CN102690759A - Separation and purification method of endophytic fungi of solidago canadensis - Google Patents

Abstract

The invention relates to a separation and purification method of endophytic fungi of solidago canadensis. The method comprises the following steps of: 1, collecting stems and leaves of the solidago canadensis; 2, carrying out solid culture and sterility test on the stems and the leaves; and 3, carrying out isolated culture on the endophytic fungi of tissues of the solidago canadensis to obtain the endophytic fungi of the solidago canadensis. A fungus colony has silk velvet texture and a villous surface, does not generate pigment and is light olivaceous and black brown at the back; and the gene complex of the endophytic fungi ITS of the solidago canadensis is composed of 602 basic groups (bp). The geomicrobiology status of a bacterial strain of chaetomiumglobosumyt03 with broad-spectrum antibacterial activity is determined, and the endophytic fungi is found to have relatively remarkable role in inhibiting mycelial growth of six provided plant pathogenic fungi through antibacterial test of the fermentation liquid of the chaetomiumglobosumyt03.

Description

The separation purification method of solidago canadesis endogenetic fungus

Technical field

The invention belongs to microbial technology field, particularly a kind of its fermented product has the separation purification method of the active mikrobe that suppresses plant pathogenic fungi, the i.e. separation purification method of solidago canadesis endogenetic fungus.

Background technology

The main means of control crop diseases and pest crop smothering are chemical pesticide controls in agricultural production process, and it guarantees the good harvest of grain high yield to alleviating the harm of disease Chinese caterpillar fungus, plays a positive role.But owing to interdependence between the nature biotechnology, mutual restriction; The undue toxic action that relies on chemical pesticide; Produce some areas the blindly phenomenon of lavishment agricultural chemicals takes place; The agricultural chemicals that particularly some longevities of residure are long and toxicity is big uses, and causes a series of food safeties, human health and living environment problem.The harm of chemical prevention at present progressively is familiar with by people, and development safety, novel agrochemical efficient, environmental protection have become the direction of development and the theme of research.Biological pesticide is difficult for producing advantages such as resistance because of low dangerous, safe in utilization, environmentally friendly to people, animal, becomes the focus and emphasis of novel pesticide industry development.

Plant endogenesis epiphyte (Endophytic fungi) is meant a certain stage life of life within plant tissue or in the life history within plant tissue; Plant tissue is not caused the fungi of considerable change; Comprise that a certain stage in those life history can build the saprophytic microorganism of hypergene life, the host temporarily not have the latent cause of disease bacterium (Latent pathogens) and the mycorhiza bacterium (Mycorhiza fungi) that injure.Plant endogenesis epiphyte has tangible variety and ubiquity.In that all were studied in the plant of endogenetic fungus at present, its existence is all arranged, tens kinds at least, nearly hundred kinds at most.In the secular symbiosis process of plant endogenesis epiphyte and host plant, make up the special growing environment of endogenetic fungus, made endogenetic fungus can produce natural compounds many and novel structure, physiologically active uniqueness.Have safety, noresidue, not in advantage such as people and animals' cylinder accumulation, control effect be single-minded, for rosy prospect has been opened up in the research and the application of agricultural chemicals lead compound and novel biopesticide.Currently utilize in the plant isolating endogenetic fungus to obtain physiologically active substance to have become the research focus, existing multinomial research shows that plant endogenesis epiphyte can produce the material of novel bacteriostatic activity.As White etc. from grass isolating endogenetic fungus branch top spore ( Acremonium coenophilum) the crop disease fungal pathogens of multiple vitro culture there is restraining effect.Findlay from Picea mariana (Picea mariana), be separated to endogenetic fungus ( Conoplea elegantala) liquid fermentation production in isolate two new benzopyrrole active components, to by rhizoctonia ( Rhizoctonia spp.) and sickle-like bacteria ( Fusarium spp.) disease that causes has good preventive effect, diseases such as rice seedling blight, bakanae disease have good preventive and therapeutic effect.Gu Aiguo from the celastrus angulatus endogenetic fungus ( Fusarium proliferatum) in be separated to enniatine (eniatinns) compounds, it all has remarkable restraining effect to plant pathogenic fungi mycelial growth and spore germination.

