CN108060088A - A kind of culture medium for being used for Pyricularia oryzae culture, producing spore and preparation method thereof - Google Patents
A kind of culture medium for being used for Pyricularia oryzae culture, producing spore and preparation method thereof Download PDFInfo
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- CN108060088A CN108060088A CN201810026740.0A CN201810026740A CN108060088A CN 108060088 A CN108060088 A CN 108060088A CN 201810026740 A CN201810026740 A CN 201810026740A CN 108060088 A CN108060088 A CN 108060088A
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- 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
Abstract
The present invention provides a kind of for Pyricularia oryzae culture, the culture medium for producing spore and preparation method thereof.The technical solution has carried out innovative design by experimental method to medium component, is according to the influence for having investigated a variety of compounding ingredients to Growth of Magnaporthe Grisea with growth rate, sporulation quantity etc..The experimental results showed that there is significant growth promoting function to Pyricularia oryzae by isometric culture medium for mixing gained with PDA culture medium by rice straw leachate, while produce spore efficiency and be also obviously improved.Culture medium of the present invention is good to the culture effect of Pyricularia oryzae, sporulation quantity protrudes, while material is easy to get, prepares simplicity, has good using effect.
Description
Technical field
The present invention relates to field of microbial culture technology, and in particular to a kind of for Pyricularia oryzae culture, the culture of production spore
Base and preparation method thereof.
Background technology
Rice blast is the primary disease of rice, and pathogenic microorganism is grey pears spore (Phyricularia grisea), is belonged to
Fungi Imperfecti, Hyphomycetes；Its sexual generation claims the big mouth coccus of grey (Magnaporthe grisea).It is ground in rice varieties disease resistance
Study carefully, physiological races of rice blast fungus identification etc. is in experiment works, Spawn incubation and conidial obtain are necessary sport technique segments,
Thus the culture effect of Pyricularia oryzae, production spore efficiency, which have experimental work, directly affects.
In the prior art, for the culture medium of Pyricularia oryzae culture and production spore generally with sorghum, rice straw, barley, wheat etc.
The direct mixture of ingredient obtains, such culture medium not only prepare it is cumbersome, but also pathogen growth compared with it is slow, production spore effect is undesirable, simultaneously
The problem of there is being easily bacterial contamination.Although also there is part researcher using medium oatmeal, rice bran culture medium, red dog
Culture medium, maize powder medium, yeast starch culture-medium etc. generally to be paid no attention to for producing spore experiment there are still spore effect is produced
Think or the technical problems such as material is not easy to obtain.
The content of the invention
It is contemplated that for the technological deficiency of the prior art, provide a kind of for Pyricularia oryzae culture, the culture of production spore
Base and preparation method thereof, with solve in the prior art conventional medium it is bad to the culture effect of Pyricularia oryzae, production spore efficiency compared with
The technical issues of low.
For realization more than technical purpose, the present invention uses following technical scheme
A kind of culture medium for being used for Pyricularia oryzae culture, producing spore, is to be mixed by isometric rice straw leachate with PDA culture medium
Close what is formed；Wherein described rice straw leachate is rice straw and water by 3:100(g:ML after ratio mixing), 16~institute for 24 hours is impregnated
The soak obtained；The PDA culture medium includes the ingredient of following parts by weight：200 parts of potato, 20 parts of glucose, 1000 parts of water.
Preferably, state the agar that PDA culture medium further includes 18 parts by weight.
Preferably, the Pyricularia oryzae is Chinese rice blast fungus microspecies ZB13.
Meanwhile the present invention provides above-mentioned for Pyricularia oryzae culture, the preparation method for the culture medium for producing spore, including following
Step：
1) take rice straw with water by 3:100(g:ML ratio) mixes the two, and immersion 16~for 24 hours, obtain rice straw leachate；
2) 15 points are boiled after the water of the potato of 200 parts by weight, the glucose of 20 parts by weight, 1000 parts by weight is mixed
Clock with filtered through gauze, takes filtrate；
3) filtrate obtained by the rice straw leachate obtained by step 1) and step 2) is pressed 1:1 volume ratio mixes, to the mixing
The agar of water weight 18 ‰ used in step 2) is added in object, is dissolved by heating.
In above technical scheme, the Pyricularia oryzae refers to that grey pears spore (Phyricularia grisea) or grey are big
Mouth coccus (Magnaporthe grisea).
