the collection date of the chaetomium globosum FSR-74 is September 19, 2014 and the collection number of the chaetomium globosum FSR-74 is CGMCC No. 9690. The strain is relatively slow in growth and has light brown colonies and an irregular wave-like edge when subjected to plate culture at 25 DEG C in a potato dextrose agar (PDA) culture medium. FSR-74 ascocarps are supergene and fixed on the surface of a matrix by rhizoids; top hair is wavy, brown and has intervals, and dense wart points are arranged on the surface; 8 ascospores are arranged in an ascus; the ascospores are shaped like lemons and have single apical bud holes. The chaetomium globosum FSR-74 has an efficient broad-spectrum bacteriostatic effect against main pathogenic bacteria causing ginseng rust rot, blight, sclerotinia rot, rust rot, blackspot, sheath blight and gray mold. The invention further provides a biological control mechanism and application of the chaetomium globosum FSR-74 in prevention and treatment of plant fungal diseases, as well as application in preparation of a microbial agent for preventing and treating the plant fungal diseases.
Description
Technical field
The present invention relates to strain chaetomium globosum and an application thereof.
Background technology
Ginseng (Panax ginseng C.A. Mey) is the negative dicotyledonous medicinal plant of Araliaceae Panax perennial root, is the rare medicinal herbs of China, has the good reputation of " kings of hundred grass ".Ginseng has establishing in large scale in countries such as China, Korea S, Korea, Russia, and wherein, special Jilin Province, Northeast China is the main producing region of ginseng, has become one of mainstay industry of locality.The long-term artificial growth of ginseng and breed breeding difficulty cause greatly that ginseng germplasm is degenerated, disease is serious, of poor quality, yield poorly.A large amount of uses of chemical pesticide, cause pesticide residue and environmental pollution, reduce security and the commodity value of ginseng crude drug.Disease prevention and control problem has become one of significant problem of restriction ginseng industry Sustainable development.
Ginseng diseases about has 20 ~ 40 kinds, wherein, 7 class pathogenic bacterias are the main pathogenic fungi causing ginseng Common Diseases and limit ginseng industry development below: the general sickness rate of ginseng maize ear rot caused by fusarium solani (Fusarium solani) is about 30%, time serious, 6 years strangers join the mortality ratio that root rot causes and can reach 50% ~ 80%, main harm seedling in spring root and rhizome portion (below earth's surface stem), the ginseng root rotted is chocolate web rot shape, the rotten shape of later stage grain, only deposits the root skin of hollow; When the Ginseng Blight In China that Phytophthora cactorum bacterium (Phytophthora cactorum) causes is serious, sickness rate can reach 70%, its symptom is for blade producing irregular dark green color spot when falling ill light, and severe one cauline leaf is withered, butt rot, plant is withered in flakes, and the underproduction is serious; The ginseng sclerotium disease main harm that Sclerotinia ginseng (Sclerotinia schinseng) causes life in 3 years is above joins root, site of pathological change is bud bud, root welding technology, after ginseng velamen evil, initial stage is raw a little white fluffy mycelium on surface, after, inner corrupt, softening rapidly, cell is all cleared up totally, only leave downright bad exterior skin, the inside and outside sclerotium forming many mouse excrement shapes of epidermis; The general sickness rate 20%-30% of black fleck disease of ginseng that ginseng alternaric bacteria (Alternaria panax) causes, reaches 100% time serious, causes early leaf fall, and plant withers, shaky, ginseng root and the underproduction of ginseng seed; The Ginseng Rhizoctonia Solani that dry thread Pyrenomycetes (Rhizoctonia solani) causes is one of ginseng Major Diseases in seedling stage, under the condition of low temperature high humidity, developmenting spread is very rapid, general sickness rate is 8 ~ 30%, severe patient can reach about 40%, germ make the stem of seedling 3 ~ 5 centimetres of dry wet soil interfaces on ground hang contracting, rot, cut off transfusion tissue, cause seedling to lodge; When the ginseng rust maize ear rot that destruction post spore bacterium (Cylindrocarpon destructans) causes is serious, sickness rate reaches more than 70%, this disease betides each position of root, and scab is rust, diffuses to full root by point to face, large, ventilative bad, the soil ulmin thickness of soil humidity, morbidity is heavy; There is brown spot in the ginseng gray mold their early stage that the pathogen of Botrytis cinerea (Botrytis cinerea Pers.) causes, do not see unusual phenomenon in appearance from ginseng root main root on the brood cell of ginseng root, but when pinching with hand, ginseng root inside organize deliquescing.The morbidity later stage above-mentionedly both shows as soft rotten symptom, and at the mould layer of fine hair shape that sick portion produces grey, sometimes can form the sclerotium of black in old complaint.
For a long time by using the Chemical control methods control ginseng diseases such as chemical pesticide and falling flat, and the excessive use of chemical pesticide not only can spoiled soil micro-ecological environment, weighting ring environment pollution, also make toxic substance accumulate in a large number in ginseng root, reduce safety in utilization and the commodity value of ginseng.Therefore, disease control emphasis progressively turns in biological control and cultural control measure.In crop plants protection field; the healthy tree rhizosphere soil of crop is utilized to screen soil microorganisms pathogenic bacteria to remarkable biocontrol effect; and make one of important means that microbiobacterial agent is biological prevention and control research, be also the important channel of beneficial microorganism resources development and utilization.
Biological control is based on ecological principle, utilizes the interaction between living species, with a kind of or a class biological inhibition another kind of or another kind of biology.The maximum advantage of biological control is free from environmental pollution, does not have pesticide residue, this be the non-biological methods prevention and elimination of disease and pests such as agricultural chemicals incomparable.Utilizing Biocontrol microorganism to prevent and treat pathogenic micro-organism is the important component part of biological prevention.It avoid a large amount of use chemical pesticide to bring a series of plant protection, environment and energy aspect problem, avoid the harm of pesticide residue to people and animals, the more important thing is and facilitate agricultural sustainable development.Chaetomium (Chaetomium sp.) fungi is distributed widely in soil and plant materials.This quasi-microorganism can produce the Multiple Classes of Antibiotics such as chaetocin, ball chaetocin, and the biological control agents as phytopathogen is widely studied.The antagonism chaetomium kind of current research is less, and a strain chaetomium globosum (Chaetomium globosum) has been separated and has obtained and be used to controlling plant diseases in plant materials, and utilizes chaetomium globosum control ginseng fungal disease to have no report.
Summary of the invention
The object of the invention is to: the main fungal venereal disease evil in producing for ginseng, especially ginseng soil-borne disease occurring area expanding day, Agro-chemicals control is except easily causing environmental pollution and pesticide residue, the practical situation that preventive effect is also undesirable, propose a strain from Soil of Ginseng Rhizomsphere, be separated chaetomium globosum (Chaetomium globosum) the FSR-74 bacterial strain that obtains and causing ginseng maize ear rot respectively, rust rot, black spot, damping-off, epidemic disease, the fusarium solani (F. solani) of sclerotium disease and gray mold, destroy post spore bacterium (C. destructans), ginseng alternaric bacteria (A. panax), dry thread Pyrenomycetes (Rh. solani), Phytophthora cactorum bacterium (Ph. cactorum), application in Sclerotinia ginseng (S. schinseng) and the pathogen of Botrytis cinerea (B. cinerea) 7 kinds of pathogenic bacteria prevention and control.