Solidago canadesis ( Solidgo canadensis) as a kind of alien species, reproductivity is extremely strong, velocity of propagation is fast, and growth vigor is obvious, and ecological suitability is wide, strives sunlight with surrounding plants, strives fertilizer, and is dead until other plant, thereby species diversity is constituted a serious threat.Solidago canadesis can be done and view and admire cut-flower, feed, nectar source, can be used for the production of natural pigment simultaneously and refines essential oil.At present, have been found that the flavones ingredient that has stronger anti-oxidant, radical eliminate activity and multiple bacteriostatic activity in the solidago canadesis body.The plant vat liquor of reporting solidago canadesis in addition in addition has significant allelopathy to the seed germination and the growth of seedling of many weeds (like bare headed barnyard grass and Herba Setariae viridis) and various vegetables (alfalfa, red three-coloured amaranth, wheat, soybean and cotton).According to the plant endogenesis epiphyte endosymbiotic theory, solidago canadesis probably exists special endogenetic fungus, through producing the special secondary metabolite with anti-microbial activity.

Summary of the invention

The object of the present invention is to provide a kind of therefrom state Hefei City, Anhui Province solidago canadesis ( Solidgo canadensis) in the plant living body, separate its tunning and have the stronger active mikrobe of inhibition plant pathogenic fungi, the i.e. separation purification method of solidago canadesis endogenetic fungus.

Solidago canadesis endogenetic fungus called after ball hair shell yt03 according to the invention ( Chaetomium globosumYt03), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on July 20th, 2011, deposit number is CGMCC No.5088;

The concrete separation and purification operation steps of solidago canadesis endogenetic fungus is following:

(1). gather the stem and the leaf of solidago canadesis;

(2). the surface sterilization of said stem and leaf and aseptic detection: the stem that step (1) is gathered soaks 50s successively, in the chlorine bleach liquor of concentration 0.2%, soaks 90s in the alcohol of concentration 70%; The blade of gathering is soaked 40s successively, in the chlorine bleach liquor of concentration 0.2%, soaks 60s in the alcohol of concentration 70%; The stem that soaked and leaf with aseptic water washing 3 times, are dried in the shade stem after being sterilized and leaf; Stem after the sterilization and leaf are cut into 0.5 cm * 0.5 cm big or small stem piece and leaf piece respectively; Then a part of stem piece and leaf piece are directly planted on first potato dextrose agar solid medium flat board, 24～28 ℃ of constant temperature culture of temperature were cultivated 3～7 days;

Another part stem piece and leaf piece are carried out aseptic detection: adopt and organize blotting; Stem piece and leaf piece placed roll gently on second potato dextrose agar solid medium flat board or be close to substratum and place 2 min; Then remove stem piece and leaf piece; In 28 ℃ of constant temperature culture of temperature, cultivated 7 days, there are not stem piece surface and leaf piece surface sterile after assorted bacterium grows i.e. explanation sterilization;

(3). solidago canadesis is organized the separation and Culture of endogenetic fungus: when growing mycelia around the stem piece of solidago canadesis on first potato dextrose agar solid medium flat board of step (2) and the leaf piece; Adopt most advanced and sophisticated mycelia picking method; Be inoculated on the 3rd potato dextrose agar flat board, 24～28 ℃ of constant temperature culture of temperature were cultivated 3～10 days; Grow complete bacterium colony, be the solidago canadesis endogenetic fungus; The bacterium colony quality velvet shape of solidago canadesis endogenetic fungus, the bacterium colony surface is velvet-like, citrine, non-pigment produces, back side band chocolate;

Said first potato dextrose agar solid culture based formulas, second potato dextrose agar solid culture based formulas and the 3rd potato dextrose agar solid culture based formulas are: 1 liter of yam 200 gram, glucose 20 grams, agar 18 grams, zero(ppm) water; Potato dextrose agar solid medium 30 min that under 121 ℃ of temperature, pressure 0.1MPa, sterilize before using;