On this basis, The present invention gives the application that the culture medium is used for Involved in Sporulation in Magnaporthe grisea, comprise the following steps：It chooses
Pyricularia oryzae inoculation is taken to culture medium flat plate, in 28 DEG C of constant temperature incubations, after mycelia on tablet is covered with (about 7d), dislocation
In black light lamp illumination cultivation 3d on indoor tissue culture frame, 25~28 DEG C of room temperature；After fully production spore, per ware 15mL distilled water
Lower spore is washed, spore quantity is calculated under 100 power microscopes.
The present invention provides a kind of for Pyricularia oryzae culture, the culture medium for producing spore and preparation method thereof.The technical solution
Innovative design has been carried out to medium component by experimental method, has been a variety of according to having investigated with growth rate, sporulation quantity etc.
Compound influence of the ingredient to Growth of Magnaporthe Grisea.It is mixed in equal volume the experimental results showed that being pressed by rice straw leachate with PDA culture medium
The culture medium of gained has significant growth promoting function to Pyricularia oryzae, while sporulation quantity is also at higher level；Comparison discovery,
Reduced levels are in using sporulation quantity when oligomict rice straw leachate or PDA culture medium culture Pyricularia oryzae, and by the two
With the culture medium of the present invention of special ratios mixing gained, Involved in Sporulation in Magnaporthe grisea, which has measured, to be obviously improved, it can be seen that, the present invention
Specific compounding mode to promote Involved in Sporulation in Magnaporthe grisea generate exact synergistic effect.Culture medium of the present invention is to Pyricularia oryzae
Culture effect is good, sporulation quantity protrudes, while material is easy to get, prepares simplicity, has good using effect.
Description of the drawings
Fig. 1 is the comparison of same rice blast bacterial strain speed of growth on different culture media in the specific embodiment of the invention
Figure.
Fig. 2 is the comparison figure of same rice blast bacterial strain sporulation quantity on different culture media in the specific embodiment of the invention.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.In addition to being defined, institute in following embodiment
Technical and scientific term has the identical meanings being commonly understood by with those skilled in the art of the invention.
1st, materials and methods
1.1 bacterial strain
Pyricularia oryzae 1-10 (ZB13), academy of agricultural sciences of Jiangxi Province harmful organism control stress point laboratory preserve.
1.2 for examination culture medium
Rice straw leachate：30g rice straws are shredded impregnates 16-24h with 1000mL clear water；
Potato leachate：The chopping of 200g potato decortications impregnates 2h with 1000mL clear water；
Rice straw maize powder medium：Rice straw 30g, corn flour 20g, agar 18g, clear water 1000mL；
PDA culture medium：Potato 200g, glucose 20g, agar 18g, clear water 1000mL；
Medium oatmeal:Oatmeal 50g, sucrose 20g, agar 16g, water 1000mL；
Combo 1：Rice straw leachate+potato leaches (1:1) (agar 18g is added per 1000mL)；
Combo 2：Rice straw leachate+PDA culture medium (1:1)；
Combo 3：Rice straw maize powder medium+potato leachate (1:1)；
Combo 4：Rice straw maize powder medium+PDA culture medium (1:1)；
Combo 5：Rice straw maize powder medium；
Combo 6：PDA culture medium；
Combo 7：Rice straw leaches liquid culture medium (adding agar 18g per 1000mL)；
Combo 8：Medium oatmeal.
1.3 production spore methods
The fungus block of the strains tested picking same size of pure culture is inoculated into the culture dish for 8 kinds of formula culture mediums of examination
On (9cm), it is placed in 28 DEG C of insulating boxs and cultivates.After mycelia on tablet is covered with (about 7d), tissue culture frame disposed within is moved
Upper black light lamp illumination cultivation 3d, 25~28 DEG C of room temperature.After fully producing spore, spore under being washed per ware with 15mL distillations, 100 times
Spore quantity is calculated under microscope.
The comparison of 1.4 same bacterial strain sporulation quantities on different formulations culture medium
After strains tested 1-10 (being preserved by this laboratory) is activated in PDA culture medium, with card punch obtain diameter 5mm into
The consistent bacterium dish of ripe degree is inoculated in different formulations culture medium central, 28 DEG C of culture 7d, then is placed under black light lamp and irradiates 3d, uses
15mL sterilizing washing lower surface mycelia, Buddhist nun's suede filtered through gauze obtain rice blast fungus conidial suspension, with 100 power microscopes
Observation calculates the quantity of spore under a visual field, is repeated 3 times, is averaged and is compared.