The invention provides a kind of to above-mentioned cause 7 of ginseng maize ear rot, rust rot, black spot, damping-off, epidemic disease, sclerotium disease and gray mold kinds of pathogenic fungies all have the chaetomium globosum of good prevention and control effect (
chaetomium globosum) FSR-74, the preservation name of this bacterial strain is called ball hair shell FSR-74, and Classification And Nomenclature is ball hair shell
chaetomium globosumdepositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on September 19th, 2014, deposit number: CGMCC No.9690, preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
The chaetomium globosum FSR-74 identification of strains that the present invention proposes adopts traditional form authenticate technology and modern rDNA-ITS gene sequence analysis.FSR-74 potato dextrose agar (PDA) dull and stereotyped upper 25 DEG C cultivate time growth comparatively slow, bacterium colony is shallow brown, the brown or brown of olive, tool canescence or olive brown aerial hyphae, back side brown, normal tool olive brown exudate, colony edge often splits shape in irregular ripple.The microstructure of FSR-74 is at 10 × 50 optical microphotograph Microscopic observations, and ascoma table is raw, is bonded to stromal surface with brown rhizoid, and be that olive is green to olive brown under reflected light, obovate is extremely subsphaeroidal, diameter 160 ~ 240 μm, height about 220 ~ 300 μm; Ascoma wall is made up of dun textura intricata; Top setation is a large amount of and intensive, meander-like, wavy or curling slightly in the shape of a spiral, brown, tool every, base portion barrier film is comparatively obvious, not branch, the intensive wart point of surperficial tool, and base portion is wide about 2.5 ~ 4 μm; Side setation meander-like, separates obviously; Ascus club-like, 27 ~ 36 × 11 ~ 15 μm, inside have 8 thecaspores, ascus wall is easily cleared up; Thecaspore lemon shape, two is flat-sided, two ends each tool one apicule, brown time ripe, 8.5 ~ 11 × 7 ~ 8.5 μm, the single leaden hole of tool.Described potato dextrose agar (PDA) formula is: potato 200 g/L, glucose 10 g/L, agar 15 g/L, distilled water 1000 mL, 2 μ g/mL penicillin, pH is 6.8 ~ 7.2.
The rDNA-ITS sequence length of the chaetomium globosum FSR-74 bacterial strain that the present invention proposes is 604 bp, specifically as shown in SEQ ID No:l.The softwares such as application BLAST and DNAMAN are analyzed, and by the ITS sequence of FSR-74 bacterial strain by BLAST comparison, can find the close strain sequence that homology is very high in GenBank.With bacterial strain FSR-74 similarity the highest be Chaetomium globosum, homology reaches 99%.Find with UPGMA method phylogenetic tree construction according to MEGA5.10Beta2 software, FSR-74 and Chaetomium globosum belongs to a hereditary branch together, and sibship is very close, and pro-borne reaches 99%.Combining form credit class and molecular biology identification result, can confirm that bacterial strain FSR-74 of the present invention is chaetomium globosum (Chaetomium globosum).
The present invention relates to the application of chaetomium globosum FSR-74 in control fungal diseases of plants, described plant optimization is ginseng, described fungi preferably causes the fusarium solani (F. solani) of ginseng maize ear rot, cause the destruction post spore bacterium (C. destructans) of ginseng rust maize ear rot, cause the ginseng alternaric bacteria (A. panax) of black fleck disease of ginseng, cause the dry thread Pyrenomycetes (R. solani) of Ginseng Rhizoctonia Solani, cause the Phytophthora cactorum bacterium (P. cactorum) of Ginseng Blight In China, cause the Sclerotinia ginseng of ginseng sclerotium disease (S. schinseng) and cause in these 7 kinds of pathogenic bacterias of the pathogen of Botrytis cinerea (B. cinerea) of ginseng gray mold one or more.
The invention still further relates to the mechanisms of control wheat scab of chaetomium globosum FSR-74 in control fungal diseases of plants, described plant optimization is ginseng, described fungi preferably causes the fusarium solani (F. solani) of ginseng maize ear rot, cause the destruction post spore bacterium (C. destructans) of ginseng rust maize ear rot, cause the ginseng alternaric bacteria (A. panax) of black fleck disease of ginseng, cause the dry thread Pyrenomycetes (R. solani) of Ginseng Rhizoctonia Solani, cause the Phytophthora cactorum bacterium of Ginseng Blight In China (P. cactorum) and cause the pathogen of Botrytis cinerea (B. cinerea) of ginseng gray mold.Chaetomium globosum FSR-74 seizes locus by Competition and nutritive substance effectively suppresses ginseng pathogenic bacteria mycelial growth; Discharge Substance cause pathogenic bacteria mycelia and conidium deformity by hyperparasitism, suppress growth of pathogenic bacteria and spore germination.
The invention still further relates to the microbial preparation of a kind of chaetomium globosum FSR-74, it prepares by following methods:
(1) chaetomium globosum FSR-74 test tube slant kind is activated, get 25 mm pure culture biscuits involvng inoculations in 100mL potato glucose (PDB) liquid nutrient medium bottled with 250 mL triangles, at 170 r/min, at 25 DEG C, cultivate 48 h, obtain seed liquor;
(2) by chaetomium globosum FSR-74 seed liquor with 10%(volume ratio) be inoculated in fermentation culture and cultivate, at 170 r/min, at 25 DEG C, cultivate 96 h, obtain nutrient solution;
(3) filtered through 2 layers of sterile gauze by nutrient solution, filtrate counts through blood counting chamber, spore suspension is dispersed to 6 gL
-1in Xylo-Mucine (CMC) solution, obtain spore suspension, spore content is 2 × 10
5cfumL
-1;
(4) by chaetomium globosum FSR-74 nutrient solution at centrifugal 20 min of 8000 r/mim, filter, supernatant is fermented liquid.
Chaetomium globosum FSR-74 of the present invention and fermented liquid thereof have field diseases prevention and somatotrophic effect to ginseng.
The invention has the advantages that, preserving number be CGMCC No.9690 chaetomium globosum FSR-74 to cause respectively ginseng maize ear rot, rust rot, black spot, damping-off, epidemic disease, sclerotium disease and gray mold fusarium solani (
f. solani), destroy post spore bacterium (
c. destructans), ginseng alternaric bacteria (
a. panax), dry thread Pyrenomycetes (
r. solani), Phytophthora cactorum bacterium (
p. cactorum), Sclerotinia ginseng (
s. schinseng) and the pathogen of Botrytis cinerea (
b. cinerea) etc. 7 kinds of pathogenic bacterias there is prevention effect in various degree, during opposite culture, 46.67% ~ 73.33% is reached to ginseng growth of pathogenic bacteria inhibiting rate, fermented liquid reaches 41.11% ~ 61.45% to ginseng growth of pathogenic bacteria inhibiting rate when cultivating, the fermented liquid of FSR-74 has growth promoting function to ginseng, nontoxic no pathogenicity, to person poultry safety, free from environmental pollution; Simultaneously, biocontrol strain FSR-74 spore suspension is applied directly in soil and carries out filling root to plant or be coated with leaf process playing its antibacterial, germicidal action, can significantly improve Ginseng Rhizosphere biological community structure, form a bio-diversity Soil of Ginseng Rhizomsphere micro-ecological environment, thus effectively control the generation of ginseng fungal disease enduringly.
Accompanying drawing explanation
Fig. 1: chaetomium globosum FSR-74 bacterial strain of the present invention to destruction post spore bacterium (
cylindrocarpon destructans) the dull and stereotyped restraining effect of growth of mycelia.
Fig. 2: chaetomium globosum FSR-74 of the present invention strain to fusarium solani (
fusarium solani) the dull and stereotyped restraining effect of growth of mycelia.
Fig. 3: chaetomium globosum FSR-74 bacterial strain of the present invention to ginseng alternaric bacteria (
alternaria panax) the dull and stereotyped restraining effect of growth of mycelia.
Fig. 4: chaetomium globosum FSR-74 bacterial strain of the present invention to Sclerotinia ginseng (
sclerotinia schinseng) the dull and stereotyped restraining effect of growth of mycelia.
Fig. 5: chaetomium globosum FSR-74 bacterial strain Rhizoctonia solani of the present invention (
rhizoctonia solani) the dull and stereotyped restraining effect of growth of mycelia.
Fig. 6: chaetomium globosum FSR-74 bacterial strain of the present invention to Phytophthora cactorum bacterium (
phytophthora cactorum) the dull and stereotyped restraining effect of growth of mycelia.
Fig. 7: chaetomium globosum FSR-74 bacterial strain of the present invention to the pathogen of Botrytis cinerea (
botrytis cinerea) the dull and stereotyped restraining effect of growth of mycelia.
Fig. 8: chaetomium globosum FSR-74 bacterial strain suppression fusarium solani of the present invention (
fusarium solani) microscopic examination of mycelial growth.