Said solidago canadesis endogenetic fungal bacterial strain ITS gene is serial as follows:

1 TCCTCCGGCC?TTATTGATAT?GCTTAAGTTC?AGCGGGTCTT?CCTACCTGAT

51 CCGAGGTCAA?CCTTGGGTTA?AAAGGTGGTT?TAACGGCCGG?AACCCGCGGC

101 GCGACCAGAG?CGAGATGTAT?GCTACTACGC?TCGGTGCGAC?AGCGAGCCCG

151 CCACTGCTTT?TCAGGGCCTG?CGGCAGCCGC?AGGTCCCCAA?CACAAGCCCG

201 GGGGCTTGAT?GGTTGAAATG?ACGCTCGAAC?AGGCATGCCC?GCCAGAATGC

251 TGGCGGGCGC?AATGTGCGTT?CAAAGATTCG?ATGATTCACT?GAATTCTGCA

301 ATTCACATTA?CTTATCGCAT?TTCGCTGCGT?TCTTCATCGA?TGCCAGAACC

351 AAGAGATCCG?TTGTTGAAAG?TTTTGACTTA?TTCAGTACAG?AAGACTCAGA

401 GAGGCCATAA?ATTATCAAGA?GTTTGGTGAC?CTCCGGCGGG?CGCCCGCGGT

451 GGGGCCCAGG?GGCGCCCGGG?GGGTAAACCC?CGGGGCCGCC?CGCCGAAGCA

501 ACGGTATAGG?TAACGTTCAC?AATGGTTTAG?GGAGTTTTGC?AACTCTGTAA

551 TGATCCCTCC?GCTGGTTCAC?CAACGGAGAC?CTTGTTACGA?CTTTTTACTT

601 CC

Solidago canadesis endogenetic fungal bacterial strain ITS genome is made up of 602 bases (bp).

With ITS gene order homology is the phylogenetic tree that fundamental construction forms the relevant fungi comprise ball hair shell yt03 bacterial strain, and the result finds out, Chaetomium globosumYt03 with Chaetomium globosumThe ITS sequence homology of ATCC 6205 reaches 100%.The difference that does not have base.

Solidago canadesis endogenetic fungus bacterium colony cultural characteristic

Solidago canadesis endogenetic fungus temperature on the potato dextrose agar solid medium was cultivated 7 days for 25 ℃, and colony diameter reaches 6.6cm, quality velvet shape, and the bacterium colony surface is velvet-like, citrine, non-pigment produces, back side band chocolate.See Fig. 1.

Solidago canadesis endogenetic fungus morphological specificity

Scattered or all living creatures of the perithecium of solidago canadesis endogenetic fungus, spherical or subsphaeroidal, difference in size is bigger, and big person's diameter can reach 400 μ m, and little person 70 μ m are chocolate; Ruff does not have barrier film, and chocolate is straight or spirrillum is crooked, diameter 3-4 μ m, and there is spinelet on the surface, sees Fig. 2.Ascus is broken in early days and discharges a large amount of thecaspores, and thecaspore is inferior sphere or lemon shape usually, 6-10 * 4-7 μ m, and wall is smooth.See Fig. 3.

The molecular biological characteristics of solidago canadesis endogenetic fungus

Adopt the molecular biology round pcr, the dna sequencing analysis, the ITS gene of endogenetic fungus is by 602 based compositions.

Correlated series among the dna sequence dna of solidago canadesis endogenetic fungus and the Genbank is than right 602 bases, and homology is 100%.With the ITS sequence is the phylogenetic tree of fundamental construction, chaetomium globosum yt03 with Chaetomium globosumThe evolutionary distance of ATCC 6205 (EF524036) is nearest.See Fig. 4, adopt MEGA4.1 software, ortho position connection method display ball chaetomium yt03 and relevant ITS rDNA phylogenetic tree of planting carry out 1000 times similarity double counting, grow tree node among the figure and only show that the Bootstrap value is greater than 50% numerical value.