The comparison of 1.5 same bacterial strain speeds of growth in different product spore culture mediums
After strains tested 1-10 cultivates 28 DEG C of culture 7d of constant incubator on different formulations culture medium, it is straight to measure bacterium colony
Footpath calculates the rice blast fungus speed of growth, is repeated 3 times, is averaged and is compared.
The calculating of 1.6 colony growth rates
Colony diameter (cm)=colony diameter total length (cm)-inoculation bacterium dish diameter (5mm)
2 results and analysis
The comparison of 2.1 same bacterial strain growth rates on different formulations culture medium
After strains tested 1-10 is activated in PDA culture medium, the consistent bacterium of diameter 5mm maturity is obtained with card punch
Dish is inoculated in the culture medium central of different combo ingredients, and colony diameter is measured after 28 DEG C of constant temperature incubation 7d as a result, more identical bacterium
Growing state of the strain on different combo culture mediums, experimental result are shown in Table 1, Fig. 1.
By table 1, Fig. 1 is shown, there were significant differences for the culture speed of the culture mediums of 8 kinds of different combos to Pyricularia oryzae.For examination
Bacterial strain 1-10 28 DEG C of constant temperature incubations in culture medium combo 1, combo 2, combo 3, combo 4, combo 5, combo 6, combo 7, combo 8
The average colony diameter of 7d be respectively 7.66cm, 7.62cm, 7.38cm, 7.51cm, 6.45cm, 7.39cm, 5.27cm,
7.36cm, combo 1, combo 2 and 4 strain growth of combo are best, are trained between three without significant difference, combo 3, combo 6 and combo 8
Bacteria strain speed is taken second place, and is thirdly combo 5, combo 7 is worst.
Speed of growth comparative result of the 1 same bacterial strain of table on different combo culture mediums
Note：Significance difference analysis result of the lowercase letter under 5% probability in table
The comparison of 2.2 same bacterial strain sporulation quantities on different formulations culture medium
After strains tested 1-10 is activated in PDA culture medium, the consistent bacterium of diameter 5mm maturity is obtained with card punch
Dish is inoculated in different formulations culture medium central, 28 DEG C of culture 7d, then is placed under black light lamp and irradiates 3d, under 15mL sterilizing washings
Surface mycelia in one culture dish, Buddhist nun's suede filtered through gauze obtain rice blast fungus conidial suspension, seen with 100 power microscopes
It examines, calculates the quantity of spore under a visual field, be repeated 3 times, be averaged and be compared.
Experimental result is from table 2, Fig. 2, strains tested 1-10 sporulation quantity difference ratios on the culture medium of 8 kinds of different combos
Larger, otherness is notable.It produces spore using culture medium combo 1, combo 2, combo 3, combo 4, combo 5, combo 6 and micro- is regarded at one
Average spore count under wild is respectively 35.3,50.3 43.3 35.6,44.6,19.3,22.6,37.3.
It is best that combo 2 produces spore, is thirdly combo 1 secondly for combo 3 and combo 5, combo 4 and combo 8, combo 6 produce spore with combo 7
It is worst.
Sporulation quantity comparative result of the 2 same bacterial strain of table on different combo culture mediums
Note：Significance difference analysis result of the lowercase letter under 5% probability in table
2.3 combos 2 are compared with medium oatmeal, barley corn medium culture, the advantage of production spore
Medium oatmeal, barley corn medium culture are presently most used Sporogenous Medium For Pyricularia Oryzaes, using with
Group 2, medium oatmeal, barley corn culture medium respectively cultivate strains tested 1-10 and produce spore, relatively entirely produce spore cycle length, production
Spore amount, financial cost.That 50 cultures are made is flat for each 1L culture mediums of medium oatmeal that are compared with of combo 2, wherein sporulation quantity
Plate culture Pyricularia oryzae simultaneously produces spore, and under the sterile washings of 1000mL on all tablets mycelia, spore, filtered with double gauze
Spore suspension is obtained, microscopy spore count under 100 power microscopes records the spore quantity under a visual field, is repeated 3 times, is averaged
Value.The barley corn that 1L is measured with container makes barley corn culture medium, and inoculation is washed for examination Involved in Sporulation in Magnaporthe grisea after producing spore with 1000mL
Magnaporthe grisea spore on lower barley corn, double gauze filter, and microscopy spore count under 100 power microscopes is recorded under a visual field
Spore quantity, be repeated 3 times, be averaged.
