CN104745488A - Method for obtaining marigold black spot pathogenic bacteria single spore system by virtue of inoculation and isolation - Google Patents
Method for obtaining marigold black spot pathogenic bacteria single spore system by virtue of inoculation and isolation Download PDFInfo
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Abstract
The invention belongs to the field of isolation and identification of pathogenic bacteria and in particular relates to a method for obtaining marigold black spot pathogenic bacteria single spore system by virtue of inoculation and isolation. The method comprises the following steps: collecting sick plants, preparing spore suspension, inoculating, performing bacterial parasite separation, producing spores and performing single spore system isolation. According to the bacterial parasite separation step, the adopted plants are not sick field plants, but naturally pathogenic plants obtained from the manual inoculation step; only one-step dish transfer is performed after the bacterial parasite separation step, the single spores can be subjected to ultra-low temperature storage, and a single spore system with good activity is obtained. The needed time of obtaining a batch of bacterial systems (comprising 7-9 single spore systems) is shortened from half a year to 1 month, and the speed is obviously improved compared with the previous speed; and moreover, the existence of saprophytic bacteria is effectively removed, and the marigold black spot pathogenic bacteria can be accurately obtained.
Description
Technical field
The invention belongs to the isolation identification field of pathogenic bacteria, particularly a kind of method being obtained Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system by inoculation and isolation.
Background technology
Flower of Aztec Marigold (Tagetes erecta L.) is composite family (Compusitae) Tagetes (Tagetes) annual herb plant, not only can be used for the landscaping in city, be also the important source material extracting natural carotenol simultaneously.Along with continuous popularization and the expansion of cultivated area, the generation of Flower of Aztec Marigold blackspot disease is day by day serious, and particularly the climatic anomaly time is as cloudy weather for several days running sky or high temperature and drought etc.The marigold with pigment of Yanqing County of Beijing area establishing in large scale, the serious generation of 2010 and 2011 2 years diseases has become its limiting factor developed.In the production of development Flower of Aztec Marigold, disease to have become one of important limiting factor.
Adopt traditional classification authentication method to the qualification of Flower of Aztec Marigold blackspot pathogenic bacteria at present, because of the difference of form, region, conclusion is also inconsistent more.China has on some documents and had report to Flower of Aztec Marigold black spot, they have used different kind names: as noble and unsullied (noble and unsullied, Bai Qingrong, Dong Ran, Deng generation and the pathogen identification [J] of .2002. marigold bacterial leaf spot. Jilin Auto Industry, 24 (2): 94-96,107) etc. by morphology, Physiological-biochemical Characters and Ecological Characteristics, the pathogenic bacterium of marigold bacterial leaf spot are defined as pseudomonas syringae Flower of Aztec Marigold pvs oryzae and oryzicola (Pseudomonas syringae pv.tagetes); The Pathogen identification of Gansu province Leaf of Aztec Marigold pinta is then Alternaria (Alternaria sp.) species by Wang Long etc.; The pathogen identification of Leaf of Aztec Marigold pinta is Alternaria tenuissima (A.tenuissima) by identification of morphology by Wang Ting etc.
Time needed for aforesaid method is longer, complex operation, and obtaining a collection of fungus strain (7 ~ 9 monospore systems) needs half a year.Further, traditional classification authentication method often cannot reject saprophytic microorganism effectively when bacterial isolate bacterium, limited accuracy, causes the qualification result of different people different.Therefore, a kind of separation method of easy and simple to handle, the time is short, accuracy is high Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system is needed in current scientific research and practice.
Summary of the invention
The invention provides a kind of method being obtained Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system by inoculation and isolation.
The present invention is achieved by the following technical solutions:
Obtained a method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system by inoculation and isolation, comprise the following steps:
Invalid plant acquisition step:
Gather the invalid plant of the Flower of Aztec Marigold black spot in field;
Spore suspension preparation steps:
Get the black mould in the stem section of described invalid plant, be mixed with spore suspension;
Tieback step:
Described spore suspension is inoculated in Flower of Aztec Marigold seedling, obtains the plant of natural occurrence;
Bacterial parasite separating step:
Choose the part with scab of the plant being grown on described natural occurrence, after sterilization, cultivate the bacterium colony obtaining doubtful bacterial parasite;
Produce spore step:
The bacterium colony of described doubtful bacterial parasite is carried out expansion numerous, bring out generation spore; Again described spore inoculating is verified parasitics in Flower of Aztec Marigold seedling, choose the bacterial strain having parasitics;
Monospore system separating step:
There is the bacterial strain of parasitics to carry out monospore system separating treatment by described, obtain Alternaria alternata caused occurrence monospore system.
Aforesaid method preferred embodiment in, in described invalid plant acquisition step, also described invalid plant is carried out moisturizing process.
Aforesaid method preferred embodiment in, described moisturizing is treated to: described invalid plant is soaked 3-6 hour in clear water, be preferably 4 hours, then put into humidity be 100% enclosed space in moisturizing 40-60 hour, be preferably 48 hours.
Aforesaid method preferred embodiment in, in described spore suspension preparation steps, the spore concentration of described spore suspension is 5000-15000 spore/milliliter, be preferably 10000 spore/milliliters.
Aforesaid method preferred embodiment in, in described tieback step, described inoculation is: by sprinkling have the Flower of Aztec Marigold seedling of described spore suspension to be placed in closed moisture-keeping container, the atmospheric moisture in described moisture-keeping container is 100%.
Aforesaid method preferred embodiment in, in described tieback step, in described moisture-keeping container, 40-60 DEG C is housed, is preferably the water of 50 DEG C; Preferably, in described tieback step, in described moisture-keeping container, the height of the water surface reaches the 0.3-0.8 of the height of the container that Flower of Aztec Marigold seedling is housed, and more preferably 0.6.
Aforesaid method preferred embodiment in, in described bacterial parasite separating step, adopt PDA substratum; In described PDA substratum, the concentration of PDA culture medium powder is 30-50g/L, more preferably 39g/L.
Aforesaid method preferred embodiment in, in described product spore step, adopt corn culture medium; In described corn culture medium, the concentration of corn culture medium powder is 70-80g/L, more preferably 75g/L.
Aforesaid method preferred embodiment in, in described monospore system separating step, described monospore system separating treatment is the generation spore having the bacterial strain of parasitics described in getting, and coats on water agar, picking spore after observing; Again the spore of picking is cultivated 5-8 days on PDA substratum, obtain described monospore system.
Aforesaid method preferred embodiment in, in described monospore system separating step, in water agar, agar powder concentration is 20-40g/L, more preferably 30g/L.
Compared to existing technology, the present invention has following beneficial effect:
1, the plant that bacterial parasite separating step of the present invention adopts is the plant of the natural occurrence obtained in artificial tieback step, instead of adopt field diseased plant, merely through once turning ware and available monospore carries out Cryopreservation after bacterial parasite separating step, obtain the unit cell subsystem with good vigor.
2, the present invention obtains a collection of fungus strain (7 ~ 9 monospore systems) the required time and will shorten to 1 month from half a year, and the speed be separated is comparatively significantly improved.
3, the present invention effectively eliminate saprophytic microorganism existence, can accurately obtain Flower of Aztec Marigold Alternaria alternata caused occurrence.
Accompanying drawing explanation
Fig. 1: embodiment 1 middle period portion is with the photo of the plant of large-scale scab.
Fig. 2: embodiment 1 middle period portion is with the photo of the plant of small-sized scab.
Fig. 3: in embodiment 1, stem is with the photo of the plant of scab.
Fig. 4: the bacterium colony of the doubtful bacterial parasite in embodiment 1 on PDA culture medium flat plate.
Fig. 5: the photo not inoculating successful spore (40x10) seen under microscope (40x10) in embodiment 1.
Fig. 6: the photo of the successful spore of inoculation (40x10) seen under microscope (40x10) in embodiment 1.
Fig. 7: the photo of the bacterium colony grown at PDA culture medium flat plate as the spore of monospore system in embodiment 1.
Fig. 8: the photo of Flower of Aztec Marigold black spot field disease symptom in embodiment 1; Wherein, (a) (b) is blade disease symptom, and (c) is stem disease symptom, and (d) is the field morbidity middle and later periods.
Fig. 9: the photo of Flower of Aztec Marigold Alternaria alternata caused occurrence qualification in embodiment 1; Wherein, a () is the pathogenic bacteria front colonial morphology on PDA substratum, b () is the pathogenic bacteria reverse side colonial morphology on PDA substratum, c () is mycelia, spore shape (10 × 20), d () is spore shape (10 × 40), e () is Flower of Aztec Marigold seedling leaves disease symptom after tieback, (f) is Flower of Aztec Marigold seedling plants disease symptom after tieback.
Embodiment
Obtained a method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system by inoculation and isolation, comprise the following steps:
Step one, invalid plant gather: the invalid plant in field gathering the black spot of Flower of Aztec Marigold cross-fertilize seed seedling, kept dry under normal temperature.
Before the inoculation of step 3, moisturizing can bring out the bacterial parasite being mixed with miscellaneous bacteria; What this moisturizing was brought out is operating as: this invalid plant is soaked in sterilized water 3-6 hour (such as: the scope between any or any in 3 hours, 5 hours, 6 hours, be preferably 4 hours), make invalid plant completely moistening, again moistening invalid plant to be placed in enclosed space moisturizing 40-60 hour (such as: in 40 hours, 50 hours, 60 hours arbitrarily or arbitrarily between scope, be preferably 4 hours), humidity is 100%.
Step 2, spore suspension are prepared: choose the invalid plant that black mould is obvious, black mould (being namely mixed with the bacterial parasite of miscellaneous bacteria) in its stem section of scraping, the spore suspension that spore concentration is 5000-15000 spore/milliliter (such as: the scope between any or any in 5000 spore/milliliters, 8000 spore/milliliters, 12000 spore/milliliters, 15000 spore/milliliters, is preferably 10000 spore/milliliters) is mixed with distilled water.
Step 3, tieback (being preferably artificial tieback): this spore suspension is inoculated in Flower of Aztec Marigold seedling, obtains the plant of natural occurrence.
Being operating as of above-mentioned inoculation:
Get spore suspension, be sprayed on healthy Flower of Aztec Marigold seedling with micro-sprayer; To spray the Flower of Aztec Marigold seedling of the health of sterilized water for contrast, all seedling after sprinkling are put into moisturizing bucket, for ensureing that blade face is for a long time by moisture film is covered, 40-60 DEG C is filled (such as: the scope between any or any in 40 DEG C, 45 DEG C, 55 DEG C, 60 DEG C in bucket, be preferably 50 DEG C) sterilized water, water surface elevation reach kind to be implanted with the nutrition pot height of Flower of Aztec Marigold seedling a 0.3-0.8 (such as: 0.3,0.4,0.45,0.5, in 0.7.0.8 arbitrarily or arbitrarily between scope, be preferably 0.6); Again air humidifier is put into moisturizing bucket, then moisturizing bucket is obturaged, make atmospheric moisture in bucket reach 100%; Treat 30-50 hour (such as: the scope between any or any in 30 hours, 25 hours, 45 hours, 48 hours, 50 hours, preferably 40 hours) after, from moisturizing bucket, take out all Flower of Aztec Marigold seedling, allow plant natural occurrence, obtain the plant of natural occurrence.
Above-mentioned moisturizing bucket is preferably the plastics box of 60L.
Step 4, bacterial parasite are separated: the part with scab choosing the plant being grown on this natural occurrence, be inoculated in after sterilization PDA culture medium powder concentration be 30-50g/L (such as: in 30g/L, 35g/L, 40g/L, 45g/L, 50g/L arbitrarily or arbitrarily between scope, be preferably 39g/L), PDA substratum cultivate; After 7-10 days, obtain the bacterium colony of doubtful bacterial parasite.
The preparation method of above-mentioned PDA substratum is:
Take PDA culture medium powder, use distilled water heating for dissolving, constant volume, after sterilizing, cooled and solidified becomes PDA culture medium flat plate, and 4 DEG C of preservations are stand-by;
Being operating as of above-mentioned inoculation:
Get the blade of the plant of natural occurrence, with clear water punching twice, thieving paper suck dry moisture, chooses more typical scab, is cut into the fritter of an about centimeter square; Wash 30 seconds with 75% spirituous solution again, and then use the chlorine bleach liquor of 15% to sterilize 1 ~ 3 minute; After using rinsed with sterile water twice again, be cut into about 2 ~ 3 millimeters of square fritters, then with aseptic washing once, be placed on respectively on PDA culture medium flat plate and cultivate.
The plant that this step adopts is the plant of the natural occurrence obtained in artificial tieback step, instead of adopts field diseased plant, afterwards merely through once turning ware and available monospore carries out Cryopreservation, can obtain the unit cell subsystem with good vigor.
Step 5, produce spore: the bacterium colony of this doubtful bacterial parasite is forwarded to corn culture medium powder concn be 70-80g/L (such as: in 70g/L, 72g/L, 76g/L, 78g/L, 80g/L arbitrarily or arbitrarily between scope, be preferably 75g/L), it is numerous that corn culture medium carries out expansion, bring out generation spore, by spore inoculating in Flower of Aztec Marigold seedling checking parasitics, choose the bacterial strain having parasitics.
Above-mentioned verification method, according to section He Shi rule, carries out pathogenicbacteria separation again and again inoculates, to determine that whether isolate is for pathogenic bacterium from the plant of inoculation sequela.The preparation method of this corn culture medium is:
Take corn culture medium powder, use distilled water heating for dissolving, constant volume, after sterilizing, cooled and solidified becomes corn culture medium dull and stereotyped, and 4 DEG C of preservations are stand-by;
Above-mentionedly bring out spore-bearing being operating as: cover with after corn culture medium until bacterium colony, adopt " scraping method off " to bring out generation spore, namely aseptically strike off bacterium colony surface fungus strain, drive ware and cultivate, cultivate and can produce spore in 2-6 days.
Step 6, monospore system are separated: this had the bacterial strain of parasitics to carry out monospore system separating treatment, obtain Alternaria alternata caused occurrence monospore system, preserve in subzero 80 DEG C of paper using dish methods.
Being operating as of this monospore system separating treatment:
On Bechtop, will bring out in spore-bearing corn culture medium flat board and add sterilized water, with spore under sterilized painting brush, obtain spore liquid; Spore liquid is drawn again with liquid-transfering gun, be added in agar powder concentration be 20-40g/L (such as: in 20g/L, 25g/L, 28g/L, 35g/L, 40g/L arbitrarily or arbitrarily between scope, be preferably 30g/L) water agar flat board on, with triangle glass rod, spore liquid is smoothened; Then observe under microscopical low power lens (4 times): if the spore in each visual field is no more than 1, and when there is no other spores around checking in the visual field, this in the visual field 1 spore is chosen down together with substratum by available pin of choosing, implant other PDA substratum (wherein, PDA culture medium powder concentration is 35-45g/L, can be in 30g/L, 35g/L, 40g/L, 45g/L, 50g/L arbitrarily or arbitrarily between scope, be preferably 39g/L) innerly cultivate 5-8 days, be preferably 7 days, namely obtain 1 monospore system.
The preparation method of above-mentioned water agar plate is:
Take agar powder, use distilled water heating for dissolving, constant volume, after sterilizing, cooled and solidified becomes water agar plate, and 4 DEG C of preservations are stand-by.
Above method obtains a collection of fungus strain (7 ~ 9 monospore systems) the required time and will shorten to 1 month from half a year, and the speed be separated is comparatively significantly improved; Above method can effectively eliminate saprophytic microorganism existence, can accurately obtain Flower of Aztec Marigold Alternaria alternata caused occurrence.
Embodiment 1:
The method of the acquisition Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system of the present embodiment, comprises the following steps:
(1), invalid plant gathers: the invalid plant in field gathering the black spot of Flower of Aztec Marigold cross-fertilize seed seedling, kept dry under normal temperature.
As shown in Figure 8, Fig. 8-a, Fig. 8-b are the blade disease symptom of field Flower of Aztec Marigold cross-fertilize seed seedling, and Fig. 8-c is the stem disease symptom of field Flower of Aztec Marigold cross-fertilize seed seedling, and Fig. 8-d is the field Flower of Aztec Marigold cross-fertilize seed seedling morbidity middle and later periods.
(2), moisturizing is brought out: this invalid plant is soaked 4 hours in sterilized water, make invalid plant completely moistening, again moistening invalid plant is placed in encloses container, is moisturizing 48 hours under the condition of 100% in humidity, obtains the invalid plant inducing black mould.
(3), spore suspension preparation: choose the invalid plant that black mould is obvious, the black mould (being namely mixed with the bacterial parasite of miscellaneous bacteria) in its stem section of scraping, is mixed with sterilized water the spore suspension that spore concentration is 10000 spore/milliliters.
(4), artificial tieback: the spore suspension getting about 15ml, be sprayed on the Flower of Aztec Marigold seedling of 5 strain health with micro-sprayer, spray the Flower of Aztec Marigold seedling of the health of sterilized water with 5 other strains for contrast, all Flower of Aztec Marigold seedling are all planted in nutrition pot; All seedling after sprinkling are put into the plastics box of 60L, and for ensureing that blade face is for a long time by moisture film is covered, fill the sterilized water of 50 DEG C in case, water surface elevation reaches 0.6 of nutrition pot height; Again air humidifier is put into plastics box, then plastics box is obturaged, make atmospheric moisture in case reach 100%; From plastics box, take out all Flower of Aztec Marigold seedling after 40 hours, allow plant natural occurrence, obtain the plant of natural occurrence.The appearance of the plant of natural occurrence as shown in Figure 1, Figure 2, shown in Fig. 3, Fig. 9-e, Fig. 9-f: the leaf portion of the plant of Fig. 1 is with large-scale scab, the leaf portion of the plant of Fig. 2 is with small-sized scab, the stem of the plant of Fig. 3 is with scab, Fig. 9-e is Flower of Aztec Marigold seedling leaves disease symptom after tieback, and Fig. 9-f is Flower of Aztec Marigold seedling plants disease symptom after tieback.
(5), bacterial parasite be separated: the blade getting the plant of natural occurrence, with clear water punching twice, thieving paper suck dry moisture, chooses more typical scab, is cut into the fritter of an about centimeter square; Wash 30 seconds with 75% spirituous solution again, and then use the chlorine bleach liquor of 15% to sterilize 1 ~ 3 minute; After using rinsed with sterile water twice again, be cut into about 2 ~ 3 millimeters of square fritters, again with aseptic washing once, (preparation method is: take 9.75g PDA culture medium powder to be placed on the PDA culture medium flat plate that PDA culture medium powder concentration is 39g/L respectively, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes PDA culture medium flat plate, 4 DEG C of preservations are stand-by) above cultivate after 7-10 days, obtain the bacterium colony of doubtful bacterial parasite.
The feature of the bacterium colony of doubtful bacterial parasite for: as shown in Fig. 4, Fig. 9-a, Fig. 9-b, bacterial strain growth regulation 2-3 days on PDA culture medium flat plate is white, and gray area appears in central authorities afterwards, white mycelium fine hair shape; As shown in Fig. 9-c, Fig. 9-d, microscopy finds that its conidiophore list is raw or grow thickly, tawny; As shown in Fig. 6, Fig. 9-c and Fig. 9-d, conidium is single raw or concatenate, brown, and spore body hangs contracting obviously, size (65 ~ 140) μm × (15 ~ 42) μm, tabula number 3 ~ 8, mediastinum number 0 ~ 7; As shown in Fig. 6, Fig. 9-d, beak and spore body are distinguished obviously, size (7 ~ 110) μm × (3 ~ 10) μm, tabula number 0 ~ 3.
(6), spore is produced: (preparation method is: take 18.75g corn culture medium powder the bacterium colony of this doubtful bacterial parasite to be forwarded to the corn culture medium flat board that corn culture medium powder concn is 75g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes corn culture medium dull and stereotyped, 4 DEG C of preservations are stand-by) to carry out expansion numerous, cover with after corn culture medium until bacterium colony, " scraping method off " is adopted to bring out generation spore, namely bacterium colony surface fungus strain is aseptically struck off, drive ware to cultivate, cultivate and can produce spore in 2-6 days; The spore inoculating induced is verified parasitics in Flower of Aztec Marigold seedling, chooses according to section's He Shi rule the bacterial strain having parasitics.
(7), monospore system is separated: on Bechtop, growth is had the sterilized water adding 2ml in the corn culture medium flat board of the bacterial classification of parasitics, with spore under sterilized painting brush, obtains spore liquid, the spore liquid of 50 μ l is drawn again with liquid-transfering gun, (preparation method is: take 7.5g agar powder to be added in the water agar flat board that agar powder concentration is 30g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes water agar plate, 4 DEG C of preservations are stand-by) on, with triangle glass rod, spore liquid is smoothened, then observe under microscopical low power lens (4 times) at once: if the spore in each visual field is no more than 1, and when there is no other spores around checking in the visual field, this in the visual field 1 spore is chosen down together with substratum by available pin of choosing, implant other PDA substratum (PDA culture medium powder concentration is 39g/L) inner cultivation 7 days, namely 1 monospore system is obtained, this monospore is lain in subzero 80 DEG C of paper using dish methods to preserve.
The sporocyst of step (5) the microscopy doubtful Alternaria alternata caused occurrence monospore system of the present embodiment is described as: spore body hangs contracting obviously: long 65 ~ 140 microns, wide 15 ~ 42 microns, tabula number 3 ~ 8, and rake is every 0 ~ 7; Beak: distinguish obviously with spore body, long 7 ~ 110 microns, wide 3 ~ 10 microns, tabula number 0 ~ 3.(be recited as according to Zhang Tianyu: spore body: 72.5 ~ 109x15.5 ~ 25.0 micron, spore body: diaphragm 8 ~ 13, rake is every 0 ~ 5; Beak: light brown ~ colourless, 46.5 ~ 133.0x2.5 ~ 4.5 micron).From measurement numerically, we with " Chinese fungi will " upper Zhang Tianyu (Zhang Tianyu .2003. China fungi will the 16 volume Alternaria [M]. Science Press: that 86) reports is close.Situation in conjunction with inoculation morbidity proves, the germ be separated in the present embodiment should be exactly the pathogenic bacteria (as Fig. 9-c, Fig. 9-d institute diagram) of the Flower of Aztec Marigold black spot of the record of " Chinese fungi will ".
During step (7) monospore system is separated, the bacterium colony for growing after cultivating 8 days as the spore of monospore system on other PDA substratum in Fig. 7; Step (6) is produced in spore, Fig. 5 be see under microscope (40x10) do not inoculate successful spore (40x10), Fig. 6 is the successful spore of inoculation (40x10) seen under microscope (40x10).
Table 1: the measuring result (unit of length is micron) of the black class germ Altrnaria tagitica size that the present embodiment is separated to
The existing document of China there is report to Flower of Aztec Marigold black spot, Wang Long (Wang Long, Zhang Xiaoling, He Dongyun, Deng generation and the Pathogen identification [J] of .2007. Leaf of Aztec Marigold pinta. agriculture in south (landscape flower version), 2 (4): 7-9) only say that they obtain Flower of Aztec Marigold pathogenic bacteria and " Chinese fungi will " report different, therefore not named (Alternaria sp.).Wang Ting (Wang Ting, Wang Long, the flourish .2010. Leaf of Aztec Marigold pinta Pathogen identification of Wang Sheng and biological characteristic research [J] thereof. Gansu Agriculture University's journal, 3:66-68) be decided to be toVerticilliumdahliaActivitie Activitie S of Relative [Alternaria tenuissima (Fr) Wiltshire].In addition, Jilin agricultural university in 2002 Wu Xinying (Wu Xinying .2002. Flower of Aztec Marigold rod method leaf spot research [D]. Jilin Agriculture University: 1-2) Master's thesis named as Alternaria dianthicola (Alternaria gypsophilae).All with the present embodiment, not inoculate successful saprophytic microorganism similar viewed from the Photomicrograph that they provide; Therefore, not probably the cause of disease of desired Flower of Aztec Marigold black spot in the present embodiment.
The Flower of Aztec Marigold black spot size that the Flower of Aztec Marigold alternaria spore document that table 2, the present embodiment obtain is recorded compares
Embodiment 2:
The method of the acquisition Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system of the present embodiment, comprises the following steps:
(1), invalid plant gathers: the invalid plant in field gathering the black spot of Flower of Aztec Marigold cross-fertilize seed seedling, kept dry under normal temperature.
(2), moisturizing is brought out: this invalid plant is soaked 3 hours in sterilized water, make invalid plant completely moistening, again moistening invalid plant is placed in encloses container, is moisturizing 40 hours under the condition of 100% in humidity, obtains the invalid plant inducing black mould.
(3), spore suspension preparation: choose the invalid plant that black mould is obvious, the black mould (being namely mixed with the bacterial parasite of miscellaneous bacteria) in its stem section of scraping, is mixed with sterilized water the spore suspension that spore concentration is 5000 spore/milliliters.
(4), artificial tieback: the spore suspension getting about 15ml, be sprayed on the Flower of Aztec Marigold seedling of 5 strain health with micro-sprayer, spray the Flower of Aztec Marigold seedling of the health of sterilized water with 5 other strains for contrast, all Flower of Aztec Marigold seedling are all planted in nutrition pot; All seedling after sprinkling are put into the plastics box of 60L, and for ensureing that blade face is for a long time by moisture film is covered, fill the sterilized water of 40 DEG C in case, water surface elevation reaches 0.3 of nutrition pot height; Again air humidifier is put into plastics box, then plastics box is obturaged, make atmospheric moisture in case reach 100%; From plastics box, take out all Flower of Aztec Marigold seedling after 40 hours, allow plant natural occurrence, obtain the plant of natural occurrence.
(5), bacterial parasite be separated: the blade getting the plant of natural occurrence, with clear water punching twice, thieving paper suck dry moisture, chooses more typical scab, is cut into the fritter of an about centimeter square; Wash 30 seconds with 75% spirituous solution again, and then use the chlorine bleach liquor of 15% to sterilize 1 ~ 3 minute; After using rinsed with sterile water twice again, be cut into about 2 ~ 3 millimeters of square fritters, again with aseptic washing once, (preparation method is: take 9.75g PDA culture medium powder to be placed on the PDA culture medium flat plate that PDA culture medium powder concentration is 39g/L respectively, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes PDA culture medium flat plate, 4 DEG C of preservations are stand-by) above cultivate after 7-10 days, obtain the bacterium colony of doubtful bacterial parasite.
This bacterium colony of microscopy, the sporocyst of doubtful Alternaria alternata caused occurrence monospore system is described as: spore body hangs contracting obviously: long 110 ~ 120 microns, wide 30 ~ 35 microns, tabula number 6 ~ 9, and rake is every 3 ~ 6; Beak: distinguish obviously with spore body, long 7 ~ 110 microns, wide 5 ~ 10 microns, tabula number 0 ~ 3.
(6), spore is produced: (preparation method is: take 17.5g corn culture medium powder the bacterium colony of this doubtful bacterial parasite to be forwarded to the corn culture medium flat board that corn culture medium powder concn is 70g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes corn culture medium dull and stereotyped, 4 DEG C of preservations are stand-by) to carry out expansion numerous, cover with after corn culture medium until bacterium colony, " scraping method off " is adopted to bring out generation spore, namely bacterium colony surface fungus strain is aseptically struck off, drive ware to cultivate, cultivate and can produce spore in 2-6 days; The spore inoculating induced is verified parasitics in Flower of Aztec Marigold seedling, chooses according to section's He Shi rule the bacterial strain having parasitics.
(7), monospore system is separated: on Bechtop, growth is had the sterilized water adding 2ml in the corn culture medium flat board of the bacterial classification of parasitics, with spore under sterilized painting brush, obtains spore liquid, the spore liquid of 50 μ l is drawn again with liquid-transfering gun, (preparation method is: take 5g agar powder to be added in the water agar flat board that agar powder concentration is 20g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes water agar plate, 4 DEG C of preservations are stand-by) on, with triangle glass rod, spore liquid is smoothened, then observe under microscopical low power lens (4 times) at once: if the spore in each visual field is no more than 1, and when there is no other spores around checking in the visual field, this in the visual field 1 spore is chosen down together with substratum by available pin of choosing, implant other PDA substratum (PDA culture medium powder concentration is 39g/L) inner cultivation 7 days, namely 1 monospore system is obtained, this monospore is lain in subzero 80 DEG C of paper using dish methods to preserve.
Embodiment 3:
The method of the acquisition Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system of the present embodiment, comprises the following steps:
(1), invalid plant gathers: the invalid plant in field gathering the black spot of Flower of Aztec Marigold cross-fertilize seed seedling, kept dry under normal temperature.
(2), moisturizing is brought out: this invalid plant is soaked 6 hours in sterilized water, make invalid plant completely moistening, again moistening invalid plant is placed in encloses container, is moisturizing 60 hours under the condition of 100% in humidity, obtains the invalid plant inducing black mould.
(3), spore suspension preparation: choose the invalid plant that black mould is obvious, the black mould (being namely mixed with the bacterial parasite of miscellaneous bacteria) in its stem section of scraping, is mixed with sterilized water the spore suspension that spore concentration is 15000 spore/milliliters.
(4), artificial tieback: the spore suspension getting about 15ml, be sprayed on the Flower of Aztec Marigold seedling of 5 strain health with micro-sprayer, spray the Flower of Aztec Marigold seedling of the health of sterilized water with 5 other strains for contrast, all Flower of Aztec Marigold seedling are all planted in nutrition pot; All seedling after sprinkling are put into the plastics box of 60L, and for ensureing that blade face is for a long time by moisture film is covered, fill the sterilized water of 60 DEG C in case, water surface elevation reaches 0.8 of nutrition pot height; Again air humidifier is put into plastics box, then plastics box is obturaged, make atmospheric moisture in case reach 100%; From plastics box, take out all Flower of Aztec Marigold seedling after 40 hours, allow plant natural occurrence, obtain the plant of natural occurrence.
(5), bacterial parasite be separated: the blade getting the plant of natural occurrence, with clear water punching twice, thieving paper suck dry moisture, chooses more typical scab, is cut into the fritter of an about centimeter square; Wash 30 seconds with 75% spirituous solution again, and then use the chlorine bleach liquor of 15% to sterilize 1 ~ 3 minute; After using rinsed with sterile water twice again, be cut into about 2 ~ 3 millimeters of square fritters, again with aseptic washing once, (preparation method is: take 9.75g PDA culture medium powder to be placed on the PDA culture medium flat plate that PDA culture medium powder concentration is 39g/L respectively, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes PDA culture medium flat plate, 4 DEG C of preservations are stand-by) above cultivate after 7-10 days, obtain the bacterium colony of doubtful bacterial parasite.
This bacterium colony of microscopy, the sporocyst of doubtful Alternaria alternata caused occurrence monospore system is described as: spore body hangs contracting obviously: long 65 ~ 140 microns, wide 15 ~ 42 microns, tabula number 3 ~ 8, and rake is every 0 ~ 7; Beak: distinguish obviously with spore body, long 7 ~ 110 microns, wide 3 ~ 10 microns, tabula number 0 ~ 4.
(6), spore is produced: (preparation method is: take 20g corn culture medium powder the bacterium colony of this doubtful bacterial parasite to be forwarded to the corn culture medium flat board that corn culture medium powder concn is 80g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes corn culture medium dull and stereotyped, 4 DEG C of preservations are stand-by) to carry out expansion numerous, cover with after corn culture medium until bacterium colony, " scraping method off " is adopted to bring out generation spore, namely bacterium colony surface fungus strain is aseptically struck off, drive ware to cultivate, cultivate and can produce spore in 2-6 days; The spore inoculating induced is verified parasitics in Flower of Aztec Marigold seedling, chooses according to section's He Shi rule the bacterial strain having parasitics.
(7), monospore system is separated: on Bechtop, growth is had the sterilized water adding 2ml in the corn culture medium flat board of the bacterial classification of parasitics, with spore under sterilized painting brush, obtains spore liquid, the spore liquid of 50 μ l is drawn again with liquid-transfering gun, (preparation method is: take 10g agar powder to be added in the water agar flat board that agar powder concentration is 40g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes water agar plate, 4 DEG C of preservations are stand-by) on, with triangle glass rod, spore liquid is smoothened, then observe under microscopical low power lens (4 times) at once: if the spore in each visual field is no more than 1, and when there is no other spores around checking in the visual field, this in the visual field 1 spore is chosen down together with substratum by available pin of choosing, implant other PDA substratum (PDA culture medium powder concentration is 39g/L) inner cultivation 7 days, namely 1 monospore system is obtained, this monospore is lain in subzero 80 DEG C of paper using dish methods to preserve.
Embodiment 4:
The method of the acquisition Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system of the present embodiment, comprises the following steps:
(1), invalid plant gathers: the invalid plant in field gathering the black spot of Flower of Aztec Marigold cross-fertilize seed seedling, kept dry under normal temperature.
(2), moisturizing is brought out: this invalid plant is soaked 3 hours in sterilized water, make invalid plant completely moistening, again moistening invalid plant is placed in encloses container, is moisturizing 60 hours under the condition of 100% in humidity, obtains the invalid plant inducing black mould.
(3), spore suspension preparation: choose the invalid plant that black mould is obvious, the black mould (being namely mixed with the bacterial parasite of miscellaneous bacteria) in its stem section of scraping, is mixed with sterilized water the spore suspension that spore concentration is 12000 spore/milliliters.
(4), artificial tieback: the spore suspension getting about 15ml, be sprayed on the Flower of Aztec Marigold seedling of 5 strain health with micro-sprayer, spray the Flower of Aztec Marigold seedling of the health of sterilized water with 5 other strains for contrast, all Flower of Aztec Marigold seedling are all planted in nutrition pot; All seedling after sprinkling are put into the plastics box of 60L, and for ensureing that blade face is for a long time by moisture film is covered, fill the sterilized water of 40 DEG C in case, water surface elevation reaches 0.8 of nutrition pot height; Again air humidifier is put into plastics box, then plastics box is obturaged, make atmospheric moisture in case reach 100%; From plastics box, take out all Flower of Aztec Marigold seedling after 40 hours, allow plant natural occurrence, obtain the plant of natural occurrence.
(5), bacterial parasite be separated: the blade getting the plant of natural occurrence, with clear water punching twice, thieving paper suck dry moisture, chooses more typical scab, is cut into the fritter of an about centimeter square; Wash 30 seconds with 75% spirituous solution again, and then use the chlorine bleach liquor of 15% to sterilize 1 ~ 3 minute; After using rinsed with sterile water twice again, be cut into about 2 ~ 3 millimeters of square fritters, again with aseptic washing once, (preparation method is: take 9.75g PDA culture medium powder to be placed on the PDA culture medium flat plate that PDA culture medium powder concentration is 39g/L respectively, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes PDA culture medium flat plate, 4 DEG C of preservations are stand-by) above cultivate after 7-10 days, obtain the bacterium colony of doubtful bacterial parasite.
This bacterium colony of microscopy, the sporocyst of doubtful Alternaria alternata caused occurrence monospore system is described as: spore body hangs contracting obviously: long 65 ~ 140 microns, wide 15 ~ 42 microns, tabula number 3 ~ 8, and rake is every 0 ~ 7; Beak: distinguish obviously with spore body, long 7 ~ 110 microns, wide 3 ~ 10 microns, tabula number 0 ~ 4.
(6), spore is produced: (preparation method is: take 19.25g corn culture medium powder the bacterium colony of this doubtful bacterial parasite to be forwarded to the corn culture medium flat board that corn culture medium powder concn is 77g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes corn culture medium dull and stereotyped, 4 DEG C of preservations are stand-by) to carry out expansion numerous, cover with after corn culture medium until bacterium colony, " scraping method off " is adopted to bring out generation spore, namely bacterium colony surface fungus strain is aseptically struck off, drive ware to cultivate, cultivate and can produce spore in 2-6 days; The spore inoculating induced is verified parasitics in Flower of Aztec Marigold seedling, chooses according to section's He Shi rule the bacterial strain having parasitics.
(7), monospore system is separated: on Bechtop, growth is had the sterilized water adding 2ml in the corn culture medium flat board of the bacterial classification of parasitics, with spore under sterilized painting brush, obtains spore liquid, the spore liquid of 50 μ l is drawn again with liquid-transfering gun, (preparation method is: take 8.75g agar powder to be added in the water agar flat board that agar powder concentration is 35g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes water agar plate, 4 DEG C of preservations are stand-by) on, with triangle glass rod, spore liquid is smoothened, then observe under microscopical low power lens (4 times) at once: if the spore in each visual field is no more than 1, and when there is no other spores around checking in the visual field, this in the visual field 1 spore is chosen down together with substratum by available pin of choosing, implant other PDA substratum (PDA culture medium powder concentration is 39g/L) inner cultivation 7 days, namely 1 monospore system is obtained, this monospore is lain in subzero 80 DEG C of paper using dish methods to preserve.
Embodiment 5:
The method of the acquisition Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system of the present embodiment, comprises the following steps:
(1), invalid plant gathers: the invalid plant in field gathering the black spot of Flower of Aztec Marigold cross-fertilize seed seedling, kept dry under normal temperature.
(2), moisturizing is brought out: this invalid plant is soaked 6 hours in sterilized water, make invalid plant completely moistening, again moistening invalid plant is placed in encloses container, is moisturizing 40 hours under the condition of 100% in humidity, obtains the invalid plant inducing black mould.
(3), spore suspension preparation: choose the invalid plant that black mould is obvious, the black mould (being namely mixed with the bacterial parasite of miscellaneous bacteria) in its stem section of scraping, is mixed with sterilized water the spore suspension that spore concentration is 8000 spore/milliliters.
(4), artificial tieback: the spore suspension getting about 15ml, is sprayed on the Flower of Aztec Marigold seedling of 5 strain health with micro-sprayer, spray the Flower of Aztec Marigold seedling of the health of sterilized water with 5 strains for contrast, all Flower of Aztec Marigold seedling are all planted in nutrition pot; All seedling after sprinkling are put into the plastics box of 60L, and for ensureing that blade face is for a long time by moisture film is covered, fill the sterilized water of 45 DEG C in case, water surface elevation reaches 0.7 of nutrition pot height; Again air humidifier is put into plastics box, then plastics box is obturaged, make atmospheric moisture in case reach 100%; From plastics box, take out all Flower of Aztec Marigold seedling after 40 hours, allow plant natural occurrence, obtain the plant of natural occurrence.
(5), bacterial parasite be separated: the blade getting the plant of natural occurrence, with clear water punching twice, thieving paper suck dry moisture, chooses more typical scab, is cut into the fritter of an about centimeter square; Wash 30 seconds with 75% spirituous solution again, and then use the chlorine bleach liquor of 15% to sterilize 1 ~ 3 minute; After using rinsed with sterile water twice again, be cut into about 2 ~ 3 millimeters of square fritters, again with aseptic washing once, (preparation method is: take 9.75g PDA culture medium powder to be placed on the PDA culture medium flat plate that PDA culture medium powder concentration is 39g/L respectively, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes PDA culture medium flat plate, 4 DEG C of preservations are stand-by) above cultivate after 7-10 days, obtain the bacterium colony of doubtful bacterial parasite.
This bacterium colony of microscopy, the sporocyst of doubtful Alternaria alternata caused occurrence monospore system is described as: spore body hangs contracting obviously: long 78 ~ 135 microns, wide 18 ~ 25 microns, tabula number 3 ~ 8, and rake is every 1 ~ 7; Beak: distinguish obviously with spore body, long 55 ~ 100 microns, wide 4 ~ 9 microns, tabula number 1 ~ 2.
(6), spore is produced: (preparation method is: take 18g corn culture medium powder the bacterium colony of this doubtful bacterial parasite to be forwarded to the corn culture medium flat board that corn culture medium powder concn is 72g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes corn culture medium dull and stereotyped, 4 DEG C of preservations are stand-by) to carry out expansion numerous, cover with after corn culture medium until bacterium colony, " scraping method off " is adopted to bring out generation spore, namely bacterium colony surface fungus strain is aseptically struck off, drive ware to cultivate, cultivate and can produce spore in 2-6 days; The spore inoculating induced is verified parasitics in Flower of Aztec Marigold seedling, chooses according to section's He Shi rule the bacterial strain having parasitics.
(7), monospore system is separated: on Bechtop, growth is had the sterilized water adding 2ml in the corn culture medium flat board of the bacterial classification of parasitics, with spore under sterilized painting brush, obtains spore liquid, the spore liquid of 50 μ l is drawn again with liquid-transfering gun, (preparation method is: take 8g agar powder to be added in the water agar flat board that agar powder concentration is 32g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes water agar plate, 4 DEG C of preservations are stand-by) on, with triangle glass rod, spore liquid is smoothened, then observe under microscopical low power lens (4 times) at once: if the spore in each visual field is no more than 1, and when there is no other spores around checking in the visual field, this in the visual field 1 spore is chosen down together with substratum by available pin of choosing, implant other PDA substratum (PDA culture medium powder concentration is 39g/L) inner cultivation 7 days, namely 1 monospore system is obtained, this monospore is lain in subzero 80 DEG C of paper using dish methods to preserve.
Embodiment 6:
The method of the acquisition Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system of the present embodiment, comprises the following steps:
(1), invalid plant gathers: the invalid plant in field gathering the black spot of Flower of Aztec Marigold cross-fertilize seed seedling, kept dry under normal temperature.
(2), moisturizing is brought out: this invalid plant is soaked 5 hours in sterilized water, make invalid plant completely moistening, again moistening invalid plant is placed in encloses container, is moisturizing 50 hours under the condition of 100% in humidity, obtains the invalid plant inducing black mould.
(3), spore suspension preparation: choose the invalid plant that black mould is obvious, the black mould (being namely mixed with the bacterial parasite of miscellaneous bacteria) in its stem section of scraping, is mixed with sterilized water the spore suspension that spore concentration is 14000 spore/milliliters.
(4), artificial tieback: the spore suspension getting about 15ml, is sprayed on the Flower of Aztec Marigold seedling of 5 strain health with micro-sprayer, spray the Flower of Aztec Marigold seedling of the health of sterilized water with 5 strains for contrast, all Flower of Aztec Marigold seedling are all planted in nutrition pot; All seedling after sprinkling are put into the plastics box of 60L, and for ensureing that blade face is for a long time by moisture film is covered, fill the sterilized water of 45 DEG C in case, water surface elevation reaches 0.45 of nutrition pot height; Again air humidifier is put into plastics box, then plastics box is obturaged, make atmospheric moisture in case reach 100%; From plastics box, take out all Flower of Aztec Marigold seedling after 40 hours, allow plant natural occurrence, obtain the plant of natural occurrence.
(5), bacterial parasite be separated: the blade getting the plant of natural occurrence, with clear water punching twice, thieving paper suck dry moisture, chooses more typical scab, is cut into the fritter of an about centimeter square; Wash 30 seconds with 75% spirituous solution again, and then use the chlorine bleach liquor of 15% to sterilize 1 ~ 3 minute; After using rinsed with sterile water twice again, be cut into about 2 ~ 3 millimeters of square fritters, again with aseptic washing once, (preparation method is: take 9.75g PDA culture medium powder to be placed on the PDA culture medium flat plate that PDA culture medium powder concentration is 39g/L respectively, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes PDA culture medium flat plate, 4 DEG C of preservations are stand-by) above cultivate after 7-10 days, obtain the bacterium colony of doubtful bacterial parasite.
This bacterium colony of microscopy, the sporocyst of doubtful Alternaria alternata caused occurrence monospore system is described as: spore body hangs contracting obviously: long 65 ~ 100 microns, wide 20 ~ 40 microns, tabula number 3 ~ 5, and rake is every 2 ~ 5; Beak: distinguish obviously with spore body, long 8 ~ 100 microns, wide 6 ~ 10 microns, tabula number 0 ~ 3.
(6), spore is produced: (preparation method is: take 19g corn culture medium powder the bacterium colony of this doubtful bacterial parasite to be forwarded to the corn culture medium flat board that corn culture medium powder concn is 76g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes corn culture medium dull and stereotyped, 4 DEG C of preservations are stand-by) to carry out expansion numerous, cover with after corn culture medium until bacterium colony, " scraping method off " is adopted to bring out generation spore, namely bacterium colony surface fungus strain is aseptically struck off, drive ware to cultivate, cultivate and can produce spore in 2-6 days; The spore inoculating induced is verified parasitics in Flower of Aztec Marigold seedling, chooses according to section's He Shi rule the bacterial strain having parasitics.
(7), monospore system is separated: on Bechtop, growth is had the sterilized water adding 2ml in the corn culture medium flat board of the bacterial classification of parasitics, with spore under sterilized painting brush, obtains spore liquid, the spore liquid of 50 μ l is drawn again with liquid-transfering gun, (preparation method is: take 9.5g agar powder to be added in the water agar flat board that agar powder concentration is 38g/L, use distilled water heating for dissolving, be settled to 250ml, after sterilizing, cooled and solidified becomes water agar plate, 4 DEG C of preservations are stand-by) on, with triangle glass rod, spore liquid is smoothened, then observe under microscopical low power lens (4 times) at once: if the spore in each visual field is no more than 1, and when there is no other spores around checking in the visual field, this in the visual field 1 spore is chosen down together with substratum by available pin of choosing, implant other PDA substratum (PDA culture medium powder concentration is 39g/L) inner cultivation 7 days, namely 1 monospore system is obtained, this monospore is lain in subzero 80 DEG C of paper using dish methods to preserve.
Claims (10)
1. obtained a method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system by inoculation and isolation, it is characterized in that: the method comprises the following steps:
Invalid plant acquisition step:
Gather the invalid plant of the Flower of Aztec Marigold black spot in field;
Spore suspension preparation steps:
Get the black mould in the stem section of described invalid plant, be mixed with spore suspension;
Tieback step:
Described spore suspension is inoculated in Flower of Aztec Marigold seedling, obtains the plant of natural occurrence;
Bacterial parasite separating step:
Choose the part with scab of the plant being grown on described natural occurrence, after sterilization, cultivate the bacterium colony obtaining doubtful bacterial parasite;
Produce spore step:
The bacterium colony of described doubtful bacterial parasite is carried out expansion numerous, bring out generation spore; Again described spore inoculating is verified parasitics in Flower of Aztec Marigold seedling, choose the bacterial strain having parasitics;
Monospore system separating step:
There is the bacterial strain of parasitics to carry out monospore system separating treatment by described, obtain Alternaria alternata caused occurrence monospore system.
2. obtained the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system according to claim 1 by inoculation and isolation, it is characterized in that: in described invalid plant acquisition step, also described invalid plant is carried out moisturizing process.
3. the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system is obtained according to claim 2 by inoculation and isolation, it is characterized in that: described moisturizing is treated to: described invalid plant is soaked 3-6 hour in clear water, be preferably 4 hours, put into again humidity be 100% enclosed space in moisturizing 40-60 hour, be preferably 48 hours.
4. the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system is obtained according to claim 1 by inoculation and isolation, it is characterized in that: in described spore suspension preparation steps, the spore concentration of described spore suspension is 5000-15000 spore/milliliter, is preferably 10000 spore/milliliters.
5. the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system is obtained according to claim 1 by inoculation and isolation, it is characterized in that: in described tieback step, described inoculation is: have the Flower of Aztec Marigold seedling of described spore suspension to be placed in closed moisture-keeping container sprinkling, the atmospheric moisture in described moisture-keeping container is 100%.
6. obtained the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system according to claim 5 by inoculation and isolation, it is characterized in that: in described tieback step, in described moisture-keeping container, 40-60 DEG C is housed, be preferably the water of 50 DEG C;
Preferably, in described tieback step, in described moisture-keeping container, the height of the water surface reaches the 0.3-0.8 of the height of the container that Flower of Aztec Marigold seedling is housed, and is preferably 0.6.
7. obtained the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system according to claim 1 by inoculation and isolation, it is characterized in that: in described bacterial parasite separating step, adopt PDA substratum; In described PDA substratum, the concentration of PDA culture medium powder is 30-50g/L, is preferably 39g/L.
8. obtained the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system according to claim 1 by inoculation and isolation, it is characterized in that: in described product spore step, adopt corn culture medium; In described corn culture medium, the concentration of corn culture medium powder is 70-80g/L, is preferably 75g/L.
9. the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system is obtained according to claim 1 by inoculation and isolation, it is characterized in that: in described monospore system separating step, described monospore system separating treatment is the generation spore having the bacterial strain of parasitics described in getting, coat on water agar, picking spore after observing; Again the spore of picking is cultivated 5-8 days on PDA substratum, obtain described monospore system.
10. obtained the method for Flower of Aztec Marigold Alternaria alternata caused occurrence monospore system according to claim 9 by inoculation and isolation, it is characterized in that: in described monospore system separating step, in water agar, agar powder concentration is 20-40g/L, be preferably 30g/L.
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| CN111088316A (en) * | 2019-12-04 | 2020-05-01 | 北京农业生物技术研究中心 | Method for observing phenotypes before and after inoculation of pathogenic bacteria on marigold |
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