Fig. 9: chaetomium globosum FSR-74 bacterial strain suppression Phytophthora cactorum bacterium of the present invention (
phytophthora cactorum) microscopic examination of mycelial growth.
Figure 10: chaetomium globosum FSR-74 bacterial strain suppression dry thread Pyrenomycetes of the present invention (
rhizoctonia solani) microscopic examination of mycelial growth.
Figure 11: chaetomium globosum FSR-74 bacterial strain suppression destruction post spore bacterium of the present invention (
cylindrocarpon destructans) microscopic examination of mycelial growth.
Figure 12: chaetomium globosum FSR-74 bacterial strain suppression ginseng alternaric bacteria of the present invention (
alternaria panax) microscopic examination of mycelial growth.
Figure 13: chaetomium globosum FSR-74 bacterial strain suppression the pathogen of Botrytis cinerea of the present invention (
botrytis cinerea) microscopic examination of mycelial growth.
Figure 14: chaetomium globosum FSR-74 bacterial strain fermentation liquor of the present invention to fusarium solani (
fusarium solani) restraining effect of mycelial growth.
Figure 15: chaetomium globosum FSR-74 bacterial strain fermentation liquor of the present invention to Phytophthora cactorum bacterium (
phytophthora cactorum) restraining effect of mycelial growth.
Figure 16: chaetomium globosum FSR-74 bacterial strain fermentation liquor of the present invention to ginseng alternaric bacteria (
alternaria panax) restraining effect of mycelial growth.
Figure 17: chaetomium globosum FSR-74 bacterial strain fermentation liquor of the present invention to the pathogen of Botrytis cinerea (
botrytis cinerea) restraining effect of mycelial growth.
Figure 18: chaetomium globosum FSR-74 bacterial strain fermentation liquor suppression destruction post spore bacterium of the present invention (
cylindrocarpon destructans) microscopic examination of mycelial growth.
Figure 19: chaetomium globosum FSR-74 bacterial strain fermentation liquor suppression fusarium solani of the present invention (
fusarium solani) microscopic examination of mycelial growth.
Figure 20: chaetomium globosum FSR-74 bacterial strain fermentation liquor suppression ginseng alternaric bacteria of the present invention (
alternaria panax) microscopic examination of mycelial growth.
Figure 21: chaetomium globosum FSR-74 bacterial strain fermentation liquor suppression Phytophthora cactorum bacterium of the present invention (
phytophthora cactorum) microscopic examination of mycelial growth.
Figure 22: chaetomium globosum FSR-74 bacterial strain fermentation liquor of the present invention to the pathogen of Botrytis cinerea (
botrytis cinerea) microscopic examination of mycelial growth.
Figure 23: chaetomium globosum FSR-74 bacterial strain fermentation liquor suppression ginseng alternaric bacteria of the present invention (
alternaria panax) microscopic examination of spore germination.
Figure 24: chaetomium globosum FSR-74 bacterial strain of the present invention is to the growth promoting function (A: blank solution substratum, B: cast chaetomium globosum FSR-74 fermented liquid) of ginseng.
Figure 25: chaetomium globosum FSR-74 bacterial strain of the present invention is to the prevention effect (A: sterilized water fills with root, B: chaetomium globosum FSR-74 spore suspension liquid irrigating root) of destroying the microbial ginseng rust maize ear rot of post spore.
Figure 26: chaetomium globosum FSR-74 bacterial strain of the present invention is to the prevention effect (A: sterilized water smears blade, B: chaetomium globosum FSR-74 spore suspension smears blade) of the microbial black fleck disease of ginseng of ginseng rod method.
Embodiment
Embodiment 1
The separation of chaetomium globosum (Chaetomium globosum) FSR-74 bacterial strain and preservation
This bacterial strain is separated from the rhizosphere soil of the perennial ginseng healthy tree in Jilin Province's Fusong County elm culture of ginseng ground and obtains.Gather above-mentioned pedotheque, after removing surperficial litter, cross 2 mm sieves.Take fresh soil samples 10 g, put into the triangular flask that granulated glass sphere and 90 mL stroke-physiological saline solution are housed, fully vibration 30 min, make sample mix with sterilized water, obtained soil suspension liquid.Aseptically get 1 mL vibration liquid, add 9 mL stroke-physiological saline solution, make 10 successively by gradient
-2, 10
-3, 10
-4diluent.Drawing each diluent of 100 μ L respectively joins on potato dextrose agar (PDA) flat board, adopts plate dilution method even spread, and often process 3 times and repeat, 7 d cultivated by 25 DEG C of incubators.Picking individual colonies is transferred to after potato dextrose agar (PDA) slat chain conveyor grows bacterium colony, and adopt single spore separation method to carry out separation and purification, purifying bacterial strain is in 4 DEG C of preservations.
Potato dextrose agar (PDA) formula is: potato 200 g/L, glucose 10 g/L, agar 15 g/L, distilled water 1000 mL, 2 μ g/mL penicillin, pH is 6.8 ~ 7.2.
This bacterial strain potato dextrose agar (PDA) dull and stereotyped upper 25 DEG C cultivate time growth comparatively slow, bacterium colony is shallow brown, the brown or brown of olive, tool canescence or olive brown aerial hyphae, back side brown, normal tool olive brown exudate, colony edge often splits shape in irregular ripple.The ascoma table of FSR-74 bacterial strain is raw, is bonded to stromal surface with brown rhizoid, and be that olive is green to olive brown under reflected light, obovate is extremely subsphaeroidal, diameter 160 ~ 240 μm, height about 220 ~ 300 μm; Ascoma wall is made up of dun textura intricata; Top setation is a large amount of and intensive, meander-like, wavy or curling slightly in the shape of a spiral, brown, tool every, base portion barrier film is comparatively obvious, not branch, the intensive wart point of surperficial tool, and base portion is wide about 2.5 ~ 4 μm; Side setation meander-like, separates obviously; Ascus club-like, 27 ~ 36 × 11 ~ 15 μm, inside have 8 thecaspores, ascus wall is easily cleared up; Thecaspore lemon shape, two is flat-sided, two ends each tool one apicule, brown time ripe, 8.5 ~ 11 × 7 ~ 8.5 μm, the single leaden hole of tool.
This bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 19th, 2014, and preserving number is CGMCC No.9690, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Our called after FSR-74 of this bacterial strain, Classification And Nomenclature is: chaetomium globosum Chaetomium globosum.
Embodiment 2
Chaetomium globosum FSR-74 bacterial strain is to the restraining effect of ginseng fungal disease growth of pathogenic bacteria
Adopt 3 cups and dishes face-offs, growth rate method measures bacterial strain FSR-74 to the restraining effect of ginseng 7 kinds of fungal disease pathogenic bacterias: the bacterium cake with the punch tool of diameter 5mm, the ginseng pathogenic bacteria bacterium colony activated being made corresponding size, aseptic inoculation is to the centre of potato dextrose agar (PDA) dull and stereotyped (diameter 90 mm), the bacterium cake of a bacterium to be measured is respectively being accessed apart from 3 sides at pathogenic bacteria 2.5 cm place, another 1 side is blank, as treatment group, each process repeats 3 times; Separately one piece of PDA dull and stereotyped only inoculation ginseng pathogenic bacteria, as a control group, be all placed in 25 DEG C of incubators and cultivate 7 d, treat that control group pathogenic bacteria bacterium colony covers with flat board, measurement processing group pathogenic bacteria colony diameter (unit: mm), and according to following formulae discovery bacteriostasis rate.Often kind of pathogenic bacteria repeats 3 times, results averaged.Bacteriostasis rate (%)=(contrast pathogenic bacteria bacterium colony expansion radius-expand radius with the pathogenic bacteria bacterium colony of biocontrol fungi opposite culture)/contrast pathogenic bacteria bacterium colony expands radius × 100%
Potato dextrose agar (PDA) formula is: potato 200 g/L, glucose 10 g/L, agar 15 g/L, distilled water 1000 mL, 2 μ g/mL penicillin, pH is 6.8 ~ 7.2.
