CN105567569A - Culture method for mycosphaerella arachidicola of dendrobium sw. - Google Patents

Culture method for mycosphaerella arachidicola of dendrobium sw. Download PDF

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CN105567569A
CN105567569A CN201510918364.2A CN201510918364A CN105567569A CN 105567569 A CN105567569 A CN 105567569A CN 201510918364 A CN201510918364 A CN 201510918364A CN 105567569 A CN105567569 A CN 105567569A
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strawberry
substratum
herba dendrobii
sucrose
brown
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CN105567569B (en
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王汉荣
王连平
方丽
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Zhejiang Academy of Agricultural Sciences
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Zhejiang Academy of Agricultural Sciences
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/14Fungi; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/70Undefined extracts
    • C12N2500/76Undefined extracts from plants

Abstract

The invention provides a culture method for mycosphaerella arachidicola of dendrobium sw.. The method comprises the following step of providing a strawberry decoction culture medium, wherein every 1,000 milliliters of strawberry decoction culture medium comprises: 200 grams of strawberry stems and leaves, 20 grams of sucrose, 20 grams of agar and the balance of water. By the culture medium and the culture method, a large number of spores of the mycosphaerella arachidicola can be obtained.

Description

A kind of cultural method of Herba Dendrobii brown patch germ
Technical field
The invention belongs to microorganism field, especially, belong to and adopt cultural method to cultivate the cultural method of Herba Dendrobii brown patch germ.
Background technology
Herba Dendrobii (DendrobiumSw.) has another name called Herba hedyotis costatae, Yunnan iron sheet, and being the perennial herbaceous plant that grows nonparasitically upon another plant of the orchid family (Orchidaceae), is a kind of Chinese medicinal materials of preciousness.Just on the books in Shennong's Herbal, there is nourishing Yin and clearing heat, the beneficial stomach that promotes the production of body fluid, moisten the lung and relieve the cough, improving eyesight is kept fit, effect of easing pain and diminishing inflammation.Simultaneously modern study shows, stem of noble dendrobium medicinal material except have that book on Chinese herbal medicine records etc. except effect, also there is the effects such as antitumor, radioprotective, anti-platelet aggregation and enhancing body immunizing power.The demand of stem of noble dendrobium medicinal material constantly increases in recent years, and constantly bringing forth new ideas of cultivation technique makes the area of the artificial growth stem of noble dendrobium constantly increase, but along with the increase of concentrated cultivated area, produced problem is also on the increase, there is multiple different disease, such as Herba Dendrobii brown spot in growing dendrobium.Stem of noble dendrobium blade catch an illness after scab just in the filbert point to chocolate, rear point expands as the scab of about 5 ~ 6mm gradually.This not only affects the quality of the stem of noble dendrobium, also affects the output of the stem of noble dendrobium, and tool statistics brown spot causes production loss 5%-10%.Prevent the generation of brown spot and amplification from contributing to improving the yield and quality of stem of noble dendrobium production, guarantee the development that stem of noble dendrobium industry is healthy and orderly.In order to study the brown spot of how to prevent and treat Herba Dendrobii, just need first to cultivate brown spot to carry out the research of some aspects, such as study the biological characteristics of this germ, the test of pesticide effectiveness etc. filter out suitable medicine and prevent and treat.But this germ is difficult to cultivate, and yields poorly, for this reason, need to provide the research how high efficiency cultivation brown spot germ carries out related fields.
Summary of the invention
In order to overcome the above problems, the invention provides a kind of method of cultivating Herba Dendrobii brown patch germ, the method comprises: provide strawberry to decoct juice substratum, wherein this strawberry decocts consisting of of juice substratum: strawberry cauline leaf 200g, sucrose 20g, agar 20g, adds and adds water to 1000ml.
Preferably, strawberry cauline leaf 200g is prepared through following process: in frying pan, put into 1000mL distilled water and 200g strawberry cauline leaf, after boiling 5min, filters and removes cauline leaf, add 20g sucrose, be settled to 1000mL.
Preferably, the pH value of cultivation is 7.
Preferably, the temperature of cultivation is 25-30 degree Celsius.
Preferably, described Herba Dendrobii brown patch germ is many beaks Phoma sp (Phomamultirostrata (Mathuretal.) Dorenboseh & Boerema).