The antibacterial tests of the antibiotic fermented liquid of solidago canadesis endogenetic fungus:

1. plant pathogenic fungi:

The Phytophthora capsici germ ( Phytophthora capsici), the tomato wilt bacterium ( Fusarium oxysporum), Valsa mali ( Cytospora mandshurica), apple anthrax bacteria ( Colletotrichum gloeosporioides), the jujube anthrax bacteria (Gloelsporium fructigenum), fusarium graminearum ( Fusarium graminearum).

2. the cultivation of endogenetic fungus:

The solidago canadesis endogenetic fungus is inserted in 250 milliliters of triangular flasks (50 milliliters of liquid substratum are housed), placed on (28 ± 2) ℃ shaking table (160 rev/mins) rotating and culturing 7 days, remove by filter mycelium under the aseptic condition, fermentation broth sample is subsequent use.

3. the cultivation of pathogenic micro-organism

The slant culture of getting pathogenic bacteria inserts the dull and stereotyped activation of potato dextrose agar solid medium respectively, in (28 ± 2) ℃ thermostat container, cultivates 5 days.

4. suppress the mycelial growth rate method and measure anti-microbial activity

The substratum that in 100 milliliters aseptic triangular flask, adds 6 milliliters of antibiotic fermentation broth samples and 54 milliliters of aseptic thawings of potato dextrose agar; Shake up; Respectively getting 10 milliliters respectively, to place diameter be to process flat board in 9 centimetres of sterile petri dish; The cooling back is put into 1 and is supplied examination pathogenic bacteria bacterium cake (diameter is 6 millimeters) on each substratum plane, the mycelia face of bacterium cake is attached to media surface, places 28 ± 1 ℃ to cultivate 96 hours flat board.Adopt the right-angled intersection method to measure the colony growth diameter, calculate inhibiting rate with following formula:

Table 1: chaetomium globosum yt03 of the present invention is to the The anti-bacterial result of six kind of plant pathogenic micro-organisms

The present invention separates the solidago canadesis endogenetic fungus, and clear and definite chaetomium globosum yt03 with broad spectrum antibiotic activity ( Chaetomium globosumYt03) the microbiology status of bacterial strain is through right Chaetomium globosum yt03The antibacterial tests of antibiotic fermented liquid find this bacterium to supply examination six kind of plant pathogenic fungies (be the Phytophthora capsici germ ( Phytophthora capsici), the tomato wilt bacterium ( Fusarium oxysporum), Valsa mali (Cytospora mandshurica),Apple anthrax bacteria (Colletotrichum gloeosporioides),The jujube anthrax bacteria (Gloelsporium fructigenum),Fusarium graminearum ( Fusarium graminearum)) have comparatively obvious suppression mycelial growth effect, see table 1.The present invention utilizes the plant endogenesis epiphyte resource, in the hope of obtaining anti-plant pathogenic fungi disease novel agricultural antibiotics natural component.

Description of drawings

Fig. 1 is the bacterium colony picture (on the potato dextrose agar solid medium) of chaetomium globosum yt03 of the present invention.

Fig. 2 is the microgram (perithecium) of chaetomium globosum yt03 of the present invention.

Fig. 3 is the microgram (thecaspore) of chaetomium globosum yt03 of the present invention.

Fig. 4 is the phylogeny tree graph of chaetomium globosum yt03 homology of the present invention.

Embodiment

Below in conjunction with embodiment, the present invention is done explanation further.

Embodiment:

Solidago canadesis endogenetic fungus called after ball hair shell yt03 according to the invention ( Chaetomium globosumYt03), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on July 20th, 2011, deposit number is CGMCC No.5088;

The concrete separation and purification operation steps of solidago canadesis endogenetic fungus is following:

(1). gather the stem and the leaf of solidago canadesis;

(2). the solid culture of stem and leaf and aseptic detection: the stem that step (1) is gathered soaks 50s successively, in the chlorine bleach liquor of concentration 0.2%, soaks 90s in the alcohol of concentration 70%; The blade of gathering is soaked 40s successively, in the chlorine bleach liquor of concentration 0.2%, soaks 60s in the alcohol of concentration 70%; The stem that soaked and leaf with aseptic water washing 3 times, are dried in the shade stem after being sterilized and leaf; Stem after will sterilizing with the sterile surgical cutter and leaf are cut into the stem piece and the leaf piece of 0.5 cm * 0.5 cm size respectively; Then a part of stem piece and leaf piece are directly planted on first potato dextrose agar solid medium flat board, 24～28 ℃ of constant temperature culture of temperature were cultivated 3～7 days;

Another part stem piece and leaf piece are carried out aseptic detection: adopt and organize blotting; Stem piece and leaf piece placed roll gently on second potato dextrose agar solid medium flat board or be close to substratum and place 2 min; Then remove stem piece and leaf piece; In 28 ℃ of constant temperature culture of temperature, cultivated 7 days, there are not stem piece surface and leaf piece surface sterile after assorted bacterium grows i.e. explanation sterilization;

(3). solidago canadesis is organized the separation and Culture of endogenetic fungus: when growing mycelia around the stem piece of solidago canadesis on first potato dextrose agar solid medium flat board of step (2) and the leaf piece; See Fig. 1, adopt most advanced and sophisticated mycelia picking method, be inoculated on the 3rd potato dextrose agar flat board; 24～28 ℃ of constant temperature culture of temperature; Cultivated 3～10 days, and grew complete bacterium colony, be the solidago canadesis endogenetic fungus; The bacterium colony quality velvet shape of solidago canadesis endogenetic fungus, the bacterium colony surface is velvet-like, citrine, non-pigment produces, back side band chocolate;

Said first potato dextrose agar solid culture based formulas, second potato dextrose agar solid culture based formulas and the 3rd potato dextrose agar solid culture based formulas are: 1 liter of yam 200 gram, glucose 20 grams, agar 18 grams, zero(ppm) water; Potato dextrose agar solid medium 30 min that under 121 ℃ of temperature, pressure 0.1MPa, sterilize before using;

Solidago canadesis endogenetic fungal bacterial strain ITS gene order is following:

1 TCCTCCGGCC?TTATTGATAT?GCTTAAGTTC?AGCGGGTCTT?CCTACCTGAT

51 CCGAGGTCAA?CCTTGGGTTA?AAAGGTGGTT?TAACGGCCGG?AACCCGCGGC

101 GCGACCAGAG?CGAGATGTAT?GCTACTACGC?TCGGTGCGAC?AGCGAGCCCG

151 CCACTGCTTT?TCAGGGCCTG?CGGCAGCCGC?AGGTCCCCAA?CACAAGCCCG

201 GGGGCTTGAT?GGTTGAAATG?ACGCTCGAAC?AGGCATGCCC?GCCAGAATGC

251 TGGCGGGCGC?AATGTGCGTT?CAAAGATTCG?ATGATTCACT?GAATTCTGCA

301 ATTCACATTA?CTTATCGCAT?TTCGCTGCGT?TCTTCATCGA?TGCCAGAACC

351 AAGAGATCCG?TTGTTGAAAG?TTTTGACTTA?TTCAGTACAG?AAGACTCAGA

401 GAGGCCATAA?ATTATCAAGA?GTTTGGTGAC?CTCCGGCGGG?CGCCCGCGGT

451 GGGGCCCAGG?GGCGCCCGGG?GGGTAAACCC?CGGGGCCGCC?CGCCGAAGCA

501 ACGGTATAGG?TAACGTTCAC?AATGGTTTAG?GGAGTTTTGC?AACTCTGTAA

551 TGATCCCTCC?GCTGGTTCAC?CAACGGAGAC?CTTGTTACGA?CTTTTTACTT

601 CC

Solidago canadesis endogenetic fungal bacterial strain ITS genome is made up of 602 bases (bp).

Claims (1)

1. the separation purification method of solidago canadesis endogenetic fungus is characterized in that: said solidago canadesis endogenetic fungus called after ball hair shell yt03 ( Chaetomium globosumYt03), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on July 20th, 2011, deposit number is CGMCC No.5088;

Concrete separation and purification operation steps is following:

(1). gather the stem and the leaf of solidago canadesis;

(2). the surface sterilization of said stem and leaf and aseptic detection: the stem that step (1) is gathered soaks 50s successively, in the chlorine bleach liquor of concentration 0.2%, soaks 90s in the alcohol of concentration 70%; The blade of gathering is soaked 40s successively, in the chlorine bleach liquor of concentration 0.2%, soaks 60s in the alcohol of concentration 70%; The stem that soaked and leaf with aseptic water washing 3 times, are dried in the shade stem after being sterilized and leaf; Stem after the sterilization and leaf are cut into 0.5 cm * 0.5 cm big or small stem piece and leaf piece respectively; Then a part of stem piece and leaf piece are directly planted on first potato dextrose agar solid medium flat board, 24～28 ℃ of constant temperature culture of temperature were cultivated 3～7 days;

Another part stem piece and leaf piece are carried out aseptic detection: adopt and organize blotting; Stem piece and leaf piece placed roll gently on second potato dextrose agar solid medium flat board or be close to substratum and place 2 min; Then remove stem piece and leaf piece; In 28 ℃ of constant temperature culture of temperature, cultivated 7 days, there are not stem piece surface and leaf piece surface sterile after assorted bacterium grows i.e. explanation sterilization;

(3). solidago canadesis is organized the separation and Culture of endogenetic fungus: when growing mycelia around the stem piece of solidago canadesis on first potato dextrose agar solid medium flat board of step (2) and the leaf piece; Adopt most advanced and sophisticated mycelia picking method; Be inoculated on the 3rd potato dextrose agar flat board, 24～28 ℃ of constant temperature culture of temperature were cultivated 3～10 days; Grow complete bacterium colony, be the solidago canadesis endogenetic fungus; The bacterium colony quality velvet shape of solidago canadesis endogenetic fungus, the bacterium colony surface is velvet-like, citrine, non-pigment produces, back side band chocolate;

Said first potato dextrose agar solid culture based formulas, second potato dextrose agar solid culture based formulas and the 3rd potato dextrose agar solid culture based formulas are: 1 liter of yam 200 gram, glucose 20 grams, agar 18 grams, zero(ppm) water; Potato dextrose agar solid medium 30 min that under 121 ℃ of temperature, pressure 0.1MPa, sterilize before using;

Said solidago canadesis endogenetic fungal bacterial strain ITS gene is serial as follows:

1 TCCTCCGGCC?TTATTGATAT?GCTTAAGTTC?AGCGGGTCTT?CCTACCTGAT

51 CCGAGGTCAA?CCTTGGGTTA?AAAGGTGGTT?TAACGGCCGG?AACCCGCGGC

101 GCGACCAGAG?CGAGATGTAT?GCTACTACGC?TCGGTGCGAC?AGCGAGCCCG

151 CCACTGCTTT?TCAGGGCCTG?CGGCAGCCGC?AGGTCCCCAA?CACAAGCCCG

201 GGGGCTTGAT?GGTTGAAATG?ACGCTCGAAC?AGGCATGCCC?GCCAGAATGC

251 TGGCGGGCGC?AATGTGCGTT?CAAAGATTCG?ATGATTCACT?GAATTCTGCA

301 ATTCACATTA?CTTATCGCAT?TTCGCTGCGT?TCTTCATCGA?TGCCAGAACC

351 AAGAGATCCG?TTGTTGAAAG?TTTTGACTTA?TTCAGTACAG?AAGACTCAGA

401 GAGGCCATAA?ATTATCAAGA?GTTTGGTGAC?CTCCGGCGGG?CGCCCGCGGT

451 GGGGCCCAGG?GGCGCCCGGG?GGGTAAACCC?CGGGGCCGCC?CGCCGAAGCA

501 ACGGTATAGG?TAACGTTCAC?AATGGTTTAG?GGAGTTTTGC?AACTCTGTAA

551 TGATCCCTCC?GCTGGTTCAC?CAACGGAGAC?CTTGTTACGA?CTTTTTACTT

601 CC

Solidago canadesis endogenetic fungal bacterial strain ITS genome is made up of 602 bases (bp).

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