Result is as shown in table 1, and bacterial strain FSR-74 all has obvious restraining effect to causing the fusarium solani of ginseng maize ear rot, rust rot, black spot, damping-off, epidemic disease, sclerotium disease and gray mold (F. solani), destruction post spore bacterium (C. destructans), ginseng alternaric bacteria (A. panax), dry thread Pyrenomycetes (R. solani), Phytophthora cactorum bacterium (P. cactorum), Sclerotinia ginseng (S. schinseng) and the pathogen of Botrytis cinerea (B. cinerea) 7 kinds of pathogenic bacterias respectively.Particularly 67.48% is reached to the bacteriostasis rate of the fusarium solani causing ginseng maize ear rot, to causing the dry thread Pyrenomycetes of Ginseng Rhizoctonia Solani and causing the bacteriostasis rate of the Sclerotinia ginseng of ginseng sclerotium disease to be about 62%, be 50% ~ 53% to the bacteriostasis rate of the pathogen of Botrytis cinerea of the destruction post spore bacterium and ginseng gray mold that cause ginseng rust maize ear rot, also 46.67% is reached to the bacteriostasis rate of the Phytophthora cactorum bacterium of the ginseng rod method and Ginseng Blight In China that cause black spot, reflects that the preventive and therapeutic effect of bacterial strain FSR-74 to ginseng 7 kinds of main pathogen fungies has broad spectrum (table 1).
Table 1 chaetomium globosum FSR-74 is to the restraining effect (%) of ginseng pathogenic fungi
Embodiment 3 chaetomium globosum FSR-74 nutrient solution, the preparation of spore suspension and zymocyte liquid
Chaetomium globosum FSR-74 test tube slant kind is activated, gets 25 mm pure culture biscuits involvng inoculations in the 100 mL potato dextrose broth (PDB) bottled with 250 mL triangles, at 170 r/min, at 25 DEG C, cultivate 48 h, obtain seed liquor; By chaetomium globosum FSR-74 seed liquor with 10%(volume ratio) be inoculated in fermentation culture and cultivate, at 170 r/min, at 25 DEG C, cultivate 96 h, obtain nutrient solution; Filtered through 2 layers of sterile gauze by chaetomium globosum FSR-74 nutrient solution, filtrate counts through blood counting chamber, spore suspension is dispersed to 6 gL
-1in Xylo-Mucine (CMC) solution, obtain spore suspension, spore content is 2 × 10
5cfumL
-1; By chaetomium globosum FSR-74 strain cultured solution at centrifugal 20 min of 8000 r/mim, supernatant is fermented liquid; Fermentation liquor 0.45 μm of filtering with microporous membrane, obtains chaetomium globosum FSR-74 without fermented liquid.
The preparation method of potato dextrose broth (PDB): potato 200 g/L, glucose 10 g/L, distilled water 1000 mL, pH are 6.8 ~ 7.2.In 1000 mL triangular flasks, loading amount is 300 mL nutrient solutions, seals triangle bottleneck with double-deck sealed membrane, 121 DEG C of moist heat sterilization 30 min, for subsequent use after cooling.
Embodiment 4
Chaetomium globosum FSR-74 bacterial strain fermentation liquor is to the restraining effect of ginseng pathogenic bacteria
Toxic medium is adopted to cultivate, growth rate method measures chaetomium globosum FSR-74 fermented liquid to the restraining effect of ginseng fungal disease germ: prepare chaetomium globosum FSR-74 bacterial strain without fermented liquid by embodiment 3, FSR-74 bacterial strain is mixed in 1:4 ratio with potato dextrose agar (PDA) without fermented liquid, be mixed with pastille substratum, then ginseng pathogenic bacteria bacterium cake is placed in pastille substratum central authorities, compare to mix sterilized water, each process repeats for 5 times, constant temperature culture at 25 DEG C, measures colony radius after 6 d and calculates bacteriostasis rate.Method of calculation are with embodiment 2.
Result is as shown in table 2, chaetomium globosum FSR-74 fermented liquid to causing the fusarium solani (F. solani) of ginseng maize ear rot, rust rot, black spot, damping-off, epidemic disease and gray mold respectively, destroy post spore bacterium (C. destructans), ginseng alternaric bacteria (A. panax), dry thread Pyrenomycetes (R. solani), Phytophthora cactorum bacterium (P. cactorum) and the pathogen of Botrytis cinerea (B. cinerea) 6 kinds of pathogenic bacterias generations restraining effect in various degree.Wherein, 61.45% is reached to causing the destruction post spore bacterium bacteriostasis rate of ginseng rust maize ear rot, the bacteriostasis rate of Rhizoctonia solani and Phytophthora cactorum bacterium is about 50%, minimum to the inhibiting rate of ginseng alternaric bacteria and the pathogen of Botrytis cinerea, be about 41% ~ 44%, illustrate that the control of chaetomium globosum FSR-74 fermented liquid to 6 kinds of ginseng pathogenic bacterias has broad spectrum (table 2, Fig. 8-16).
Table 2 chaetomium globosum FSR-74 fermented liquid is to the bacteriostatic action (%) of ginseng pathogenic fungi
Embodiment 5 chaetomium globosum FSR-74 bacterial strain suppresses the Biocontrol Mechanism of ginseng fungal disease growth of pathogenic bacteria
Opposite culture method is adopted to study the Biocontrol Mechanism of many chaetomium globosums FSR-74 control ginseng fungal disease pathogenic bacteria: respectively to access 15 mm chaetomium globosum FSR-74 bacterium cake and ginseng pathogenic bacteria bacterium cake at diameter 9 cm potato dextrose agar (PDA) dull and stereotyped relative position respectively, two vaccinations are at a distance of 3 cm, be inoculated in separately potato dextrose agar (PDA) with ginseng pathogenic bacteria or biocontrol fungi to compare, each process repeats for 5 times, constant temperature culture at 25 DEG C, in opposite culture process, when ginseng pathogenic bacteria mycelia front end occurs suppressing phenomenon, cut mycelia on antagonism band, adopt Keyemce to surpass depth of field 3 D stereo microscope (VHX-600) and observe hypha form and the situation that influences each other.
Result shows, chaetomium globosum FSR-74 has certain restraining effect to ginseng pathogenic bacteria mycelial growth.On potato dextrose agar (PDA) substratum, chaetomium globosum FSR-74 and fusarium solani, the pathogen of Botrytis cinerea and the ginseng alternaric bacteria speed of growth suitable; During opposite culture, chaetomium globosum FSR-74 is close to growth of pathogenic bacteria, and form obvious antagonism band in bacterium colony joint, thin and have intertwinement near the pathogenic bacteria mycelia at chaetomium globosum FSR-74 edge, mycelium darkens with yellow or tawny secretory substance generation, and more shallow apart from the colony colour that chaetomium globosum FSR-74 is far away, show to interact two bacterial strain antagonism growths; Chaetomium globosum FSR-74 is than the dry thread Pyrenomycetes poor growth causing Ginseng Rhizoctonia Solani, with opposite culture time lengthening, mycelium near chaetomium globosum FSR-74 edge is thicker, darken, opposite culture later stage dry thread Pyrenomycetes edge produces aerial hyphae hardly, and and between chaetomium globosum FSR-74, form obvious antagonism band; Chaetomium globosum FSR-74 is than Phytophthora cactorum bacterium, Sclerotinia ginseng is fast with destruction post spore bacteria growing speed, obvious restraining effect is produced to 3 kinds of pathogenic bacterias, show that pathogenic bacteria edge produces aerial hyphae hardly and produces brown secretory product in bacterium colony junction, opposite culture later stage pathogenic bacteria bacterium colony is besieged, bacterium colony is atrophy gradually, illustrates that bacterial strain FSR-74 has stronger competitive edge (Fig. 1-7) thus.
