CN100392103C - Molecular detection method for Fusarium circinatum - Google Patents
Molecular detection method for Fusarium circinatum Download PDFInfo
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Abstract
The present invention relates to a molecular detection method for fusarium circinatum. A primer is used for designing software Prime5.1, a pair of primers (a universal primer G1/G2 and a specific primer S1/S2) arranged on a fusarium IGS fragment is designed, and fusarium sample DNA extract liquid is used as a template for a first round of universal primer PCR amplification region; subsequently, PCR amplification products are diluted into 100 times; 1 mul is takn as a template for a second round of the specificity primer PCR amplification reaction; amplification products carry out electrophoresis detection; when 873 bp DNA specificity strips exist, detected germs are fusarium circinatum. When the method of the present invention is used for detecting the fusarium circinatum, only one workaday is needed, the detection sensitivity reaches the minimum excessive germ DNA concentration of 5*10<-3>pg/mul, and the conidiospore concentration is 10 per 100 mul. The method of the present invention can be used for fast, sensitively and accurately detecting the fusarium circinatum in soil, lumber and seeds, and is suitable for occasions with high chronergy requirements, such as port quarantine, etc. to use.
Description
Technical field
The present invention relates to relate to a kind of be suitable for that departments such as the Animal or Plant Quarantine, production of forestry and plant protection use to fusarium circinatum bacterium (Fusarium circinatum Nirenberg ﹠amp; O ' Donnell, the perfect stage is Gibberellacircinata Nirenberg ﹠amp; O ' detection method Donnell) particularly simply, molecular detecting method fast, belongs to biological field.
Background technology
Fusarium circinatum is the world today's one of severe diseases of pine tree that causes harm.Nineteen forty-six, the fusarium circinatum evil is found on southeastern US North Carolina State pinon pine (P.virginiana) first, causes that mainly trunk, branch produce symptoms such as resin flux, ulcer.The California finds that the earliest this disease is 1986, and distributing in the locality subsequently constantly enlarges, and the host of causing harm also is on the increase, and comprises multiple seeds such as Pinus, Pseudotsuga menziesii (Mirbel) Franco.In addition, Japan 1989, Mexico 1991, South Africa 1994, Chile also report the generation of this disease calendar year 2001 in succession.Fusarium circinatum presents a kind of gesture of rapid spread, serious day by day to forest harm, especially fusarium circinatum bacterium in 1986 makes the pine of California, USA middle part seashore suffer crushing blow, has caused the extensive concern of state (area) governments such as Australia, New Zealand and European Union.At present, though the report that this disease of China Shang Weiyou takes place, the quarantine of should strengthen conscientiously entering the territory seed, nursery stock, timber and Wooden package prevents that the fusarium circinatum bacterium from importing within the border.For this reason, set up fusarium circinatum bacterium economy, detection technique is significant apace.
Be considered to the kind of Li Se group sickle-like bacteria Fusarium section Liseola in the classification of fusarium circinatum bacterium, its perfect stage is Gibberella fujikuroi (sawada) Ito; Mating continum H among the Kimura.Except that above-mentioned mating continum, G.fujikuroi at least also comprises other 7 diverse mating continums in heredity, respectively with A, B, C, D, E, F, G name.Different mating continums have the tendency of growing surely in definitive host.Carry out fusarium circinatum bacterium quarantine identify usually need to carry out simultaneously pathogenic mensuration or with the sexual hybridization of known standard bacterial strain, conventional quarantine identification method is wasted time and energy, the result is also inaccurate sometimes.Viljoen A, Sreenkarnp E T and Schweigkofler W etc. once tried to disclose difference between fusarium circinatum bacterium and other sibling species from the gene angle, had set up RAPD, PCR-RFLP and real time PCR detection method in succession.Yet take a broad view of aforesaid method, the experimental repeatability that has is not strong, and the operation that has is loaded down with trivial details, and the testing cost costliness all fails to solve a difficult problem that directly detects the fusarium circinatum bacterium from sample well, and it is bigger that difficulty is directly used at the port.
Summary of the invention
The purpose of this invention is to provide a kind of economy, quick, easy, high specificity and highly sensitive fusarium circinatum bacterium molecule detection method, carry out inward seed, nursery stock, the quarantine of diseased wood timber for each port, be strictly on guard against that the fusarium circinatum bacterium is provided into by the reliable technique means that provide.
The present invention extracts the DNA of germ from mycelia, conidium and the seed of germ, soil, timber, with the sample DNA is template, carry out the pcr amplification of first round universal primer, the product of reaction is a template with the first round, carry out second pcr amplification of taking turns special primer, amplified production carries out electrophoresis detection.
Concrete detection method is as follows:
1.. extract the DNA of pine tree germ sample, preserve standby down for-20 ℃;
2.. announce that according to GenBank Fusarium belongs to IGS sequence (accession number: AY249392, AY249389, AY249397, AY249403, AY249402, AY249399, AY249398, AY249401, AY249400, AY249393, AY249395, AY249385, AY249386, AY249382, AY249384, AY249383, AY249380, AY249387, AY249391, AY249394, AY249390, AY249366, AY249388, AY249381, AY249379, AY249405, AY249404, AY249408, AY249406, AY249407, AY249409), utilize primer-design software (Prime 5.1), design the universal primer G1/G2 on transcribed spacer (IGS) fragment in a pair of sickle-like bacteria ribosomal gene, its sequence is:
G1：5’-GCGGTGTCGGTGTGCTTGTA-3’
G2：5’-ACTCACGGCCACCAAACCA-3’
3.. according to fusarium circinatum bacterium and sibling species thereof, kind of the IGS difference of being correlated with, design a pair of Auele Specific Primer S1/S2 that can differentiate the fusarium circinatum bacterium, its sequence is:
S1：5’-CTTACCTTGGCTCGAGAAGG-3’
S2：5’-CCTACCCTACACCTCTCACT-3’
4.. utilize Auele Specific Primer S1/S2 and universal primer G1/G2, it is as follows to carry out fusarium circinatum bacterium nest-type PRC amplification step:
First round amplification: universal primer PCR amplification
The reaction system cumulative volume is 25 μ l, and reactive component is: 10 * reaction buffer, 2.5 μ l, 2.5mmol/L MgCl
21.5 μ l, each 0.5 μ l of 10pmol/L upstream and downstream universal primer G1, G2,10mmol/L dNTP2 μ l, 5u/ μ l Taq enzyme 0.25 μ l, DMSO1.25 μ l, template fusarium circinatum bacterium sample DNA extracting solution 1 μ l, aqua sterilisa is supplied 25 μ l; The amplified reaction program is: 95 ℃ of sex change 5min, enter circulation, and 94 ℃ of sex change 30s, 56 ℃ of annealing 45s, 72 ℃ are extended 1min, and 20 circulations are extended 10min for back 72 ℃.
Second takes turns amplification: the special primer pcr amplification
The reaction system cumulative volume is 25 μ l, takes turns pcr amplification product with the 1st and dilutes 100 times, gets 1 μ l as template; Other reactive component is: 10 * reaction buffer, 2.5 μ l, 2.5mmol/L MgCl
21.5 μ l, each 0.5 μ l of 10pmol/L upstream primer S1, downstream primer S2,10mmol/L dNTP2 μ l, 5u/ μ l Taq enzyme 0.25 μ l, DMSO1.25 μ l, aqua sterilisa supply 25 μ l; Response procedures is: 94 ℃ of sex change 3min, enter circulation, and 94 ℃ of sex change 35s, 66 ℃ of annealing 55s, 72 ℃ are extended 50s, and 40 circulations are extended 12min for back 72 ℃.
5.. amplified production carries out electrophoresis detection
Get 10 μ l amplified productions, adopt 1.2% sepharose, under ultraviolet lamp, detect after 20～40 minutes at electrophoresis under the voltage 80V, if there is the DNA specific band of 873bp, then determine to be detected germ be the fusarium circinatum bacterium.
In sum, the present invention has successfully set up the nested PCR method of a cover detection fusarium circinatum bacterium.Use this method to detect the fusarium circinatum bacterium, only need a workaday time, it is 5 * 10 that detection sensitivity reaches the minimum germ DNA concentration of limiting the quantity of
-3Pg/ μ l, conidium 10/100 μ l.Overcome morphology and identified shortcomings such as length consuming time, the low and existing Molecular Detection repeatability of accuracy is not strong, operation is loaded down with trivial details, testing cost is expensive, detection sensitivity is low; Adopt detection method of the present invention, can be directly from soil, timber and seed fast, sensitive, accurately detect the fusarium circinatum bacterium, be particularly suitable for the strong especially occasion of ageing requirement such as port quarantine and use.
Embodiment
Below by example, the present invention is described in further details.
The Molecular Detection of embodiment fusarium circinatum bacterium
Strains tested and correlation properties see Table 1.
Table 1 strains tested and source
Table?1?Strains?tested?and?their?origins
NCAUR: the U.S. uses farming research center National Center for Agricultural Utilization Research (USA)
NJAU: the Nanjing Agriculture University of Agricultural University Of Nanjing
1. the extraction of sample DNA
1.1 the extraction of mycelium DNA
1.1-1 strain culturing and mycelium are collected
Bacterial strain is connected on the PDA substratum 20-25 ℃ of following dark culturing 10 days.With inoculating needle mycelium is scraped gently, place plastics tubing, lyophilize adds a little quartz sand and is ground to fine powder, preserves down for-20 ℃.
1.1-2 extract mycelia DNA with the CTAB method, concrete operations are as follows:
1) get the 0.2g hypha powder in the 2ml centrifuge tube, add cell lysis buffer solution 1ml, 10 μ g/ml Proteinase Ks, behind thorough mixing on the vortice, 55 ℃ of water-bath 1-3h;
2) with centrifuge tube 12,000rpm, centrifugal 10min;
3) get supernatant 800 μ l, add isopyknic phenol: chloroform: primary isoamyl alcohol is 25: 24: 1 a mixed solution, puts upside down mixing gently, 12, and 000rpm, centrifugal 10min;
Get upper strata water 600 μ l, add isopyknic chloroform: primary isoamyl alcohol is 24:: 1 mixed solution, put upside down mixing gently, 12,000rpm, centrifugal 10min;
4) get upper strata water 400 μ l, add the ice dehydrated alcohol of 2 times of volumes and the 3M NaAc of 1/10 volume, place-20 ℃ of precipitation 1-3h;
5) with centrifuge tube 12,000rpm, centrifugal 10min;
6) abandon supernatant, precipitation with 70% washing with alcohol once;
7) after the drying, with 100 μ l aqua sterilisas dissolvings, add RNase in 37 ℃ clear up 1h after, preserve standby down for-20 ℃.
1.2 the extraction of germ spore DNA
With the DNA of freeze-thaw method extraction germ spore, concrete operations are as follows:
1) get 100 μ l spore suspensions to the 2ml centrifuge tube, add cell lysis buffer solution 500 μ l, 10 μ g/ml Proteinase Ks, thorough mixing on vortice is put into mortar with centrifuge tube, adds an amount of liquid nitrogen flash freezer 2min;
2) centrifuge tube is put into 75 ℃ of water-bath 2min;
3) repeat above-mentioned freeze thawing 2 times, after the last quick-frozen, in 75 ℃ of water-bath 30min;
1) 7.5mol/LNH of adding 250 μ l
4Ac mixes the back gently in 12,000rpm, centrifugal 10min;
5) get supernatant 600 μ l, the 2ml centrifuge tube of Yu Yixin, the ice dehydrated alcohol of 2 times of volumes of adding ,-20 ℃ of precipitation 1h;
6) with centrifuge tube 12,000rpm, centrifugal 10min;
7) abandon supernatant, precipitation with 70% washing with alcohol once;
8) after the drying,, preserve standby down for-20 ℃ with the dissolving of 10 μ l aqua sterilisas.
1.3 the extraction of germ DNA in the infected seed
1) get the 20g seed in triangular flask, add aqua sterilisa, fully vibration washing 5min gets washings in 4000rpm, centrifugal 5min, collecting precipitation;
2) abandon supernatant, add 100 μ l aqua sterilisas, fully concussion;
3) get above-mentioned 100 μ l spore suspensions to the 2ml centrifuge tube, add cell lysis buffer solution 500 μ l, 10 μ g/ml Proteinase Ks, thorough mixing on vortice is put into mortar with centrifuge tube, adds an amount of liquid nitrogen flash freezer 2min;
4) centrifuge tube is put into 75 ℃ of water-bath 2min;
5) repeat above-mentioned freeze thawing 2 times, after the last quick-frozen, in 75 ℃ of water-bath 30min;
6) the 7.5mol/L NH of adding 250 μ l
4Ac mixes the back gently in 12,000rpm, centrifugal 10min;
7) get supernatant 600 μ l, the 2ml centrifuge tube of Yu Yixin, the ice dehydrated alcohol of 2 times of volumes of adding ,-20 ℃ of precipitation 1h;
8) with centrifuge tube 12,000rpm, centrifugal 10min;
9) abandon supernatant, precipitation with 70% washing with alcohol once;
10) after the drying,, preserve standby down for-20 ℃ with the dissolving of 10 μ l aqua sterilisas.
The extraction of germ DNA in the soil 1.4 carry disease germs
1) get 0.3g soil, oven dry is ground to fine powder.
2) after the fine powder after will grinding adds in the 2ml centrifuge tube, add 0.4% skim-milk, add cell lysis buffer solution 500 μ l, 10 μ g/ml Proteinase Ks, thorough mixing on vortice is put into mortar with centrifuge tube, adds an amount of liquid nitrogen flash freezer 2min;
3) centrifuge tube is put into 75 ℃ of water-bath 2min;
4) repeat above-mentioned freeze thawing 2 times, after the last quick-frozen, in 75 ℃ of water-bath 30min;
5) the 7.5mol/L NH of adding 250 μ l
4Ac mixes the back gently in 12,000rpm, centrifugal 10min;
6) get supernatant 600 μ l, the 2ml centrifuge tube of Yu Yixin, the ice dehydrated alcohol of 2 times of volumes of adding ,-20 ℃ of precipitation 1h;
7) with centrifuge tube 12,000rpm, centrifugal 10min;
8) abandon supernatant, precipitation with 70% washing with alcohol once;
9) after the drying,, preserve standby down for-20 ℃ with the dissolving of 10 μ l aqua sterilisas.
The extraction of germ DNA in the wood chip 1.5 carry disease germs
1) get the 0.5g wood sawdust, after adding 2ml centrifuge tube is interior, add cell lysis buffer solution 500 μ l, 10 μ g/ml Proteinase Ks, thorough mixing on vortice is put into mortar with centrifuge tube, adds an amount of liquid nitrogen flash freezer 2min;
2) centrifuge tube is put into 75 ℃ of water-bath 2min;
3) repeat above-mentioned freeze thawing 2 times, after the last quick-frozen, in 75 ℃ of water-bath 30min;
4) the 7.5mol/L NH4Ac of adding 250 μ l mixes the back gently in 12,000rpm, centrifugal 10min;
5) get supernatant 600 μ l, the 2ml centrifuge tube of Yu Yixin, the ice dehydrated alcohol of 2 times of volumes of adding ,-20 ℃ of precipitation 1h;
6) with centrifuge tube 12,000rpm, centrifugal 10min;
7) abandon supernatant, precipitation with 70% washing with alcohol once;
8) after the drying,, preserve standby down for-20 ℃ with the dissolving of 10 μ l aqua sterilisas.
2. the design of universal primer and special primer
According to login sickle-like bacteria rRNA intragenic spacer district IGS sequence (accession number: AY249392, AY249389, AY249397 among the GenBank, AY249403, AY249402, AY249399, AY249398, AY249401, AY249400, AY249393, AY249395, AY249385, AY249386, AY249382, AY249384, AY249383, AY249380, AY249387, AY249391, AY249394, AY249390, AY249366, AY249388, AY249381, AY249379, AY249405, AY249404, AY249408, AY249406, AY249407, AY249409), utilize primer-design software (Prime 5.1), design a pair of sickle-like bacteria universal primer G1/G2, with a pair of special primer S1/S2 at the fusarium circinatum bacterium, its sequence is:
G1：5’-GCGGTGTCGGTGTGCTTGTA-3’
G2：5’-ACTCACGGCCACCAAACCA-3’
S1：5’-CTTACCTTGGCTCGAGAAGG-3’
S2：5’-CCTACCCTACACCTCTCACT-3’
3. set up germ detection specificity amplification reaction condition
The reaction system volume is 25 μ l, and reactive component is: 10 * reaction buffer, 2.5 μ l, 2.5mmol/L MgCl
21.5 μ l, each 0.5 μ l of 10pmol/L upstream and downstream primer S1, S2,101mmol/L dNTP2 μ l, 5u/ μ l Taq enzyme 0.25 μ l, DMSO1.25 μ l, template DNA: 5ng, aqua sterilisa supply 25 μ l.Replace template DNA as negative control with aqua sterilisa.Mixed solution increases on the PTC-200PCRPCR instrument, and response procedures is: 94 ℃ of sex change 3min, enter circulation, and 94 ℃ of sex change 35s, 66 ℃ of annealing 55s, 72 ℃ are extended 50s, and 40 circulations are extended 12min for back 72 ℃
4. specific amplification sensitivity test
The DNA of NFUH-3 is diluted to different concentration gradients, is respectively 5 * 10
5Pg/ μ l, 5 * 10
4Pg/ μ l, 5 * 10
3Pg/ μ l, 5 * 10
2Pg/ μ l, 50pg/ μ l, 5pg/ μ l, 5 * 10
-1Pg/ μ l, 5 * 10
-2Pg/ μ l.Getting 1 μ l is template, follow procedure 3 augmentation detection sensitivity.
5. germ detects the universal primer amplification
Reaction system is 25 μ l, and reactive component is: 10 * reaction buffer, 2.5 μ l, 2.5mmol/L MgCl
21.5 μ l, each 0.5 μ l of 10pmol/L upstream and downstream primer G1, G2,10mmol/L dNTP2 μ l, 5u/ μ l Taq enzyme 0.25 μ l, DMSO1.25 μ l, template DNA extracting solution 1 μ l, aqua sterilisa is supplied 25 μ l.Replace template DNA as negative control with aqua sterilisa.Mixed solution increases on the PTC-200PCR instrument, and response procedures is: 95 ℃ of sex change 5min, enter circulation, and 94 ℃ of sex change 30s, 56 ℃ of annealing 45s, 72 ℃ are extended 1min, and 20 circulations are extended 10min for back 72 ℃.
6. the foundation of nest-type PRC Molecular Detection system
Follow procedure 5 carries out the 1st and takes turns the reaction of pcr amplification, and system is 25 μ l.
Follow procedure 3 is taken turns 100 times of pcr amplification product dilutions with the 1st, gets 1 μ l and carries out the 2nd as the 2nd template of taking turns amplification and take turns pcr amplification reaction, and the reaction system volume is 25 μ l.
7. the mensuration of nest-type PRC sensitivity
The DNA that extracts NFUH-3 is diluted to 5 * 10
5Pg/ μ l, 5 * 10
4Pg/ μ l, 5 * 10
3Pg/ μ l, 5 * 10
2Pg/ μ l, 50pg/ μ l, 5pg/ μ l, 5 * 10
-1Pg/ μ l, 5 * 10
-2Pg/ μ l, 5 * 1
-3Pg/ μ l, 5 * 10
-4Pg/ μ l, 5 * 10
-511 concentration gradients such as pg/ μ l, follow procedure 5 amplifications.
With 100 μ l H
2NFUH-3 spore quantity among the O is furnishing 1 * 10 respectively
7Individual, 1 * 10
6Individual, 1 * 10
5Individual, 1 * 10
4Individual, 1 * 10
3Individual, 1 * 10
2Individual, 10,1, centrifugal, extract DNA, follow procedure 5 amplifications.
In 20g slash pine seed, 0.3g soil and 0.5g wood chip, press 1 * 10 respectively
6Individual, 1 * 10
5Individual, 1 * 10
4Individual, 1 * 10
3Individual, 1 * 10
2Individual, 6 gradients such as 10 grades add the NFUH-3 spore liquid, extract DNA, follow procedure 3 amplifications.
8. test experience result
1) the universal primer G1/G2 with design increases to the genomic dna of strains tested, and the result shows, all can amplify the band of a 873bp size for 16 kinds of sickle-like bacteria of examination, and negative control and other reaping hooks kind beyond belonging to does not amplify this band.
2) with a pair of special primer S1/S2 that identifies the fusarium circinatum bacterium strains tested genomic dna is increased, the result shows, 4 bacterial strains of fusarium circinatum bacterium can amplify the specific band of 364bp size, and negative control and other bacterial strains do not amplify specific band.DNA increases to different templates concentration, and the result shows and uses special primer to carry out that pcr amplification is minimum can detected germ DNA concentration to be 50pg/ μ l.
3) nest-type PRC amplification electrophoresis result shows that 4 bacterial strains of fusarium circinatum bacterium can amplify the specific band of 364bp size, and negative control and other bacterial strains do not amplify specific band.Different templates concentration DNA is carried out the nest-type PRC amplification, and the result shows that it can detected germ DNA concentration be 5 * 10 that nest-type PRC increases minimum
-3Pg/ μ l, conidium quantity is 10.
4) nested PCR method is detected the fusarium circinatum bacterium in seed, nursery stock, timber and soil sensitivity is measured, the result can detect 100 spores in the 20g seed, 0.3g can detect 1000 spores in the soil, can detect 100 spores in the 0.5g wood chip.
Claims (2)
1. the molecular detecting method of fusarium circinatum bacterium is characterized in that detection method is as follows:
1.. extract the DNA of pine tree germ sample, preserve standby down for-20 ℃;
2.. announce that according to GenBank Fusarium belongs to the IGS sequence, utilize primer-design software Prime 5.1, design the universal primer G1/G2 on the transcribed spacer IGS fragment in a pair of sickle-like bacteria ribosomal gene, its sequence is:
G1：5’-GCGGTGTCGGTGTGCTTGTA-3’
G2：5’-ACTCACGGCCACCAAACCA-3’
3.. according to fusarium circinatum bacterium and sibling species thereof, kind of the IGS difference of being correlated with, design a pair of Auele Specific Primer S1/S2 that can differentiate the fusarium circinatum bacterium, its sequence is:
S1：5’-CTTACCTTGGCTCGAGAAGG-3’
S2：5’-CCTACCCTACACCTCTCACT-3’
4.. utilize Auele Specific Primer S1/S2 and universal primer G1/G2, it is as follows to carry out fusarium circinatum bacterium nest-type PRC amplification step:
The first round, the universal primer PCR amplification:
The reaction system cumulative volume is 25 μ l, and reactive component is: 10 * reaction buffer, 2.5 μ l, 2.5mmol/L MgCl
21.5 μ l, each 0.5 μ l of 10pmol/L upstream and downstream universal primer G1, G2,10mmol/L dNTP 2 μ l, 5u/ μ l Taq enzyme 0.25 μ l, DMSO 1.25 μ l, template pine tree germ sample DNA extracting solution 1 μ l, aqua sterilisa is supplied 25 μ l; The amplified reaction program is: 95 ℃ of sex change 5min, enter circulation, and 94 ℃ of sex change 30s, 56 ℃ of annealing 45s, 72 ℃ are extended 1min, and 20 circulations are extended 10min for back 72 ℃;
Second takes turns, the special primer pcr amplification:
The reaction system cumulative volume is 25 μ l, takes turns pcr amplification product with the 1st and dilutes 100 times, gets 1 μ l as template; Other reactive component is: 10 * reaction buffer, 2.5 μ l, 2.5mmol/L MgCl
21.5 μ l, each 0.5 μ l of 10pmol/L upstream primer S1, downstream primer S2,10mmol/LdNTP2 μ l, 5u/ μ l Taq enzyme 0.25 μ l, DMSO1.25 μ l, aqua sterilisa supply 25 μ l; Response procedures is: 94 ℃ of sex change 3min, enter circulation, and 94 ℃ of sex change 35s, 66 ℃ of annealing 55s, 72 ℃ are extended 50s, and 40 circulations are extended 12min for back 72 ℃;
5.. amplified production carries out electrophoresis detection:
Get 10 μ l amplified productions, adopt 1.2% sepharose, under ultraviolet lamp, detect after 20～40 minutes,, determine that then the germ that is detected is the fusarium circinatum bacterium if there is the DNA specific band of 873bp at electrophoresis under the voltage 80V.
2. the molecular detecting method of fusarium circinatum bacterium according to claim 1 is characterized in that the DNA of said pine tree germ sample extracts from mycelia, conidium, seed, soil or timber.
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| CN109182574B (en) * | 2018-09-07 | 2021-04-23 | 华南农业大学 | Nested PCR kit and method for detecting albizia falcataria ulcer germs |
| CN113528543B (en) * | 2021-07-16 | 2022-04-05 | 南京林业大学 | Pine resin ulcer disease pathogenic bacteria detection target Fcir _ CM004512.1.g2067.t1 and application |
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| Detection and quantification of airborne conidia of Fusariumcircinatum,the causal agent of pine pitch canker, from twoCalifornia sites by using a real-time PCR approach combinedwith a simple spore trapping method. Wolfgang Schweigkofler et al.Applied and Environmental Microbiology,Vol.70 No.6. 2004 * |
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