3 three kinds of different culture media culture Involved in Sporulation in Magnaporthe grisea of table compare
Note："+" represents the height of cost and the complexity in source, and "+" more multilist is shown as that this is higher, and raw material sources are got over
Difficulty or ease obtain.
As known from Table 3, required for three kinds of culture medium combos 2, medium oatmeal, barley corn culture medium entirely produce the spore cycle
Number of days be respectively 10 days, 12 days, 15 days, be respectively 42,32,35 under the average each microscopic field of sporulation quantity.It can be seen that
Combo 2 is in sporulation quantity with that will be better than medium oatmeal, barley corn culture medium, the warp of Synthetical cultivation based raw material on the production spore cycle
Cost of helping is with obtaining complexity, it is believed that combo 2 is produced on spore more than current common culture medium in Pyricularia oryzae culture
Advantage.
3rd, conclusion
In 8 kinds of the Pyricularia oryzae cultures, product spore culture mediums of this experimental formula, combo 2 has Growth of Magnaporthe Grisea fast, produces
The characteristics of spore amount is big, indices are better than other preparation culture mediums.
2 main formula of combo is the compound of combo 6 and combo 7, and combo 2 is to the culture speed of Pyricularia oryzae and combo 6
Quite, far faster than combo 7；Pyricularia oryzae has been respectively increased 160.62% in 2 sporulation quantity ratio of combo in combo 6, combo 7,
122.57%, it is seen that the combination of combo 2 organizes culture medium in production spore ability with huge synergistic effect than first wife.
The ingredient of combo 2 and production method are as follows in this experiment：Rice straw leachate：
(1) 30g rice straws are shredded impregnates 16-24h with 1000mL clear water；
(2) PDA culture medium：Potato 200g, glucose 20g, agar 18g, clear water 1000mL；First by potato 200g, grape
Sugared 20g, clear water 1000mL boil 15 minutes filtered through gauze and are configured to nutrient solution；
(3) nutrient solution that rice straw leachate and (2) are prepared is pressed 1:1 is made into mixed nutrient solution, then by per 1000mL nutrition
Liquid adds in agar 18g and dissolves by heating, is down flat plate after sterilizing, is finally completed the making of culture medium flat plate.
The embodiment of the present invention is described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., should all
It is included within protection scope of the present invention.
Claims (4)
- It is 1. a kind of for Pyricularia oryzae culture, the culture medium of production spore, it is characterised in that be by isometric rice straw leachate and PDA What culture medium mixed；Wherein described rice straw leachate is rice straw and water by 3:100(g:ML after ratio mixing), 16 are impregnated ~for 24 hours gained soak；The PDA culture medium includes the ingredient of following parts by weight：200 parts of potato, 20 parts of glucose, water 1000 parts.
- It is 2. according to claim 1 a kind of for Pyricularia oryzae culture, the culture medium of production spore, it is characterised in that state PDA trainings Foster base further includes the agar of 18 parts by weight.
- It is 3. according to claim 1 a kind of for Pyricularia oryzae culture, the culture medium of production spore, it is characterised in that the rice blast Germ is Chinese rice blast fungus microspecies ZB13.
- 4. for Pyricularia oryzae culture, the preparation method for the culture medium for producing spore described in claim 2, it is characterised in that including following Step：1) take rice straw with water by 3:100(g:ML ratio) mixes the two, and immersion 16~for 24 hours, obtain rice straw leachate；2) boil 15 minutes, use after the water of the potato of 200 parts by weight, the glucose of 20 parts by weight, 1000 parts by weight is mixed Filtered through gauze takes filtrate；3) filtrate obtained by the rice straw leachate obtained by step 1) and step 2) is pressed 1:1 volume ratio mixes, into the mixture The agar of water weight 18 ‰ used in step 2) is added in, is dissolved by heating.
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| CN108865890A (en) * | 2018-07-24 | 2018-11-23 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | The long-term preservation method of rice blast bacterial strain |
| CN110438197A (en) * | 2019-08-09 | 2019-11-12 | 福建省南平市农业科学研究所 | Carry application of the barley corn of Pyricularia oryzae in rice blast Screening of Fungicide |
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| CN110438197A (en) * | 2019-08-09 | 2019-11-12 | 福建省南平市农业科学研究所 | Carry application of the barley corn of Pyricularia oryzae in rice blast Screening of Fungicide |
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