Microscopic examination finds, very fast and the even thickness of the normal bacteria mycelial growth of ginseng pathogenic fungi, mycelia thickness and intensive, under chaetomium globosum FSR-74 effect, ginseng pathogenic bacteria mycelium branch around antagonism band increases, and mycelia top and bifurcation expand deformity, growth retardation, protoplasma condenses, and occurs mycelia distortion, phenomenon of rupture.In addition, picking opposite culture bacterium colony junction mycelia microscopy finds, chaetomium globosum FSR-74 mycelia, with parallel, be wound around the modes such as ginseng pathogenic bacteria mycelia and produce antagonistic action (Fig. 8-13) to it.
embodiment 6 chaetomium globosum FSR-74 bacterial strain fermentation liquor suppresses the Biocontrol Mechanism of ginseng fungal disease growth of pathogenic bacteria
Toxic medium is adopted to cultivate, growth rate method measures chaetomium globosum FSR-74 fermented liquid to the impact of ginseng fungal disease growth of pathogenic bacteria: obtain chaetomium globosum FSR-74 fermented liquid by embodiment 3 method, fermentation liquor 0.45 μm of filtering with microporous membrane, obtains chaetomium globosum FSR-74 without fermented liquid.Chaetomium globosum FSR-74 bacterial strain is mixed in 1:4 ratio with potato dextrose agar (PDA) without fermented liquid, be mixed with pastille substratum, cultivate ginseng pathogenic bacteria by embodiment 4 method, adopt Keyemce to surpass depth of field 3 D stereo microscope (VHX-600) after 6 d and observe hypha form.
Result shows, the speed of growth of ginseng pathogenic fungi mycelium on pastille substratum is significantly lower than potato dextrose agar (PDA) substratum, subiculum is thin, mycelium thickness is uneven with intertwinement, growth of pathogenic bacteria is suppressed, and ginseng alternaric bacteria and the pathogen of Botrytis cinerea discharge brown antagonistic substance in process of growth.There is active antibacterial material in chaetomium globosum FSR-74 fermented liquid, strong impact (Figure 14-17) can be produced on ginseng pathogenic bacteria hypha form.
Examine under a microscope, ginseng pathogenic bacteria mycelia enlarges into beading, short and thick, is gathered into Cong Zhizhuan, and some mycelia top or local deformity, break after forming the bubble that expands, intracellular organic matter is to external leakage.Substance in chaetomium globosum FSR-74 fermented liquid can cause protoplasma in ginseng pathogenic fungi mycelia to concentrate, and mycelia deformity, simultaneously by destroying hyphal cell wall, makes intracellular organic matter exosmose, and causes mycelia to rupture (Figure 18-22).
embodiment 7 chaetomium globosum FSR-74 bacterial strain fermentation liquor is on the impact of ginseng pathogenic bacteria conidia germination
Adopt carrier to sprout method and measure chaetomium globosum FSR-74 fermented liquid to the impact of ginseng fungal disease pathogenic bacteria spore: by ginseng alternaric bacteria (
a. panax), destroy post spore bacterium (
c. destructans), fusarium solani (
f. solani), Botrytis cinerea (
b. cinerea) spore prepare spore suspension (lower 100 the spore/visuals field of 100 times, microscope) 0.1 mL in clean 1.5 aseptic mL centrifuge tubes, chaetomium globosum FSR-74 bacterial strain is added without fermented liquid with doubling dilution, to drip 0.1 mL potato dextrose broth (PDB) for contrast, 25 DEG C of cultivations, the half of conidiospore diameter is exceeded as the standard sprouted using germ tube length, at the orthodontic condition of process 6 h, 12 h, 24 h and 48 h microscopy spore germination situation and sprouting spore germ tube on slide glass, and calculate Germination suppression rate.
Result is as shown in table 3, and chaetomium globosum FSR-74 is without fermented liquid to ginseng alternaric bacteria, and destroy post spore bacterium, the spore germination of fusarium solani and the pathogen of Botrytis cinerea all has restraining effect in various degree.25 DEG C of cultivation 6 h ginseng pathogenic fungi spore germination rates are low, and fusarium solani spore germination rate only has 11%, and other pathogenic bacteria spore germination rates are close to 0.With process time lengthening, spore germination rate increases gradually, and during 48 h, FSR-74 fermented liquid is to destruction post spore bacterium and the pathogen of Botrytis cinerea inhibition of germination maximum (about 55%), is about 45% to ginseng alternaric bacteria and fusarium solani inhibition of germination.From accompanying drawing 23, chaetomium globosum FSR-74 fermented liquid can cause the spore germination of ginseng pathogenic fungi to be obstructed, spore deformity, germ tube front end is expanded and is occurred lopsided foaming material, the mycelia that germ tube grows can not extending forwards, thus making spore lose invasive ability, chaetomium globosum FSR-74 may contain Substance without in fermented liquid.
Table 3 FSR-74 fermented liquid is on the impact of common ginseng pathogenic bacteria spore germination
embodiment 8 chaetomium globosum FSR-74 grows test surely
(1) chaetomium globosum FSR-74 determining in soil is grown
Adopt and mix native inoculation method and measure chaetomium globosum FSR-74 determine the amount of growing in soil, potato glucose (PDB) nutrient solution is inoculated in by with Rifampin (300 μ g/mL) labeled chaetomium globosum FSR-74,25 DEG C, 170 r/min, shaking table vibrates 6 d, obtain chaetomium globosum FSR-74 mutant strain nutrient solution, obtain chaetomium globosum FSR-74 mutant strain spore suspension by embodiment 3, and be diluted to 1 × 10
5cfu/mL, 4 DEG C of Refrigerator stores, for subsequent use.Nature soil (is not planted the new timbered soil of ginseng, use the impurity such as front Litter removal, according to volume ratio, new forest land soil is mixed in 2:1 ratio with vermiculite) to load diameter be 20 cm flowerpots, every basin fills native 1 kg, and the chaetomium globosum FSR-74 mutant strain spore suspension injecting 100 mL labeled in soil mixes soil.Ambient temperatare is put, and (soil first, after gradient dilution, gets 10 to the fungi be separated in 1 soil every 7 d
-2, 10
-3, 10
-4soil dilution liquid carry out flat board coating), calculate bacteria containing amount.
Result shows, the determining the amount of growing and can reach every gram of soil 2.73 × 10 of chaetomium globosum FSR-74 in natural soil after 35 d
4more than cfu/g.This illustrates that chaetomium globosum FSR-74 has stronger colonization ability in soil.
(2) chaetomium globosum FSR-74 ginseng cauline leaf determine grow
Adopt and fill with root inoculation method, the spore suspension preparation of chaetomium globosum FSR-74 mutant strain is with embodiment 3.Nature soil (is not planted the new timbered soil of ginseng, use the impurity such as front Litter removal, according to volume ratio, new forest land soil is mixed in 2:1 ratio with vermiculite) to load diameter be 20 cm flowerpots, every basin fills native 1 kg, in the same size and well-grown annual ginseng seedling is selected to transplant, the strain of every basin 4, each process repeats 6 basins.After transplanting 30 d, inject the spore suspension (every strain 25 mL) of mutant strain along the basal part of stem of ginseng-leaf to its root soil, field management is produced with conventional.Whole strain ginseng is gathered after inoculating 7 d, the root of ginseng, stem, leaf are carried out surface sterilization (with 50% alcohol immersion process 5 min), be placed on 10 min in the ethanolic soln of 75%, the chlorine bleach liquor putting into 1% again soaks 5 min, then uses aseptic water washing 5 times PDA flat board (be applied to by the sterilized water 100 μ L of last 1 flushing material detect surface sterilization whether thorough); Sterilizable material is got 1 g sterilizable material and is placed in sterile mortar after aseptic thieving paper blots, and adds 10 mL sterilized waters and grinds; Leave standstill 30 min after grinding to form homogenate, supernatant liquor is for subsequent use as leach liquor, and its concentration is 0.1 g/mL.Get on PDA flat board that 100 μ L supernatant liquors coat containing 300 μ g/mL Rifampins, often process repetition 3 times; Flat-plate inverted is placed in 25 DEG C of thermostat containers and cultivates 7 d, detects the growth whether having bacterium to be measured.Result shows, can be recycled to chaetomium globosum FSR-74 during 28 d at ginseng root.This illustrate chaetomium globosum FSR-74 can in ginseng body the long period exist, have certain endogeny, application potential is better.
embodiment 9 chaetomium globosum FSR-74 bacterial strain is to the growth-promoting functions of ginseng
Pot experiment method is adopted to measure chaetomium globosum FSR-74 bacterial strain to the growth promoting function of ginseng: to obtain chaetomium globosum FSR-74 nutrient solution by embodiment 4 method.It is 20 cm flowerpots that nature soil (do not plant the new timbered soil of ginseng, use the impurity such as front Litter removal, mixed by new forest land soil according to volume ratio with vermiculite in 2:1 ratio) is loaded diameter, and every basin fills native 1 kg.In the same size and well-grown annual ginseng seedling is selected to transplant, the strain of every basin 4.With chaetomium globosum FSR-74 fermented liquid, (bacteria containing amount is about 2 × 10 to test group
5cfumL
-1) 50 times of sterilized water diluent 30 mL fill with roots, control group 50 times of sterilized water diluent 30 mL of sterile potato glucose (PDB) nutrient solution fill with roots, often process 6 basins, random alignment, and field management is produced with conventional.Grow to after 30 d until ginseng-leaf, random selecting treatment group and each 5 strains of control group ginseng-leaf, carefully dig out whole plant, wash away root earth respectively, measures its plant height, root length, whole strain fresh weight and root fresh weight index.Then dry to constant weight for 108 DEG C, survey whole strain dry weight and root dry weight.