On the other hand, the invention provides strawberry and decoct the purposes of juice substratum in making cultivation Herba Dendrobii brown patch germ substratum, wherein said strawberry is decocted juice substratum and is made up of following component: strawberry cauline leaf 200g, sucrose 20g, agar 20g.
Preferably, preferably, strawberry cauline leaf 200g is prepared through following process: in frying pan, put into 1000mL distilled water and 200g strawberry cauline leaf, boil 5min, filters and removes cauline leaf, add 20g sucrose, be settled to 1000mL.
Preferably, the pH value of cultivation is 7.
Preferably, the temperature of cultivation is 25-30 degree Celsius.
Preferably, described Herba Dendrobii brown patch germ is many beaks Phoma sp (Phomamultirostrata (Mathuretal.) Dorenboseh & Boerema).
On the other hand, the invention provides a kind of method of cultivating Herba Dendrobii brown patch germ, the method comprises provides a kind of substratum, this cultivation composed as follows: nitrogen source compound 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate (MgSO47H2O) 0.5g, Repone K 0.5g, ferrous sulfate 0.01g, sugar source material 30g, agar 20g, adds and adds water to 1000mL, and wherein said sugared source material is maltose, sucrose, synanthrin, ribose, fructose, lactose, rhamnosyl, sorbyl alcohol or fiber biose.
Preferably, sugared source material is fiber biose.
Preferably, described nitrogen source compound is L-PROLINE, Valine, L-Phe, L-Trp or saltpetre.
Preferably, described nitrogen source compound is L-PROLINE, Serine and L-Trp.
A kind of substratum is for the preparation of the purposes of cultivating in Herba Dendrobii brown patch germ substratum, wherein, described substratum composed as follows: nitrogen source compound 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate (MgSO47H2O) 0.5g, Repone K 0.5g, ferrous sulfate 0.01g, sugar source material 30g, agar 20g, wherein said sugared source material is maltose, sucrose, synanthrin, ribose, fructose, lactose, rhamnosyl, sorbyl alcohol or fiber biose.
Preferably, sugared source material is fiber biose.
Preferably, described nitrogen source compound is L-PROLINE, Valine, L-Phe, L-Trp or saltpetre.
Preferably, described nitrogen source compound is L-PROLINE, Serine and L-Trp.
Preferably, described Herba Dendrobii brown patch germ is many beaks Phoma sp (Phomamultirostrata (Mathuretal.) Dorenboseh & Boerema).
Effective effect
By specific culture medium of the present invention and cultural method, high-quality, a large amount of brown patch germs can be obtained, for research brown patch germ provides useful material, particularly can provide a large amount of spores.
Specific embodiment
1 materials and methods
The separation of the sick sample collection of 1.1 Herba Dendrobii brown spots and pathogenic bacteria
Herba Dendrobii brown spot sick sample in field is collected in the iron maple hall Herba Dendrobii base, Leqing in Wild Goose and Reed Marsh Mountains town, Leqing county of Zhejiang Province, Leqing with rich Herba Dendrobii base, Lingyan Crag Herba Dendrobii base, Leqing; After sampling, standard specimen is put into freshness protection package to take back laboratory and be placed in 5 DEG C of refrigerators, stand-by.Cut Herba Dendrobii brown spot microscopy and be separated with line respectively at carrying out conventional organization separation with PSA substratum.
1.2 Pathogenicity
Adopt respectively to be separated and (be diluted to 1 × 10 from the mycelia of the sick sample isolate of Herba Dendrobii brown spot and conidium liquid 6individual/mL), spray live body wound is carried out to the stem of noble dendrobium and inoculates without wound, in the XT5401-CD275TJH intelligent biological growth cabinet of 25 DEG C, more than RH98%, cultivate after 12 h light 12 h dark inoculations, with the branch of health for contrast, repeat 3 times, timing every day observed and recorded incidence, analyzes pathogenic.
The cultural colony of 1.3 pathogenic bacterias
Pathogenic bacteria is inoculated on PSA and cultivates 7d in 25 DEG C, record colonial morphology, color; Measure 100 conidial sizes, repeat 3 times, record mean value.
1.3.1 different culture media impact that Herba Dendrobii Pathogenic Bacteria Causing Brown Blotch Disease mycelial growth and conidium are produced.
Pathogenic bacteria by the culture medium culturing in following table in 25 DEG C, often processes repetition 6 times, measures colony diameter, dry mycelial weight, pycnidial production and bacterium colony color and luster thereof etc. after 5d.
Table 1: the formula of different culture media
* decoct the set-up procedure of juice substratum: in frying pan, put into 1000mL distilled water and the corresponding article of 200g, after boiling 5min, filter reserved filtrate, add 20g sucrose, be settled to 1000mL.
1.3.2, for most preferred substratum (strawberry decocts juice substratum), we determine different pH value on Herba Dendrobii brown patch germ mycelial growth and conidial impact by the mycelia block of diameter 4.0mm the strawberry moved in the different pH value of pH value 4-12 decoct (pH value adopts pHS-9V acidometer to measure preparation) on juice substratum, be placed in 25 DEG C of cultivations, often process repetition 6 times, measure bacterium colony dry weight after 6d, calculate conidium quantity.
1.3.3 the mycelia block of diameter 4.0mm to move Herba Dendrobii brown patch germ mycelial growth and conidial impact and decocts on juice culture medium flat plate in strawberry by differing temps, be placed in 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, cultivate under 35 DEG C of differing tempss, often process repetition 6 times, after 6d, measure colony diameter and conidium quantity.
1.3.4 different carbon source is on Herba Dendrobii brown patch germ mycelial growth and conidial impact.
Through our large quantity research, find, in time decocting juice substratum with strawberry to study the impact on this Pathogen culture of optimum carbon source and nitrogenous source, find that different carbon sources is to the proterties of cultivating (bacterium colony dry weight and product spore situation) not tangible difference, that is different carbon sources or nitrogenous source seem to play same effect, do not have tangible improvement and impact, equally, on the substratum that other are similar, such as bark decocts juice substratum, sugarcane decocts juice substratum, lotus leaf decocts juice substratum, oat medium (OMA substratum) obtains decocts the similar result of juice substratum (concrete data slightly) with strawberry.
On the contrary, substratum based on Cha Shi substratum, again respectively with 20 kinds of different carbon source equivalent displacement sucrose (content is the same with sucrose) such as seminose, glucose, sucrose, soluble starch, semi-lactosi, trehalose, N.F,USP MANNITOL, wood sugar, DEAE Mierocrystalline cellulose, ethanol, ribose, fructose, sorbose, sorbyl alcohol, rhamnosyl, synanthrin, lactose, maltose, sodium cellulosate, fiber biose, cultivate at 25 DEG C, often process repetition 6 times, colony diameter is measured after 6d, observe bacterium colony dry weight and produce spore situation, obtain different results, specifically see interpretation of result.
1.3.5 different nitrogen sources is on Herba Dendrobii brown patch germ mycelial growth and conidial impact.
Substratum based on Cha Shi substratum, use L-glutamic acid respectively, glutaminase, aspargine, aspartic acid, glycine, leucine, Isoleucine, Cys, CYSTINE, tyrosine, 1B, L-threonine, L-arginine, L-Phe, L-Histidine, Serine, L-PROLINE, L-Methionine, Valine, L-Trp, ALANINE, casein hydrolysate, urea, ammonium sulfate, saltpetre, 26 kinds of nitrogenous source equivalent displacement saltpetre such as Sodium Nitrite or SODIUMNITRATE (quality is the same with saltpetre), be placed in 25 DEG C of cultivations, often process repetition 6 times, colony diameter is measured after 6d, observe bacterium colony dry weight and produce spore situation.
2..0 results and analysis
The symptom of 2.1 Herba Dendrobii brown spots
Brown spot only infects the blade of Herba Dendrobii.Blade catch an illness after scab just in the filbert point to chocolate, rear point expands gradually, maximum scab about 5 ~ 6mm.Scab is circular or oval, filbert to chocolate, and sick strong intersection often has the haloing of a circle brown sometimes.Scab intermediate recess.Time serious, blade occurs many scabs not of uniform size, general young leaves is not caught an illness.
The separation of 2.2 pathogenic bacterias and Pathogenicity
Carry out line separation to tens stem of noble dendrobium brown spot standard specimens to be separated with conventional organization, after 5d, culture dish grows a small amount of bacterium and different fungies, under these bacteriums and fungi being placed in suitable temperature and humidity conditions, the inspection of Ke He rule is carried out to the branches and leaves in identical Herba Dendrobii health, find that these bacteriums are mostly genus bacillus, be non-pathogenic bacteria; Wherein repeated isolation to a kind of fungal isolates be doubtful pathogen.Inoculation inspection is carried out at the branches and leaves of identical Herba Dendrobii health under this fungal isolates being placed in suitable temperature and humidity conditions, these fungal isolates all cause a disease to stem of noble dendrobium leaf, illness and field basically identical, be accredited as by molecular biological method and be stem of noble dendrobium Pathogenic Bacteria Causing Brown Blotch Disease.
The form of 2.3 pathogenicbacteria separation things and cultural colony
Pathogenic bacteria is on PSA, and mycelia becomes radial growth, aerial hyphae canescence, and down shape is more flourishing.Be laid on substratum, initial stage bacterium colony (especially at its edge) is light pink, transfers grey to immediately, transfer brown to by grey again, finally transfer chocolate to by brown, bacterium colony also becomes carbonaceous, the culture dish back side is chocolate radiation striated, conidium group orange.On host, under pathogenic bacteria acervulus is born in Cuticle, dispersion or symphysis, break through epidermis and expose, irregular cracking, scattered bristle after ripe, bristle brown to dun, a tool 1-3 barrier film, straight or micro-curved, gradually sharp and look light to top; Conidiophore is colourless, and conidiogenous cell is cylindrical, colourless, conidium unit cell, colourless, Long Circle or ellipse, and one end is slightly sharp, (14 ~ 15) μm × (4.5 ~ 6) μm, sometimes oily globule.Appressorium is produced, appressorium brown, long club shape or in irregular shape, (9 ~ 10) μm × (5 ~ 6) μm when mycelia or germ tube tip contact solid interface.
2.5 pathogen identification
According to the result of study (specific experiment data slightly) of the morphological feature of the pathogenicbacteria separation thing to Herba Dendrobii brown spot, cultural colony and Pathogenicity, consult monograph (Zhang Zhongyi etc., 1986; Lu Jiayun, 2001) and Wu Wenping (1991) to the research of Myrothecium 4 kinds; Determine that Herba Dendrobii brown spot is caused by Deuteromycotina (Deuteromycotina), Sphaeropsidales (Sphaeropsidales), Sphaeropsidaceae (Sphaerioidaceae), Phoma (Phoma), many beaks Phoma sp (Phomamultirostrata (Mathuretal.) Dorenboseh & Boerema).
2.6 different culture medias are on the impact (concrete structure is in table 2) of stem of noble dendrobium brown patch germ mycelial growth
Stem of noble dendrobium brown patch germ at PSA substratum, OMA substratum, Cha Shi substratum, juice substratum decocted by the stem of noble dendrobium, bark decocts juice substratum, sweet potato decocts juice substratum, strawberry decocts juice substratum, lotus leaf is decocted on 8 kinds of different culture medias such as juice substratum (table 1), grow under 25 DEG C of constant temperature.Measurement result shows that this bacterium all can grow on tested substratum, wherein stem of noble dendrobium brown patch germ decocts juice substratum sweet potato, lotus leaf decocts juice substratum, OMA substratum, strawberry decoct juice substratum grows the fastest, the speed of growth (logistic rate of increase) reaches as high as 8.013,7.553,7.594,7.956.But this bacterium colony sweet potato decoct juice substratum and lotus leaf decoct juice substratum grows thinner, biomass dry weight is obviously little than PSA substratum, the comprehensive speed of growth and increment, and this bacterium is PSA substratum, OMA substratum compared with the substratum of suitable growth, bark decocts juice substratum, and strawberry decocts juice substratum.
This bacterium all can produce the pycnidium of black on tested substratum, and wherein decoct the pycnidium quantity of juice substratum generation at most with strawberry, quantity can reach 0.85 × 10 4individual/mL, processes with other and reaches pole significant difference (making a concrete analysis of process slightly), secondly decoct juice substratum (process with other and reach significant difference) for PSA, OMA substratum and bark.Therefore OMA substratum, PSA substratum and strawberry decoct juice substratum is the suitableeest product spore substratum.Because the pycnidium producing black is an approach the most effective of effectively breeding this pathogenic bacteria, the growth being beneficial to these spores can carry out the screening of super disease carrying germ and super resistance germ, carries out fundamental research the most helpful for this disease of control.
Table 2 different culture media is on the impact of stem of noble dendrobium brown patch germ mycelial growth and illumination