CN106011220B - The method for quickly separating and detecting and used medium of sickle-like bacteria in a kind of sample - Google Patents

The method for quickly separating and detecting and used medium of sickle-like bacteria in a kind of sample Download PDF

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CN106011220B
CN106011220B CN201610618080.6A CN201610618080A CN106011220B CN 106011220 B CN106011220 B CN 106011220B CN 201610618080 A CN201610618080 A CN 201610618080A CN 106011220 B CN106011220 B CN 106011220B
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李智敏
严准
严理
高春生
余永廷
曾粮斌
陈佳
程毅
孙向平
薛召东
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Abstract

The present invention relates to the method for quickly separating and detecting and used medium of sickle-like bacteria in a kind of sample, sample is coated on isolation medium and is cultivated, it is separately cultured in based component without any nitrogen source, contains by mass percentage: 0.4%-1% carbon source, 1.5-2% agar powder, 0.15-0.3%K2HPO4, 0.1-0.3%MgSO4, 0.1-0.3%CaCl2, 0.3-0.5%CaCO3, pH 6.5-7；Also contain the antibiotic that bacterium can be inhibited without inhibiting fungi in the culture medium；Carbon source is one or more of sucrose, glucose, D- galactolipin, arabinose and mannitol；Picking white translucent aerial hyphae, microexamination judge whether it is sickle-like bacteria according to cell morphological characteristic from single colonie after culture is good.The detection to the sickle-like bacteria in all kinds of samples can be achieved in the present invention, other fungal contaminations is avoided, so that detection sensitivity is higher, the period is shorter.

Description

The method for quickly separating and detecting and used medium of sickle-like bacteria in a kind of sample
Technical field
The present invention relates to a kind of strain separating technologies, and in particular to the method for quickly separating and detecting of sickle-like bacteria in a kind of sample And used medium.
Background technique
Fusarium class fungi can lead to serious corps diseases, such as wheat scab, Fusarium and jade Rice ear rot etc..The mycetogenetic fusarium toxin of the category is that a kind of secondary metabolites can make one vomiting, diarrhea, induce big joint The serious health problems such as disease even cancer.Therefore sickle-like bacteria detection technique has in customs quarantine control, field diseases prevention and control detection Important function；It and is an important detection in grain and food safety with the detection of the fusarium toxin of sickle-like bacteria tight association Mesh.
It can only reflect the toxin concentration in sample for the detection technique of fusarium toxin content, can not be in test sample It is no that there are sickle-like bacteria living body bacterium；During the unpredictable foodstuff preservation of this method toxin whether can persistent accumulation risk.Therefore, Fusarium toxin during sickle-like bacteria content detection will be the key that prevention and control foodstuff preservation is carried out before foodstuff preservation to accumulate.
Mainly there are 4 classes to the detection method of sickle-like bacteria in field diseases or customs quarantine control at present:
1. being based on the detection technique of polymerase chain reaction (PCR)
This kind of detection technique designs a pair of generally directed to a certain segment in the genome of some or several Fusarium Species Or multiple special primers, obtain target fragment by one or many PCR amplifications, and to the segment of amplification carry out band analysis or The kind to judge thallus is sequenced.The detection technique sensitivity is still lower, and (Monitoring lower-cut is 500 cell/g samples containing sickle-like bacteria Product), band analysis false positive is high, living body and dead volume bacterium, the inadequate wide spectrum of detection range cannot be distinguished.
2. the detection technique based on antibody
Such technology has special colour developing after the specific antibody for obtaining some specific reaping hook bacterial strain, with antibody connection is some Whether the zymoprotein of reaction contains the specific reaping hook bacterial strain in test sample by chromogenic reaction.The detection technique research and development and Use cost is high, and detection range is also very narrow.
3. the sickle-like bacteria spore In vivo detection technology based on fluorescent staining
Such fluorescent staining technique is usually to carry out living body judgement to the bacterial strain for being purifying.Living body bacterium and dead bacterium can To catch different fluorescence colors, judge whether thallus is living body with this.The detection technique does not have specific aim, can not directly use Sickle-like bacteria pollution in detection grain.
4. the detection method based on sickle-like bacteria separation
Culture medium PDA or the PPA culture medium added with pentachloronitrobenzene (PCNB) are often used using fungi, are divided from all kinds of samples From sickle-like bacteria.Pure bacterial strain is obtained after multi-step continuously separates, then carries out bacterial strain identification by means such as Molecular Identifications.
This detection method is generally be directed to the material with obvious fusaridiosis.Such as Lu Weihong et al. is rotten from corncob Pure bacterial strain is obtained with the method for PDA culture medium separating for several times in sick sample, then proves that isolated layer goes out sickle by Molecular Identification (Lu Weihong, Huang Siliang, Tao Aili wait fusarium prolifertum in maize kernel rot sample to knife bacterium Fusarium proliferatum Separation and identification [J] plant protection journal, 2011,38 (03): 233-239).Li Yongjian et al. is from banana blight The Tanaka of (fusarium disease) takes rhizosphere soil, is screened after more wheel separation with the PPA culture medium with certain selection index system Prove that isolated is that (Li Yongjian, Chen Yuanfeng, Yu Guohui wait dwarf banana to sickle-like bacteria to pure bacterium colony, then with method for identifying molecules The separation of planting soil sickle-like bacteria and identification [J] guangdong agricultural science, 2014,41 (16): 74-80).
Since PDA is the culture medium of popularity, all kinds of fungies can fast-growth above it, therefore isolate and purify work It measures very big, it is difficult to which screening obtains purpose sickle-like bacteria.
PPA culture medium has certain screening effect after joined PCNB, it is possible to reduce it is some to found withered bacterium and anthrax-bacilus etc. The pollution of fungi, but it is still very big to screen isolated difficulty and workload, needs continuous multi-cycle separation screening.Such as Wang Jianfei First step isolated strains from the chu chrysanthemum continuous cropping soil for have fusaridiosis with PPA culture medium, then " picking is grown on culture medium The bacterium colony of 5d is inoculated into purifying culture 3d on potato culture, then picking colony is inoculated on potato culture, is so followed (Wang Jianfei, Zhou Yi, Gao Xiang wait separation, the mirror of Fusarium oxysporum in chu chrysanthemum continuous cropping soil to ring, until the strain purified " Fixed and variation characteristic [J] biology magazine, 2011,28 (06): 46-48).
Since such detection method is generally directed to containing a fairly large number of sample with sickle-like bacteria illness of sickle-like bacteria, and need Want multiple separation that could obtain reaping hook bacterial strain；Therefore the less sample of no sickle-like bacteria illness, thallus content is not suitable for Quickly detection.
To sum up, the detection method based on sickle-like bacteria separation, disadvantage are: cultivating used in a, current such separation method Base such as PDA or PPA etc. cause the pollution rate of other fungies high, need multiple multi-cycle separation that could obtain pure bacterial strain；Separating difficulty Greatly.B, the separation characteristic as a, this method separation cycle is too long, one separation circulation time 5 days or so, it is more than two Separation circulation need 10 days or more working times, not being suitable for quickly detecting.C, the separation characteristic as a, separation Heavy workload needs to expend biggish manpower and material resources.
Summary of the invention
The object of the present invention is to provide a kind of method for quickly separating and detecting of sickle-like bacteria in sample, it can be achieved that all kinds of samples In sickle-like bacteria detection, other fungal contaminations are avoided, so that detection sensitivity is higher, the period is shorter.
The present inventor has found during correlative study: sickle-like bacteria is a kind of fungi with nitrogen fixation, has and is not required to Want any nitrogen source, as long as having suitable carbon source and inorganic salts can normal growth physiological property；So far there are no it is any about Sickle-like bacteria can be with the report of fixed nitrogen.And the culture medium such as PDA that uses of detection method in the prior art based on sickle-like bacteria separation or The nitrogen source all rich in such as PPA, therefore the pollution rate that will lead to other fungies is high.
The technical solution of the present invention is as follows: in a kind of sample sickle-like bacteria method for quickly separating and detecting, comprising:
Sample is coated on isolation medium and is cultivated, it is described to be separately cultured in based component without any nitrogen source, by quality Percentage contains: 0.4%-1% carbon source, 1.5-2% agar powder, 0.15-0.3%K2HPO4, 0.1-0.3%MgSO4, 0.1-0.3% CaCl2, 0.3-0.5%CaCO3, pH 6.5-7；Also contain the antibiotic that bacterium can be inhibited without inhibiting fungi in the culture medium； The carbon source is one or more of sucrose, glucose, D- galactolipin, arabinose and mannitol；
Picking white translucent aerial hyphae, microexamination judge according to cell morphological characteristic from single colonie after culture is good It whether is sickle-like bacteria.
In a specific embodiment, the sample is derived from maize seed, corn stigma or wild rice stem.
In a specific embodiment, it is described can inhibit bacterium without inhibit fungi antibiotic be streptomysin and/ Or cephalosporin.
In a specific embodiment, the isolation medium thickness in plate is not less than 4 millimeters；Plate is not required to close Envelope keeps ventilative.
In a specific embodiment, the culture is placed in constant incubator, and 28~30 DEG C are cultivated 2~3 days.
In a specific embodiment, the water droplet containing 15-30% glycerol is dripped on glass slide, by the white of picking The translucent aerial hyphae of color is scattered in water droplet, and with micro- sem observation, by rule of thumb or software determines whether sickle-like bacteria.
In a specific embodiment, the kind of sickle-like bacteria is further determined that by fungal molecule identification method.Tool For body, picking white translucent aerial hyphae is inoculated in after fungi liquid culture medium further cultivates, and carries out ITS sequence amplification Sequencing.
A kind of culture medium for the quick separating detection of sickle-like bacteria in sample provided by the invention, in medium component not Containing any nitrogen source, contain by mass percentage: 0.4%-1% carbon source, 1.5-2% agar powder, 0.15-0.3%K2HPO4, 0.1- 0.3%MgSO4, 0.1-0.3%CaCl2, 0.3-0.5%CaCO3, pH 6.5-7；The carbon source is sucrose, glucose, D- gala One or more of sugar, arabinose and mannitol.
The method of the present invention is applicable to detect the sickle-like bacteria in the less sample of no sickle-like bacteria illness, thallus content, different It surely is the material with obvious fusaridiosis；Detection sensitivity is high.
It examines through many experiments, can reach using the accuracy rate containing sickle-like bacteria in the method for the present invention separation detection sample 100%；It is even lower that Monitoring lower-cut reaches 10 living cells/g；Separation detection can be completed within 2-3 days.
Compared to 1. in the detection technique of polymerase chain reaction (PCR), the present invention has the advantages that detection sensitivity is significant It improves；The detection cycle time is ideal, more economical real without other workloads during cultivating in addition to simple film-making observation With.
Compared to 2. based on the detection technique of antibody, the present invention can be improved the detection sensitivity of sickle-like bacteria, reduce testing cost.
Compared to the detection method 4. based on sickle-like bacteria separation, the present invention, can be significant other than 4. possessed economy Other fungal contaminations are reduced, the selective mechanisms period greatly shortens, and significantly improves working efficiency.
To sum up, the method for the present invention, which can be realized, carries out economic, easy and quick separating detection to the sickle-like bacteria in all kinds of samples Purpose.This method more wide spectrum, detection sensitivity is higher, the period is shorter, cost is lower.It is remarkably improved the detection effect of sickle-like bacteria Rate.Present invention can apply to sickle-like bacteria quarantine, the detection of seed sickle-like bacteria and the detections of grain sickle-like bacteria etc. in customs quarantine control.
Detailed description of the invention
Fig. 1 is the separation situation comparison of maize seed glutinous 2000 in embodiment one；
Fig. 2 is the separation situation comparison of corn stigma in embodiment one；
Fig. 3 is the doubtful sickle-like bacteria that microscope is observed in embodiment one；
Fig. 4 PCR detects sample DNA and EF1- α gene PCR gel electrophoresis in comparative test.
Specific embodiment
In a specific embodiment of the invention, the method for quickly separating and detecting of sickle-like bacteria includes following step in sample It is rapid:
1) sample treatment
Sample is without limitation, the less sample of no sickle-like bacteria illness, the thallus content of can having drawn from, such as the corn that has drawn from Kind, corn stigma, wild rice stem etc..
Fluid sample can be directly used for applying ware；Solid sample then takes 0.5-1g, adds appropriate amounts of sterilized water, aseptically grinds Juice is taken to apply ware after mill.
2) preparation of isolation medium
Isolation medium: carbon source containing 0.4%-1%, 1.5-2% agar powder by mass percentage, 0.15-0.3% K2HPO4, 0.1-0.3%MgSO4, 0.1-0.3%CaCl2, 0.3-0.5%CaCO3, pH 6.5-7, without any nitrogen source.It is described Carbon source is one or more of sucrose, glucose, D- galactolipin, arabinose and mannitol.
It is autoclaved after the culture medium bottling sealing of configuration.The culture medium to have sterilized can immediately using or put It sets stand-by.Culture medium falls to be added before ware one or more antibiotic of the inhibition bacterial growth of debita spissitudo, in preferred culture medium Every kind of antibiotic concentration is 100 mcg/mls or so.The culture medium thickness poured into plate is not less than 4mm.
3) ware culture is applied
50-200 microlitres of sample liquids is taken in being coated in above-mentioned isolation medium plate, not sealed to keep ventilative. Then plate back-off is placed in constant incubator, 28~30 DEG C are cultivated 2~3 days.
4) picking white translucent aerial hyphae microexamination
Miscellaneous bacteria is very rare in cultured plate.Upper water droplet of the drop containing 15-30% glycerol is dripped on glass slide； (on culture medium of the present invention there is this from picking white translucent aerial hyphae in single colonie in plate with sterile, tapering utensil The mycelia of feature is generally sickle-like bacteria), it is scattered on slide glass water droplet and can be used to microexamination detection.It can be with experimenter Sickle-like bacteria differentiating forms experience to determine whether sickle-like bacteria, or utilize " sickle-like bacteria Micrograph image processing and identifying system " (Wang Guobin sickle-like bacteria Micrograph image processing and identifying system exploitation [D] Agricultural University Of Hebei, 2010；Guo Ling, Zhao Yan, Liu Yong Good fortune waits research and development [J] agricultural research of sickle-like bacteria image identification system, 2011,33 (07): 122-124.) etc. it is soft The judgement of part progress sickle-like bacteria.So far, the sickle-like bacteria quick separating detection work in sample is completed.
The artificial experience or software judgment method of micro- sem observation are all reliable judgment modes；But it can also be eventually by true Bacterium molecule identification method, such as ITS, 18S and 28S DNA sequence dna sequencing etc. further determine that.
If 5) be used for other research work with the above-mentioned sickle-like bacteria being separated to
The white translucent aerial hyphae in picking single colonie is only needed to be inoculated in common fungi liquid culture medium further Culture.
The step can use common fungi liquid culture medium, or (be not added with above-mentioned liquid separation culture medium Agar, other components and proportion are identical as step 2).
6) bacterial strain is identified
In order to prove above-mentioned the 4) step it is isolated whether be sickle-like bacteria, picking mycelia through the 5) step culture extract DNA Afterwards, ITS sequence amplification sequencing is carried out.Fungi ITS sequence primer is ITS1 and ITS4 (ITS 1:5'-TCCG TAGG TGAA CCTG CGG-3 ', ITS 4:5'-TCCT CCGC TTAT TGAT ATGC-3 ').
One sickle-like bacteria separation detection optimization experiment of embodiment
Several solid mediums are configured, it is spare after high pressure-temperature sterilizing.Several medium component contents are respectively as follows:
1. PPA:D- galactolipin 1%, peptone 0.5%, KH2PO40.1%, MgSO40.05%, agar powder 2%, pH value 6.8；Pentachloronitrobenzene (PCNB), which is added, in the when of falling plate makes its final concentration of 150 mcg/ml, and streptomysin and cephalosporin is added Final concentration is respectively 100 mg/mls.
2. PDA:200g peeled potatoes are to boil water to cross filtered juice about 800ml, add 20g glucose, 20 agar powders, natural ph, Pure water is added to be settled to 1L；Streptomysin is added in the when of falling plate and cephalosporin final concentration is respectively 100 mg/mls.
3. isolation medium of the invention: 1% sucrose, 1.7% agar powder, 0.2%K2HPO4, 0.25%MgSO4, 0.25%CaCl2, 0.4%CaCO3, pH value 7.0；The when of falling plate be added streptomysin and cephalosporin final concentration be respectively 100 milligrams/ Milliliter.
Mortar toasts 2 hours through 110 DEG C, and after being cooled to room temperature, addition 3ml sterile water has enough liquid after grinding sample Body；Weighing about 0.5g maize seed respectively, " glutinous 2000 " (buying from market) and 0.5 corn stigma (from Changsha field), are respectively put into and grind It smashs to pieces, stir evenly in alms bowl.It respectively draws 200 microlitres of aqueous to be respectively coated in above-mentioned 1. -3. three kind of culture medium, covers plate lid Open-ended after son, back-off plate, which is placed in 28 DEG C of incubators, to be cultivated 3 days.
Picking aerial hyphae is scattered in containing in 15% glycerol water droplet, under the microscope microscopy.
As a result " the separation situation such as Fig. 1 of glutinous 2000 ": being covered with by other moulds 1 maize seed quickly in PDA culture medium, several Entire culture dish is covered, a few individual bacterium colony is through micro- sem observation, the bacterium colony of no sickle-like bacteria feature.PPA culture medium In have 16 filamentous fungi bacterium colonies, have 1 doubtful sickle-like bacteria bacterium colony through sediments microscope inspection.In " separation " culture medium of the invention In, only 3 bacterium colonies with sparse white translucent aerial hyphae (since background is white culture medium, do not see bacterium on figure It falls, but can visually see in real sample)；It is doubtful sickle-like bacteria through microexamination 3.
As a result the separation situation such as Fig. 2 of 2 corn stigmas: covering is covered with by a large amount of fungies in PDA culture dish, list can not be chosen Bacterium colony；The mycelia for attempting picking 10 different bacterium colonies, through micro- sem observation without doubtful sickle-like bacteria.Have in PPA culture dish big Fungi single colonie is measured, wherein filamentous fungi bacterium colony there are 69, through micro- sem observation, there are 2 doubtful sickle-like bacteria bacterium colonies.In the present invention " separation " culture medium in, 5 have a sparse white translucent aerial hyphae bacterium colony (since background is white culture medium, figure On do not see the bacterium colony of sparse white translucent aerial hyphae), be doubtful sickle-like bacteria through micro- sem observation.
To maize seed, " the separation statistical conditions of glutinous 2000 " and corn stigma such as table 1, from " success rate ", the present invention is dividing It is significantly improved from upper success rate is detected, success rate is up to 100% in our many experiments.In terms of screening process, common PDA The various fungal contaminations of fungi culture medium are very serious, it is difficult to isolate sickle-like bacteria；And although PPA has a part screening effect, but To, there are still more serious living contaminants, separative efficiency is still lower in the screening process of different materials sieve；It is and existing in terms of result Technology is compared, the present invention can the inhibition of high degree other Mycophyta miscellaneous bacterias, quick separating detects sickle-like bacteria.
The case where 1 two kinds of materials of table separate sickle-like bacteria in different medium
Table note: success rate=sickle-like bacteria clump count/filamentous fungi clump count × 100%.
From maize seed " doubtful sickle-like bacteria such as Fig. 3 a in the filamentous fungi that glutinous 2000 " are separated to.Liquid through isolation medium Body culture medium extracts DNA after expanding culture, with fungi ITS1, ITS4 primer (ITS 1:5'-TCCG TAGG TGAA CCTG CGG-3 ', ITS 4:5'-TCCT CCGC TTAT TGAT ATGC-3 ') carry out PCR amplification after be sequenced.Sequencing result is in NCBI Upper Blastn is compared, and the similitude with Fusarium verticillioides is 99%, it was demonstrated that the bacterium being separated to is exactly sickle Knife bacterium.
The sequence obtained, which is sequenced, is
>ITS Fusarium verticillioides
CTCCCAACCCCTGTGACATACCAATTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGC CCGCCAGAGGACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAA CGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAAT CATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCA AGCCCAGCTTGGTGTTGGGACTCGCGAGTCAAATCGCGTTCCCCAAATCGATTGGCGGTCACGTCGAGCTTCCATAG CGTAGTAGTAAAACCCTCGTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGG ATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATA
From such as Fig. 3 b of the doubtful sickle-like bacteria in the filamentous fungi being separated in corn stigma.Through fungi ITS1, ITS4 primer into Blastn is compared on NCBI after row amplification, and as a result the similitude with Fusarium proliferatum strain H10 is 100%, it was demonstrated that being separated to is also sickle-like bacteria.
The sequence obtained, which is sequenced, is
>ITS Fusarium proliferatum strain H10
AACCCCTGTGACATACCAATTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCC AGAGGACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGAT CTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCG AATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCC CCGGGTTTGGTGTTGGGGATCGGCGAGCCCTTGCGGCAAGCCGGCCCCGAAATCTAGTGGCGGTCTCGCTGCAGCTT CCATTGCGTAGTAGTAAAACCCTCGCAACTGGTACGCGGCGCGGCCAAGCCGTTAAACCCCCAACTTCTGAATGTTG ACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAA
It is tested as a comparison with PCR detection technique
Whether to verify the detection method of PCR suitable for maize seed " the sickle-like bacteria detection glutinous 2000 " and corn stigma.Point Indescribably take " glutinous 2000 ", corn stigma (experiment with above " embodiment one " is to sample with a batch) and Fusarium after purification The sample total DNA of verticillioides and Fusarium proliferatum.According to sickle-like bacteria EF1- α gene The pair of primers EF1- α F:5 '-of (Translation Elongation Factor 1-alpha gene) homologous sequence design ATGGGTAAGGAGGACAAGACTCAC-3 ' and EF1- α R:5 '-GAGCGACAACATACCAATGACGG-3 ', to sample DNA into Row PCR amplification, through 1% agarose gel electrophoresis testing result such as Fig. 4.
The genomic DNA of 4 samples has extracted success in Fig. 4, but the DNA is used to carry out EF1- α gene as pcr template When fragment amplification, only the DNA profiling of F.verticillioides and F.proliferatum sample can amplify purpose band, And maize seed glutinous 2000 and corn stigma sample can not amplify purpose band.Therefore, contrast and experiment further illustrates: in purpose It is not sensitive enough by the detection method of based on PCR technology in the less sample of cell (sickle-like bacteria) content, it can not detect The a small amount of sickle-like bacteria contained in sample；And method of the invention can go out the very low sickle-like bacteria of content with separation detection.
Sickle-like bacteria in two separation detection wild rice stem of embodiment
The embodiment place different from embodiment one is: the wild rice stem bought from food market is as material；It only prepares and divides Two kinds of culture mediums of solid and liquid from culture medium, component content are as follows: (fluid nutrient medium is or not 0.5% mannitol, 2% agar powder Add agar powder), 0.3%K2HPO4, 0.1%MgSO4, 0.1%CaCl2, 0.5%CaCO3, pH value 6.5.
It turns out the white translucent mycelia come and is tentatively judged as sickle-like bacteria through micro- sem observation.Through separating fluid nutrient medium Further expansion culture is sequenced after extracting DNA with fungi ITS1 and ITS4 primer amplification；The sequence of sequencing carries out on NCBI Blastn is compared, and as a result has 100% similitude with Fusarium chlamydosporum strain NZD-mf112.So far It there is no the relevant report for being reported in and sickle-like bacteria being separated or detected in wild rice stem, this example demonstrates that the present invention can be widely applied to The separation identification of sickle-like bacteria in various samples！
>ITS Fusarium chlamydosporum
CTCCCAACCCCTGTGACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACGGGACGG CCCGCCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAACAAATAAATCAAAACTTTCAACA ACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAA TCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTC AAGCTCAGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGTCGAGCTTCCATAGC GTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTAAAACCCCAACTTCTGAATGTTGACCTCGG ATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAA
As can be seen that quick separating that can be highly sensitive, inexpensive of the invention detects various samples from above-described embodiment In sickle-like bacteria.

Claims (8)

1. the method for quickly separating and detecting of sickle-like bacteria in a kind of sample, characterized by comprising:
Sample is coated on isolation medium and is cultivated, it is described to be separately cultured in based component without any nitrogen source, by quality percentage Than containing: 0.4%-1% carbon source, 1.5-2% agar powder, 0.15-0.3% K2HPO4, 0.1-0.3% MgSO4, 0.1-0.3% CaCl2, 0.3-0.5% CaCO3, pH 6.5-7；Also contain the antibiotic that bacterium can be inhibited without inhibiting fungi in the culture medium；The carbon Source is one or more of sucrose, glucose, D- galactolipin, arabinose and mannitol；
Picking white translucent aerial hyphae, microexamination judge whether according to cell morphological characteristic from single colonie after culture is good For sickle-like bacteria；
It is described that bacterium can be inhibited without inhibiting the antibiotic of fungi for streptomysin and/or cephalosporin.
2. the method for quickly separating and detecting of sickle-like bacteria in sample according to claim 1, it is characterised in that the sample takes From maize seed, corn stigma or wild rice stem.
3. the method for quickly separating and detecting of sickle-like bacteria in sample according to claim 1 or 2, it is characterised in that in plate Isolation medium thickness is not less than 4 millimeters；Plate is not required to seal, and keeps ventilative.
4. the method for quickly separating and detecting of sickle-like bacteria in sample according to claim 1 or 2, it is characterised in that the culture It is placed in constant incubator, 28 ~ 30 DEG C are cultivated 2 ~ 3 days.
5. the method for quickly separating and detecting of sickle-like bacteria in sample according to claim 1, it is characterised in that on glass slide Water droplet containing 15-30% glycerol in drop disperses the white translucent aerial hyphae of picking in water droplet, with micro- sem observation, By rule of thumb or software determines whether sickle-like bacteria.
6. according to claim 1 or 5 in sample sickle-like bacteria method for quickly separating and detecting, it is characterised in that pass through fungi Method for identifying molecules further determines that the kind of sickle-like bacteria.
7. the method for quickly separating and detecting of sickle-like bacteria in sample according to claim 6, it is characterised in that picking white half Transparent aerial hyphae is inoculated in after fungi liquid culture medium further cultivates, and carries out ITS sequence amplification sequencing.
8. a kind of culture medium for the quick separating detection of sickle-like bacteria in sample, it is characterised in that be free of and appoint in medium component What nitrogen source, contains: 0.4%-1% carbon source, 1.5-2% agar powder, 0.15-0.3% K by mass percentage2HPO4, 0.1-0.3% MgSO4, 0.1-0.3% CaCl2, 0.3-0.5% CaCO3, pH 6.5-7；The carbon source be sucrose, glucose, D- galactolipin, Ah Draw one or more of uncle's sugar and mannitol；
Also contain the antibiotic that bacterium can be inhibited without inhibiting fungi in the culture medium.
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