Results from pot experiment test shows (table 4), ginseng is planted after inoculation chaetomium globosum FSR-74, its plant plant height, whole strain fresh weight, root fresh weight, root length, whole strain dry weight and root dry weight all have increase in various degree compared with control group, wherein, after chaetomium globosum FSR-74 process, ginseng terrestrial stem leaf part dry weight and fresh weight of plant seedlings comparatively contrasts increase by 7% ~ 16%, the dry weight and fresh weight of plant seedlings of ginseng comparatively control treatment significantly increases by 33% ~ 38%, proves that chaetomium globosum FSR-74 has significant promoter action (accompanying drawing 24) to ginseng growth.Meanwhile, also illustrate that chaetomium globosum FSR-74 is safe to ginseng.
Table 4 chaetomium globosum FSR-74 is to the promoter action of Ginseng Growth
embodiment 10 chaetomium globosum FSR-74 bacterial strain is to the field controling test of ginseng fungal disease
To cause ginseng rust maize ear rot destruction post spore bacterium (
cylindrocarpon destructans) controlling experiment be arranged in Jilin Agriculture University medicinal garden ginseng experiment field in 2014 and carry out pot experiment.It is 20 cm flowerpots that nature soil (do not plant the new timbered soil of ginseng, use the impurity such as front Litter removal, mixed by new forest land soil according to volume ratio with vermiculite in 2:1 ratio) is loaded diameter, and every basin fills native 1 kg.Ginseng was transplanted April 17, selected in the same size and well-grown annual ginseng seedling to transplant, the strain of every basin 4.After transplanting 30 d, adopting and hinder root perfusion Simultaneous vaccination destruction post spore bacterium and chaetomium globosum FSR-74 spore suspension, is 2 × 10 containing spore amount
5cfumL
-1, spore suspension liquid and preparation method thereof is with embodiment 3, and each strain inoculum size of the every basin of each bacterial strain is 15 mL.Contrasting for medicament with 500 of 50% carbendazol wettable powder times of sterilized water diluents, take sterilized water as blank.Often process 6 basins, random alignment, irrigation and fertilization measurement management is produced with conventional.Add up occurring degree after inoculating 35 d, investigation disease index, investigation result is as table 5.
To cause black fleck disease of ginseng ginseng alternaric bacteria (
alternaria panax) controlling experiment be arranged in Jilin Agriculture University medicinal garden ginseng experiment field in 2014 and carry out.It is 20 cm flowerpots that nature soil (do not plant the new timbered soil of ginseng, use the impurity such as front Litter removal, mixed by new forest land soil according to volume ratio with vermiculite in 2:1 ratio) is loaded diameter, and every basin fills native 1 kg.Ginseng was transplanted April 17, selected in the same size and well-grown annual ginseng seedling to transplant, the strain of every basin 4.After transplanting 30 d, adopting acupuncture streak method Simultaneous vaccination ginseng alternaric bacteria and chaetomium globosum FSR-74 spore suspension, is 2 × 10 containing spore amount
5cfumL
-1, spore suspension liquid and preparation method thereof is with embodiment 3, and each strain inoculum size of the every basin of each bacterial strain is 15 mL.Contrasting for medicament with 500 of 50% carbendazol wettable powder times of sterilized water diluents, take sterilized water as blank.Often process 6 basins, random alignment, irrigation and fertilization measurement management is produced with conventional.Add up occurring degree after inoculating 35 d, investigation disease index, investigation result is as table 5.
Note: efficiency test method of calculation: disease index=[∑ (sick level strain number × typical value)/(total strain number × the highest sick level typical value)] × 100, relative control effect (%)=(contrast disease index-process disease index)/contrast disease index × 100
As shown in table 5, chaetomium globosum FSR-74 is to by destruction post spore microbial ginseng rust maize ear rot and have good preventive effect by the microbial black fleck disease of ginseng of ginseng rod method, and prevention effect is equal to or slightly better (accompanying drawing 25,26) with the primary medicament that contrasts of above-mentioned disease.
Table 5 chaetomium globosum FSR-74 is to the potted plant controlling experiment of above-mentioned ginseng fungal disease
the Molecular Identification of embodiment 11 FSR-74 bacterial strain
The extraction of ginseng biocontrol fungi genomic dna is carried out in application TaKaRa Universal Genomic DNA Extraction Kit Ver.3.0 test kit (precious biotechnology (Dalian) company limited, TaKaRa Code:DV811A).The ITS universal primer sequence synthesizing fungi rDNA is voluntarily ITS4:5'-TCCTCCGCTTATTGATATGC-3', ITS5:5'-GGAAGTAAAAGTCGTA ACAAGG-3', with FSR-74 phage gene group DNA for template, carries out rDNA-ITS-PCR amplification.Reaction system (20 μ L) is as follows: 2 × PCR Mix 10 μ L, ddH
2o 7.8 μ L, DNA Template 1 μ L, ITS4 0.6 μ L, ITS5 0.6 μ L.Pcr amplification program: 94 DEG C of sex change 3 min, 94 DEG C of sex change 30 s, 56 DEG C of renaturation 30 s, 72 DEG C extend 30 s, 30 circulations, and 72 DEG C extend 10 min.Pcr amplification product detects through 1% agarose gel electrophoresis, obtains the specific fragment of about 750 bp, measures this fragment sequence (adopting TIANGEN gel to reclaim test kit to check order in Sangon Biotech (Shanghai) Co., Ltd.).Result shows, the DNA of FSR-74 bacterial strain is 604 bp, specifically as shown in SEQ ID No:1.The ITS sequence application BLAST software recorded and DNAMAN software and Clustal-X are spliced software analyze, find the highlyest with FSR-74 bacterial strain similarity be
chaetomium globosum, homology reaches 99%.Utilize MEGA5.10Beta2 software with 1000 stochastic samplings of UPGMA method phylogenetic tree construction, calculate bootstrap value (Bootstrap), according to MEGA5.10Beta2 software with UPGMA method phylogenetic tree construction find, FSR-74 with
chaetomium globosumbelong to a hereditary branch together, sibship is very close, and pro-borne reaches 99%.In conjunction with traditional form credit class and modern molecular biology qualification result, can confirm bacterial strain FSR-74 of the present invention be chaetomium globosum (
chaetomium globosum).
Each case study on implementation is not to concrete restriction of the present invention above; as long as according to the scope that claim limits, under the enlightenment of this patent, in conjunction with the basic general knowledge of this area; described bacterial strain is used for, in the control of ginseng fungal disease, all belong to protection scope of the present invention.
Sequence table
SEQ ID No:l
TTGTAAGTAAAATGTCGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGATCATTACAGAGTTGCAAAACTCCCTAAACCATTGTGAACGTTACCTATACCGTTGCTTCGGCGGGCGGCCCCGGGGTTTACCCCCCGGGCGCCCCTGGGCCCCACCGCGGGCGCCCGCCGGAGGTCACCAAACTCTTGATAATTTATGGCCTCTCTGAGTCTTCTGTACTGAATAAGTCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCATCAAGCCCCCGGGCTTGTGTTGGGGACCTGCGGCTGCCGCAGGCCCTGAAAAGCAGTGGCGGGCTCGCTGTCGCACCGAGCGTAGTAGCATACATCTCGCTCTGGTCGCGCCGCGGGTTCCGGCCGTTAAACCACCTTTTAACCCAAGGTTGACCTCGGATCAGGTAGGAAGACCCGCTGAACTTAAGCATATCAATAACCCGGAGGAA
Claims (13)
1. a strain chaetomium globosum (
chaetomium globosum) FSR-74, it is characterized in that, its preserving number is CGMCC No.9690.
2. chaetomium globosum FSR-74 as claimed in claim 1, it is characterized in that, when potato dextrose agar (PDA) dull and stereotyped upper 25 DEG C of cultivations, growth is slower, bacterium colony is shallow brown, brown or the brown of olive, tool canescence or olive brown aerial hyphae, back side brown, normal tool olive brown exudate, colony edge often splits shape in irregular ripple; The microstructure of FSR-74 is at 10 × 50 optical microphotograph Microscopic observations, and ascoma table is raw, is bonded to stromal surface with brown rhizoid, and be that olive is green to olive brown under reflected light, obovate is extremely subsphaeroidal, diameter 160 ~ 240 μm, height about 220 ~ 300 μm; Ascoma wall is made up of dun textura intricata; Top setation is a large amount of and intensive, meander-like, wavy or curling slightly in the shape of a spiral, brown, tool every, base portion barrier film is comparatively obvious, not branch, the intensive wart point of surperficial tool, and base portion is wide about 2.5 ~ 4 μm; Side setation meander-like, separates obviously; Ascus club-like, 27 ~ 36 × 11 ~ 15 μm, inside have 8 thecaspores, ascus wall is easily cleared up; Thecaspore lemon shape, two is flat-sided, two ends each tool one apicule, brown time ripe, 8.5 ~ 11 × 7 ~ 8.5 μm, the single leaden hole of tool; Potato dextrose agar (PDA) formula is: potato 200 g/L, glucose 10 g/L, agar 15 g/L, distilled water 1000 mL, 2 μ g/mL penicillin, pH is 6.8 ~ 7.2.
3. chaetomium globosum FSR-74 as claimed in claim 1 or 2 is in the Biocontrol Mechanism of preventing and treating in fungal diseases of plants and application.
4. apply as claimed in claim 3, it is characterized in that, described plant is ginseng.
5. the application as described in claim 3 or 4, is characterized in that, described fungi be fusarium solani (
fusarium solani), destroy post spore bacterium (
cylindrocarpon destructans), ginseng alternaric bacteria (
alternaria panax), dry thread Pyrenomycetes (
rhizoctonia solani), Phytophthora cactorum bacterium (
phytophthora cactorum), the pathogen of Botrytis cinerea (
botrytis cinereapers.) and Sclerotinia ginseng (
sclerotinia schinseng) one or more in 7 kinds of pathogenic bacterias.
6. the application as described in claim 3-5, is characterized in that, chaetomium globosum FSR-74 is by competition, superparasitism, generation antibacterial substance and reach antibacterial object to the effect of ginseng generation disease prevention growth-promoting.
7. a microbial preparation, is characterized in that, the full nutrient solution containing chaetomium globosum FSR-74 spore and culture.
8. microbial preparation as claimed in claim 7, it is characterized in that, it is prepared by following preparation method:
(1) chaetomium globosum FSR-74 test tube slant kind is activated, get 25 mm pure culture biscuits involvng inoculations in 100 mL potato glucose (PDB) liquid nutrient mediums bottled with 250 mL triangles, at 170 r/min, at 25 DEG C, cultivate 48 h, obtain seed liquor;
(2) by chaetomium globosum FSR-74 seed liquor with 10%(volume ratio) be inoculated in fermentation culture and cultivate, at 170 r/min, at 25 DEG C, cultivate 96 h, obtain nutrient solution;
(3) filtered through 2 layers of sterile gauze by nutrient solution, filtrate counts through blood counting chamber, spore suspension is dispersed to 6 gL
-1in Xylo-Mucine (CMC) solution, obtain spore suspension, spore content is 2 × 10
5cfumL
-1;
(4) by chaetomium globosum FSR-74 nutrient solution at centrifugal 20 min of 8000 r/mim, filter, obtain fermented liquid.
9. the microbial preparation as described in claim 7-8, is characterized in that, the formula of described potato glucose (PDB) nutrient solution is: potato 200 g/L, glucose 10 g/L, distilled water 1000 mL, pH are 6.8 ~ 7.2,121 DEG C of moist heat sterilization 30 min, for subsequent use after cooling.
10. the microbial preparation as described in claim 7-9, is characterized in that,
Described PDB nutrient solution is preparation like this: put into 1000 mL water after taking 200 g potato strippings and slicings, boil to potato block deliquescing, filter to get filtrate, add 10 g glucose in filtrate fully to mix, constant volume, in 1000 mL triangular flasks, loading amount is 300 mL nutrient solutions, seals triangle bottleneck with double-deck sealed membrane, 121 DEG C of moist heat sterilization 30 min, for inoculation after cooling.
The application of 11. microbial preparations according to any one of claim 7-10 in control ginseng fungal disease.
12. apply as claimed in claim 11, it is characterized in that, described fungi for destroy post spore bacterium (
cylindrocarpon destructans) and ginseng alternaric bacteria (
alternaria panax) in one or more.
13. apply as claimed in claim 12, it is characterized in that, at ginseng fungal disease their early stage, be applied to by the diluent of described microbial preparation in soil equably or be sprayed on plant stem-leaf portion, in described diluent, microbial preparation spore content is 2 × 10
5cfumL
-1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410796936.XA CN104694397B (en) | 2014-12-22 | 2014-12-22 | One plant of chaetomium globosum and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410796936.XA CN104694397B (en) | 2014-12-22 | 2014-12-22 | One plant of chaetomium globosum and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104694397A true CN104694397A (en) | 2015-06-10 |
| CN104694397B CN104694397B (en) | 2017-12-01 |
Family
ID=53341957
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410796936.XA Active CN104694397B (en) | 2014-12-22 | 2014-12-22 | One plant of chaetomium globosum and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104694397B (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105524841A (en) * | 2015-11-09 | 2016-04-27 | 潍坊学院 | Broad-spectrum chaetomium strain applied to biological control of plant diseases and applications of broad-spectrum chaetomium strain |
| CN106754396A (en) * | 2016-11-16 | 2017-05-31 | 河南省农业科学院 | A kind of Chinese toon endogenetic fungus TS8 and its secondary metabolite, preparation method and application |
| CN106962115A (en) * | 2017-04-05 | 2017-07-21 | 周兴虎 | A kind of method for improving willow resistance against diseases |
| 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 |
| CN107475123A (en) * | 2017-08-16 | 2017-12-15 | 中国人民解放军第二军医大学 | Roxburgh anoectochilus terminal bud endogenetic fungus and its application |
| CN110257262A (en) * | 2019-07-11 | 2019-09-20 | 江苏中科亚农生物工程有限公司 | A method of preparing microbe fertilizer microbial inoculum |
| CN110551637A (en) * | 2019-09-03 | 2019-12-10 | 黑龙江省农业科学院植物保护研究所 | Echinospora echinocandii from radix astragali root for efficiently inhibiting botrytis cinerea and application thereof |
| CN111096222A (en) * | 2019-12-26 | 2020-05-05 | 天津大学 | Method for improving content of ginsenoside in adventitious roots of ginseng by using immobilized chaetomium globosum spores |
| CN111869682A (en) * | 2020-08-18 | 2020-11-03 | 河南省农业科学院植物保护研究所 | Application of chaetomium globosum in preventing and treating wheat take-all |
| CN111944699A (en) * | 2020-07-27 | 2020-11-17 | 中国科学院成都生物研究所 | Chaetomium cupreum microbial inoculum and preparation method thereof |
| CN113234602A (en) * | 2021-04-01 | 2021-08-10 | 慕恩（广州）生物科技有限公司 | Chaetomium globosum, microbial inoculum, seed soaking liquid and application |
| CN113913300A (en) * | 2021-11-11 | 2022-01-11 | 河南省农业科学院植物保护研究所 | Method for producing chaetomium globosum spore liquid by straw fermentation and application |
| CN116240113A (en) * | 2023-02-08 | 2023-06-09 | 江苏农林职业技术学院 | Biocontrol bacterium JSNL-B47 for inhibiting strawberry anthracnose and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311925A (en) * | 2011-08-03 | 2012-01-11 | 山东农业大学 | Endophytic fungi chaetomium globosum strain, microbial agent and application thereof |
| CN102742605A (en) * | 2012-06-06 | 2012-10-24 | 吉林大学 | Application of gingko endogeny Chaetomium globosum in prevention and control of plant pathogenic fungi |
-
2014
- 2014-12-22 CN CN201410796936.XA patent/CN104694397B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311925A (en) * | 2011-08-03 | 2012-01-11 | 山东农业大学 | Endophytic fungi chaetomium globosum strain, microbial agent and application thereof |
| CN102742605A (en) * | 2012-06-06 | 2012-10-24 | 吉林大学 | Application of gingko endogeny Chaetomium globosum in prevention and control of plant pathogenic fungi |
Non-Patent Citations (3)
| Title |
|---|
| 岳会敏等: "两种毛壳茵对几种植物病原菌的生防效果分析", 《哈尔滨商业大学学报(自然科学版)》 * |
| 梁炜薇等: "人参内生球毛壳菌株ＲSQMK-9 的化学成分研究", 《天然产物研究与开发》 * |
| 肖春萍等: "人参主要病原茵生防真菌的筛选及鉴定", 《西北农林科技大学学报(自然科学版)》 * |
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105524841A (en) * | 2015-11-09 | 2016-04-27 | 潍坊学院 | Broad-spectrum chaetomium strain applied to biological control of plant diseases and applications of broad-spectrum chaetomium strain |
| CN106754396A (en) * | 2016-11-16 | 2017-05-31 | 河南省农业科学院 | A kind of Chinese toon endogenetic fungus TS8 and its secondary metabolite, preparation method and application |
| CN106754396B (en) * | 2016-11-16 | 2019-08-16 | 河南省农业科学院 | A kind of Chinese toon endogenetic fungus TS8 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 |
| CN106962115A (en) * | 2017-04-05 | 2017-07-21 | 周兴虎 | A kind of method for improving willow resistance against diseases |
| CN107475123B (en) * | 2017-08-16 | 2020-02-11 | 中国人民解放军第二军医大学 | Anoectochilus roxburghii endophytic fungus and application thereof |
| CN107475123A (en) * | 2017-08-16 | 2017-12-15 | 中国人民解放军第二军医大学 | Roxburgh anoectochilus terminal bud endogenetic fungus and its application |
| CN110257262A (en) * | 2019-07-11 | 2019-09-20 | 江苏中科亚农生物工程有限公司 | A method of preparing microbe fertilizer microbial inoculum |
| CN110551637A (en) * | 2019-09-03 | 2019-12-10 | 黑龙江省农业科学院植物保护研究所 | Echinospora echinocandii from radix astragali root for efficiently inhibiting botrytis cinerea and application thereof |
| CN110551637B (en) * | 2019-09-03 | 2020-11-27 | 黑龙江省农业科学院植物保护研究所 | Echinospora echinocandii from radix astragali root for efficiently inhibiting botrytis cinerea and application thereof |
| CN111096222A (en) * | 2019-12-26 | 2020-05-05 | 天津大学 | Method for improving content of ginsenoside in adventitious roots of ginseng by using immobilized chaetomium globosum spores |
| CN111096222B (en) * | 2019-12-26 | 2022-05-17 | 天津大学 | Method for improving content of ginsenoside in adventitious roots of ginseng by using immobilized chaetomium globosum spores |
| CN111944699A (en) * | 2020-07-27 | 2020-11-17 | 中国科学院成都生物研究所 | Chaetomium cupreum microbial inoculum and preparation method thereof |
| CN111944699B (en) * | 2020-07-27 | 2022-11-01 | 中国科学院成都生物研究所 | Chaetomium cupreum microbial inoculum and preparation method thereof |
| CN111869682A (en) * | 2020-08-18 | 2020-11-03 | 河南省农业科学院植物保护研究所 | Application of chaetomium globosum in preventing and treating wheat take-all |
| CN111869682B (en) * | 2020-08-18 | 2021-12-21 | 河南省农业科学院植物保护研究所 | Application of chaetomium globosum in preventing and treating wheat take-all |
| CN113234602A (en) * | 2021-04-01 | 2021-08-10 | 慕恩（广州）生物科技有限公司 | Chaetomium globosum, microbial inoculum, seed soaking liquid and application |
| CN113913300A (en) * | 2021-11-11 | 2022-01-11 | 河南省农业科学院植物保护研究所 | Method for producing chaetomium globosum spore liquid by straw fermentation and application |
| CN113913300B (en) * | 2021-11-11 | 2023-09-22 | 河南省农业科学院植物保护研究所 | Method for producing chaetomium globosum spore liquid by straw fermentation and application |
| CN116240113A (en) * | 2023-02-08 | 2023-06-09 | 江苏农林职业技术学院 | Biocontrol bacterium JSNL-B47 for inhibiting strawberry anthracnose and application thereof |
| CN116240113B (en) * | 2023-02-08 | 2023-08-25 | 江苏农林职业技术学院 | Biocontrol bacterium JSNL-B47 for inhibiting strawberry anthracnose and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104694397B (en) | 2017-12-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104630071B (en) | Spore trichoderma and its application more than one plant | |
| CN104694397A (en) | Chaetomium globosum and application thereof | |
| CN105886428B (en) | One plant of Streptomycesalbidoflhaving and its application in microbial manure | |
| CN103789234B (en) | A kind of bacillus amyloliquefaciens and application thereof | |
| CN104498386B (en) | The preparation method and application of raw Bacillus amyloliquefaciens strain SZ23 and zymotic fluid in wild jujube | |
| CN105018366B (en) | Methylotrophic bacillus and its application | |
| CN102776130B (en) | Metarhizium anisopliae and application thereof | |
| CN106591157B (en) | The preparation and application of the Tabin aspergillus and its metabolite of one plant of disease prevention growth-promoting | |
| CN103952329A (en) | Bacillus vallismortis and application thereof | |
| CN102367421B (en) | Penicilliumgriseofulvum XJ-EP-058 strain as well as microbial preparation resisting banana vascular wilt and application thereof | |
| CN105368720B (en) | Cotton endogenetic fungus CEF-082 and its application in cotton verticillium wilt prevention and treatment | |
| CN107287130A (en) | A kind of Streptomycesalbidoflhaving bacterial strain and its application in agricultural chemicals | |
| CN104789483A (en) | Method for isolated culture of endoclyta signifer walker beauveria bassiana (bals.) vuill strain, and prevention and control of endoclyta signifer walkers | |
| CN103184161B (en) | Biocontrol strain HTC for preventing pepper phytophthora blight and application thereof | |
| CN111778173B (en) | Bacillus subtilis Pro1A2, microbial inoculum and preparation method thereof, and application of bacillus subtilis Pro1A2 in cultivation of melons | |
| CN112322560A (en) | Bacillus belgii and application thereof in prevention and control of pear diseases | |
| CN106701623A (en) | Bacillus atrophaeus antagonistic to lycium chinensis root rot and application of bacillus atrophaeus | |
| CN109971656B (en) | Ginger endogenetic trichoderma viride and application thereof | |
| CN113862160B (en) | Trichoderma pseudokoningii Tk905 strain with biocontrol and induced disease resistance effects and application thereof | |
| CN102732430A (en) | Aspergillus niger strain and application thereof | |
| CN106906171B (en) | A kind of preparation method of apple tree canker biocontrol agent | |
| CN105112304A (en) | Bjerkandera sp. Gause 15 for controlling vegetable root diseases and preparation thereof | |
| CN106754529B (en) | The bacillus amyloliquefaciens of one plant of antagonism fructus lycii root rot and its application | |
| CN111363691B (en) | Paenibacillus polymyxa and application thereof | |
| WO2002034884A1 (en) | Streptomyces kasugaensis inhibiting the fungal pathogens of plant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |