Molecular Approaches for Studying Fungi in the Environment

7/24/2019 Molecular Approaches for Studying Fungi in the Environment

1/12


2/12


3/12

Molecular Approaches for Studying Fungi in the Environment 19

The use of monoclonal antibodies to detectfungi and to study fungal interactions with hostshas been applied mainly in two different fields,namely plant pathology and biological control. Iyerand Cousin (2003) developed an indirect ELISA todetect Fusariumcontamination in foods. Antibod-

ies against F. graminearum and F. moniliforme pro-teins succeeded in identifying 15 different Fusar-iumisolates 13 species different from F. gramin-earum and F. moniliforme. The assay was tested forspecificity with 70 different moulds belonging to23 genera, and only two other strains gave a falsepositive signal.

Otten et al. (1997) describe methods toimprove the use of immunoassays for the detec-tion and quantification of the fungal soil-borneplant pathogen, Rhizoctonia solani. The authors

used a monoclonal antibody which recognisesa catechol oxidase secreted by the hyphae ofRhizoctonia. The influence of different soil typeson the retention of the antigen was tested, anda thousand-fold reduction in sensitivity of theassay was determined for clay soil, comparedto sand and loam. This detection method hasthereafter been used to monitor the influenceof the soil-borne biocontrol agent Trichodermaon population dynamics of Rhizoctonia dur-ing saprophytic growth in compost-based soils(Thornton and Gilligan 1999). For the quan-

tification of Trichoderma during antagonisticinteraction, a monoclonal antibody-based ELISAhas been developed. The antibody MF2 detectsantigens of numerous species of the generaHypocrea/Trichoderma and Gliocladium but doesnot cross-react with common soil organisms(Thornton et al. 2002). This antibody binds to thehyphae and septa of its target organisms, and wasused to stainTrichodermasp. while coiling aroundRhizoctonia during the mycoparasitic interac-tion. To specifically detect only actively growing

mycelia, two different antibodies were producedwhich selectively recognise glycoproteins secretedfrom the growing tips of either Trichoderma orRhizoctonia (Thornton 2004). The authors demon-strated that Trichoderma successfully competeswith the pathogen for nutrients and preventsthe saprophytic growth of Rhizoctonia, and nocross-reactivity was found with fungi naturallyoccurring in the soil. Other ELISA tests based onmonclonal antibodies are commercially availablefor the detection of Pythium, Phytophtora andSclerotinia (Ali-Shtayeh et al. 1991; Timmer et al.

1993; Miller 1996; Miller et al. 1997).

III. PCR-Based Methods

PCR is the exponential amplification of DNA withtwo short oligonucleotide primers which are com-plementary to the 5 and 3 ends of the target se-quence fragment. PCR is highly sensitive, and a fewtemplate strands can be amplified up to some mi-crograms of product DNA. This sensitivity, in com-bination with the specificity obtained by the twoprimers, makes PCR the most important techniquein molecular diagnostics nowadays. To further in-crease sensitivity and specificity, PCR-associatedtechniques like nested PCR have been developed.After performing the first conventional amplifica-tion step, a second PCR is added with two newprimers lying within the previously synthesizedDNA. Nested PCR allows the detection of minute

amounts of DNA, several folds lower than normalPCR.

The successful application of PCR-based diag-nostic tools generally requires three crucial steps:(1) theselection ofa suitableDNAmarker sequenceto accurately identify an organism, (2) extractionof total DNA from the sample and (3) a method toidentify the presence of the target sequence in theamplified DNA.

Selection of a Specific Target DNA SequenceA proper selection of the target fragment to be

amplified allows the detection of whole genera,in the case of primers designed across conservedDNA regions, or the identification of a singlespecies even in a background of taxonomicallyrelated organisms. One of the main targets forthe development of diagnostic PCR assays are thegenes coding for the ribosomal RNA, which arepresent in all organisms at high copy numbers. Theabundance of this type of DNA facilitates detectionand, thereby, improves the sensitivity of the assay(White et al. 1990). The fungal nuclear ribosomal

DNA consists of three genes encoding the 28S, 18Sand 5.8S subunits. These genes are separated byinternal transcribed spacer regions (ITS), and thisunit is repeated many times. The ITS region is ofparticular importance for fungal diagnostics asit consists of conserved areas and highly variablesequences as well. Fungal ITS regions can beisolated by universal primers (White et al. 1990)and, after sequencing, strain- or genus-specificprimers can be used for the identification offungi from various samples. Although Atkinset al. (2004) discriminated between two varieties

of the same fungus based on ITS sequences, in


4/12


5/12


6/12


7/12


8/12


9/12


based on immunological tests was state of the artbut this expensive technique was soon replacedby PCR-based detection systems. Nowadays, thereare many applications of PCR in diagnostics, mostof these focusing on rapid detection directly fromthe source material, without any time-consuming

cultivation steps. This technique has changed theview of molecular analysis, as now even minuteamounts of organisms, some attograms of DNA,are detectable. PCR technology has opened up newways of investigations, in particular the analysis ofwhole fungal communities from complex sources.In contrast to conventional culture-based meth-ods, even unculturable strains can be detected.The combination with other techniques, likeDGGE, RFLP, AFLP, or the use of random primers(RAPD) allow the identification of yet unknown

isolates, although the information about theirDNA sequence is limited or inexistent.Recent reverse probing technologies (microar-

rays) emerged from the hybridization techniquedeveloped by Southern in 1975. These DNA chipscan be scaled up, so that hundreds of different or-ganisms are detected simultaneously in the sample.Microarray technology has maybe advanced morethan other molecular analysis methods within thelast few years. In contrast to PCR-based technolo-gies, arrays represent a closed system technology,andtheoligonucleotide probes on thechip limit the

detection capability. A rapid adaptation to screenfor new organisms is hardly realizable. However,due to the high throughput of DNA arrays and atleast raw-quantitative results, they might be themost promising tool for commercially detectionkits in future.

References

Ali-Shtayeh MS, Macdonald JD, Kabashima J (1991)A method for using commercial ELISA tests to detectzoospores of Phytophthora and Pythium species inirrigation water. Plant Disease 75:305311

Alkan N, Gadkar V, Yarden O, Kapulnik Y (2006) Analysisof quantitative interactions between two species of ar-buscularmycorrhizal fungi, Glomus mosseae and G.in-traradices, by real-time PCR. Appl Environ Microbiol72:41924199

Anderson IC, Campbell CD, Prosser JI (2003) Diversity offungiinorganicsoilsunderamoorlandScotspine( Pi-nus sylvestrisL.) gradient. Environ Microbiol 5:11211132

Atkins SD, Clark IM (2004) Fungal molecular diagnostics:a mini review. J Appl Genet 45:315

Atkins SD, Sosnowska D, Evans VJ, Clark IM, Hirsch PR,Kerry BR (2004) Investigation of three nematophagousfungi in two potato cyst nematode suppressive soils.Bull IOLB/SROP 27(1):18

Baker BJ, Lutz MA, Dawson SC, Bond PL, Banfield JF (2004)Metabolically active eukaryotic communities in ex-tremely acidic mine drainage. Appl Environ Microbiol

70:62646271Bezuidenhout CC, Prinsloo M, Van der Walt AM (2006)

MultiplexPCR-baseddetectionofpotentialfumonisin-producingFusariumin traditional African vegetables.Environ Toxicol 21:360366

Bluhm BH, Cousin MA, Woloshuk CP (2004) Multiplexreal-time PCR detection of fumonisin-producing andtrichothecene-producing groups ofFusariumspecies.J Food Prot 67:536543

Borneman J, Chrobak M, Della Vedova G, Figueroa A,Jiang T (2001) Probe selection algorithms withapplications in the analysis of microbial communities.Bioinformatics 17 suppl 1:S39S48

Boyle B, Hamelin RC, SeguinA (2005)In vivo monitoring of

obligate biotrophic pathogen growth by kinetic PCR.Appl Environ Microbiol 71:15461552

Brandfass C, Karlovsky P (2006) Simultaneous detectionofFusarium culmorumand F. graminearum in plantmaterial by duplex PCR with melting curve analysis.BMC Microbiol 6:4

Bustin SA (2002) Quantification of mRNA using real-timereverse transcription PCR (RT-PCR): trends and prob-lems. J Mol Endocrinol 29:2339

Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitativereal-time RT-PCR a perspective. J Mol Endocrinol34:597601

Casimiro S, Moura M, Ze-Ze L, Tenreiro R, Monteiro AA(2004) Internal transcribed spacer 2 amplicon as

a molecular marker for identification ofPeronosporaparasitica(crucifer downy mildew). J Appl Microbiol96:579587

Castella G, Larsen TO, Cabanes J, Schmidt H, Alboresi A,Niessen L, Farber P, Geisen R (2002) Molecular char-acterization of ochratoxin A producing strains of thegenusPenicillium. Syst Appl Microbiol 25:7483

Chadha S, Gopalakrishna T (2006) Detection ofMagna-porthe griseain infested rice seeds using polymerasechain reaction. J Appl Microbiol 100:11471153

Chunming D, Cantor CR (2004) Quantitative analysis ofnucleic acids thelast few years of progress. J BiochemMol Biol 37:110

Clark MF, Adams AN (1977) Characteristics of the mi-

croplate method of enzyme-linked immunosorbentassay for the detection of plant viruses. J Gen Virol34:475483

Dewey FM, Thornton CR, Gilligan CA (1996) Use of mono-clonal antibodies to detect, quantify and visualizefungi in soils. Adv Bot Res 24:275308

Druzhinina IS, Kopchinskiy AG, Komon M, Bissett J, Sza-kacs G, Kubicek CP (2005) An oligonucleotide bar-code for species identification in Trichoderma andHypocrea. Fungal Genet Biol 42:813828

Dyer RB,KendraDF, Brown DW(2006)Real-time PCR assayto quantifyFusarium graminearumwild-type and re-combinant mutant DNA in plant material. J MicrobiolMethods 67:534542


10/12

26 K. Brunner, S. Zeilinger, R.L. Mach

Eng C, Vijg J (1997) Genetic testing: the problems and thepromise. Nature Biotechnol 15:422426

Filion M, St-Arnaud M, Jabaji-Hare SH (2003) Direct quan-tification of fungal DNA from soil substrate using real-time PCR. J Microbiol Methods 53:6776

Fraaije BA,Lovell DJ, Rohel EA, Hollomon DW(1999)Rapiddetection and diagnosis ofSeptoriatritici epidemics in

wheat using a polymerase chain reaction/Pico greenassay. J Appl Microbiol 86:701708

Fraaije BA, Lovell DJ, Coelho JM, Baldwin S, Hollomon DW(2001) PCR-based assays to assess wheat varietal re-sistance to blotch and rust diseases. Eur J Plant Pathol107:905917

Gachon C, Saindrenan P (2004) Real-time PCR monitoringof fungal development inArabidopsis thaliana infectedbyAlternaria brassicicola and Botrytis cinerea. PlantPhysiol Biochem 42:367371

Gadkar V, Rillig MC (2005) Suitability of genomic DNA syn-thesized by strand displacement amplification (SDA)for AFLP analysis: genotyping single spores of arbus-cular mycorrhizal (AM) fungi. J Microbiol Methods

63:157164Geisen R (2004) Molecular monitoring of environmental

conditions influencing the induction of ochratoxinA biosynthesis genes in Penicillium nordicum. MolNutr Food Res 48:532540

Ginzinger DG (2002) Gene quantification using real-timequantitative PCR: an emerging technology hits themainstream. Exp Hematol 30:503512

Gomes NC, Fagbola O, Costa R, Rumjanek NG, Buchner A,Mendona-Hagler L, Smalla K (2003) Dynamics of fun-gal communities in bulk and maize rhizosphere soil inthe tropics. Appl Environ Microbiol 69:37583766

Gryta H, Carriconde F, Charcosset JY, Jargeat P, Gardes M(2006) Population dynamics of the ectomycorrhizal

fungal speciesTricholoma populinumandTricholomascalpturatumassociated with black poplar under dif-fering environmental conditions. Environ Microbiol8:773786

Hermosa MR, Grondona I, Diaz-Minguez JM, Iturriaga EA,Monte E (2001) Development of a strain-specific SCARmarker for the detection ofTrichoderma atroviride 11,a biological control agent against soilborne fungalplant pathogens. Curr Genet 38:343350

Hirsch PR, Mauchline TH, Mendum TA (2000) Detection ofthe nematophagous fungusVerticillium chlamydospo-riumin nematode infested plant roots using PCR. My-col Res 104:435439

Ingianni A, Floris M, Palomba P, Madeddu MA, Quartuc-

cio M, Pompei R (2001) Rapid detection ofListeriamonocytogenes in foods, by a combination of PCR andDNA probe. Mol Cell Probes 15:275280

Iyer MS, Cousin MA (2003) Immunological detection ofFusariumspecies in cornmeal. J Food Prot 66:451456

Jurado M, Vazquez C, Patino B, Gonzalez-Jaen MT (2005)PCR detection assays for the trichothecene-producingspeciesFusarium graminearum,Fusarium culmorum,Fusarium poae, Fusarium equiseti and Fusariumsporotrichioides. Syst Appl Microbiol 28:562568

Klein D (2002) Quantification using real-time PCR tech-nology: applications and limitations. Trends Mol Med8:257260

Klemsdal SS, Elen O (2006) Development of a highly sen-

sitive nested-PCR method using a single closed tube

for detection ofFusarium culmorum in cereal samples.Lett Appl Microbiol 42:544548

Kostrzynska M, Bachand A (2006) Application of DNA mi-croarray technology for detection, identification, andcharacterization of food-borne pathogens. Can J Mi-crobiol 52:18

LeeSB, WardTJ (1990)Isolation ofDNA fromfungal mycelia

andsinglespores.In:InnisMA,GelfandDH,SninskyJJ,White TJ (eds) PCR Protocols. A guide to methods andapplications. Academic Press, San Diego, CA, pp 282287

Levesque CA (2001) Molecular methods for detection ofplantpathogensWhatisthefuture?CanJPlantPathol24:333336

Levesque CA, Harlton CA, de Cock AW (1998) Identificationof some oomycetes by reverse dot blot hybridization.Phytopathology 88:213222

Lievens B, Brouwer M, Vanachter AC, Levesque CA, Cam-mue BP, Thomma BP (2003) Design and developmentof a DNA array for rapid detection and identificationof multiple tomato vascular wilt pathogens. FEMS Mi-

crobiol Lett 223:113122Lievens B, Hanssen IRM, Vanachter AC, Cammune BPA,

Thomma BP (2004)Root andfoot roton tomato causedbyPhytophthora infestans detected in Belgium. PlantDisease 88:86

Lievens B, Brouwer M, Vanachter AC, Levesque CA, Cam-mue BP, Thomma BP (2005) Quantitative assessmentof phytopathogenic fungi in various substrates us-ing a DNA macroarray. Environ Microbiol 7:16981710

Lievens B, Claes L, Vanachter AC, Cammue BP, Thomma BP(2006) Detecting single nucleotide polymorphisms us-ing DNA arrays for plant pathogen diagnosis. FEMSMicrobiol Lett 255:129139

Lockhart RJ, Van Dyke MI, Beadle IR, Humphreys P, Mc-Carthy AJ (2006) Molecular biological detection ofanaerobic gut fungi (neocallimastigales) from landfillsites. Appl Environ Microbiol 72:56595661

Loppnau PA, Breuil C (2003) Species level identification ofconifer associatedCeratocystis sapstainfungi by PCR-RFLP on a beta-tubulin gene fragment. FEMS Micro-biol Lett 222:143147

LuchiN, Capretti P, Pinzani P, Orlando C, Pazzagli M (2005)Real-time PCR detection ofBiscogniauxia mediter-raneain symptomless oak tissue. Lett Appl Microbiol41:6168

Mach RL, Kullnig-Gradinger CM, Farnleitner AH, Reis-cherG,AdlerA,KubicekCP(2004)Specificdetectionof

Fusarium langsethiaeandrelatedspeciesbyDGGEandARMS-PCR of a beta-tubulin (tub1) genefragment. IntJ Food Microbiol 95:333339

Mackay IM (2004) Real-time PCR in the microbiology lab-oratory. Clin Microbiol Infect 10:190212

MacNeil L, Kauri T, Robertson W (1995) Molecular tech-niques and their potential application in monitoringthe microbiological quality of indoor air. Can J Micro-biol 41:657665

Malvick DK, Grunden E (2005) Isolation of fungal DNAfrom plant tissues and removal of DNA amplificationinhibitors. Mol Ecol Notes 5:958960

Mauchline TH, Kerry BR, Hirsch PR (2002) Quantificationin soil and the rhizosphere of the nematophagous fun-

gus Verticillium chlamydosporium by competitive PCR


11/12


and comparison with selective plating. Appl EnvironMicrobiol 68:18461853

McCartney HA, Foster SJ, Fraaije BA, Ward E (2003) Molec-ular diagnostics for fungal plant pathogens. Pest Man-age Sci 59:129142

Miller SA (1996). Detecting propagules of plant pathogenicfungi. Adv Bot Res 23:73102

Miller SA, Madden LV, Schmitthenner AF (1997) Distribu-tion ofPhytophthoraspp. in field soils determined byimmunoassay. Phytopathology 87:101107

Moeller EM, Chelkowski J, Geiger HH (1999) Species-specific PCR assays for the fungal pathogens Fusariummoniliforme and Fusarium subglutinans and theirapplication to diagnose maize ear rot. J Phytopathol147:497508

Mukoda T, Todd LA, Sobsey MD (1994) PCR and geneprobes fordetecting bioaerosols.J Aerosol Sci 25:15231532

Mule G, Gonzalez-Jaen MT, Hornok L, Nicholson P, Waal-wijk C (2005) Advances in molecular diagnosis of tox-igenic Fusariumspecies: a review. Food Addit Contam

22:316323Mutasa ES, Chwarszczynska D, Adams MJ, Ward E,

Asher MJC (1995) Development of PCR for thedetection ofPolymyxa beaein sugar beet roots and itsapplication in field studies. Physiol Mol Plant Pathol47:303313

NicholsonP,LeesA,MaurinN,ParryD,RezanoorHN(1996)PCR assay to identify and quantifyMicrodochium ni-valevarnivaleandMicrodochium nivalevarmajusinwheat. Physiol Mol Plant Pathol 48:257271

Nicholson P, Simpson DR, Weston G, Rezanoor HN, Lees A,Parry D, Joyce D (1998) Detection and quantificationofFusarium culmorum and Fusarium graminearumusing PCR assays. Physiol Mol Plant Pathol 53:1737

Nicolaisen M, Justesen AF, Thrane U, Skouboe P, Holm-strom K (2005) An oligonucleotide microarray for theidentification and differentiation of trichothecene pro-ducing and non-producing Fusarium species occur-ring on cereal grain. J Microbiol Methods 62:5769

Ong YL, Irvine A (2002) Quantitative real-time PCR: a cri-tique of method and practical considerations. Hema-tology 7:5967

Osborn AM,Moore ER, Timmis KN (2000) An evaluation ofterminal-restriction fragment length polymorphism(T-RFLP) analysis for the study of microbial commu-nity structure and dynamics. Environ Microbiol 2:3950

Otten CA, Gilligan CA, Thornton CR (1997) Quantification

of fungal antigens in soil with a monoclonal antibody-based ELISA: analysis and reduction of soil-specificbias. Phytopathology 87:730736

Pelegrinelli-Fungaro MH,Vissoto PC,Sartori D, Vioas-BoasLA (2004) A molecular method for detection ofAs-

pergillus carbonariusin coffee beans. Curr Microbiol49:123127

Providenti MA, Mautner SI, Chaudhry O, Bombardier M,Scroggins R, Gregorich E, Smith ML (2004) Determin-ingthe environmental fate of a filamentousfungus, Tri-choderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viabilityplating. Can J Microbiol 50(8):623631

Reischer GH, Lemmens M, Farnleitner A, Adler A, Mach RL

(2004) Quantification ofFusarium graminearum in in-

fected wheat by speciesspecific real-time PCRapplyinga TaqMan Probe. J Microbiol Methods 59:141146

Rubio MB, Hermosa MR, Keck E, Monte E (2005) SpecificPCRassays forthe detectionand quantificationof DNAfrom the biocontrol strain Trichoderma harzianum2413 in soil. Microbial Ecol 49:2533

Sanchez-Rangel D, SanJuan-Badillo A, Plasencia J (2005)

Fumonisin production by Fusarium verticillioidesstrains isolated from maize in Mexico and devel-opment of a polymerase chain reaction to detectpotential toxigenic strains in grains. J Agric FoodChem 53:85658571

Schesser K, Luder A, Henson JM (1991) Use of polymerasechain reaction to detect the take-all fungus, Gaeuman-nomyces graminis, in infected wheat plants. Appl Env-iron Microbiol 57:553556

Schmidt H, Ehrmann M, Vogel RF, Taniwaki MH, Niessen L(2003) Molecular typing ofAspergillus ochraceusandconstruction of species specific SCAR-primers basedon AFLP. Syst Appl Microbiol 26:138146

Schmidt H, Taniwaki MH, Vogel RF, Niessen L (2004) Uti-

lization of AFLP markers for PCR-based identificationofAspergillus carbonarius and indication of its pres-ence in green coffee samples. J Appl Microbiol 97:899909

Schnerr H, Niessen L, Vogel RF (2001) Real time detectionof the tri5 gene in Fusarium species by lightcycler-PCR using SYBR Green I for continuous fluorescencemonitoring. Int J Food Microbiol 71:5361

Sheffield VC, Cox DR, Lerman LS, Myers RM (1989) At-tachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerasechain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 86:232236

Southern EM (1975) Detection of specific sequences among

DNA fragments separated by gel electrophoresis. J MolBiol 98:503517

Taniwaki MH, Pitt JI, Teixeira AA, Iamanaka BT (2003) Thesource of ochratoxin A in Brazilian coffee and its for-mation in relation to processing methods. Int J FoodMicrobiol 82:173179

ThelwellN, Millington S, Solinas A, Booth J,Brown T (2000)Mode of action and application of Scorpion primers tomutation detection. Nucleic Acids Res 28:37523761

Thornton CR (2004) An immunological approach to quan-tifying the saprotrophic growth dynamics ofTricho-derma species during antagonistic interactions withRhizoctonia solaniin a soil-less mix. Environ Micro-biol 6:323334

Thornton CR,GilliganCA (1999) Quantificationof theeffectof the hyperparasite Trichoderma harzianum on thesaprotrophic growth dynamics ofRhizoctonia solaniin compostusing a monoclonal antibody-based ELISA.Mycol Res 103:443448

Thornton CR, Pitt D, Wakley GE, Talbot NJ (2002) Produc-tion ofa monoclonal antibodyspecificto thegenus Tri-chodermaand closely related fungi, and its use to de-tectTrichodermaspp. in naturally infested composts.Microbiology 148:12631279

Timmer LW, Menge JA, Zitko SE, Pond E, Miller SA, John-son EL V (1993) Comparison of ELISA techniques andstandard isolation methods for Phytophthora detec-tion in citrus orchards in Florida and California. Plant

Disease 77:791796


12/12

28 K. Brunner, S. Zeilinger, R.L. Mach

Tyagi S, Kramer FR (1996) Molecular beacons: probesthat fluoresce upon hybridization. Nature Biotechnol14:303308

Tyagi S, Bratu DP, Kramer FR (1998) Multicolor molecularbeacons for allele discrimination. Nature Biotechnol16:4953

Valasek MA, Repa JJ (2005) The power of real-time PCR.

Adv Physiol Educ 29:151159Valinsky L, Della Vedova G, Jiang T, Borneman J (2002)

Oligonucleotide fingerprinting of rRNA genes for ana-lysis of fungal community composition. Appl EnvironMicrobiol 68:59996004

van Elsas JD, Duarte GF, Keijzer-Wolters A, Smit E (2000)Analysis of the dynamics of fungal communities insoil via fungal-specific PCR of soil DNA followed bydenaturing gradient gel electrophoresis. J MicrobiolMethods 43:133151

Vanittanakom N, Merz WG, Sittisombut N, Khamwan C,Nelson KE, Sirisanthana T (1998) Specific identifica-tion ofPenicillium marneffei by a polymerase chainreaction/hybridization technique. Med Mycol 36:169

175Vettraino AM, Paolacci A, Vannini A (2005) Endophytism

ofSclerotinia pseudotuberosa: PCR assay for specificdetection in chestnut tissues. Mycol Res 109:96102

Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T,Hornes M, Frijters A, Pot J, Peleman J, Kuiper M et al.(1995) AFLP: a new technique for DNA fingerprinting.Nucleic Acids Res 23:44074414

Wang PH, Chang CW (2003) Detection of the low-germination-rate resting oospores of Pythiummyriotylum from soil by PCR. Lett Appl Microbiol36:157161

Ward E (1995) Improved polymerase chain reaction (PCR)detection of Gaeumannomyces graminis including

a safeguard against false negatives. Eur J Plant Pathol101:561566

Ward E (1998) Analysis of ribosomal DNA sequences ofPolymyxaspecies and related fungi and the develop-ment of genus- and species-specific PCR primers. My-col Res 102:965974

Watson RJ, Blackwell B (2000) Purification and characteri-zation of a common soil component which inhibits thepolymerase chain reaction. Can J Microbiol 46:633642

Weerasena OV, Chandrasekharan NV, Wijesundera RL,Karunanayake EH (2004) Development of a DNA

probe and a PCR based diagnostic assay forRhizoc-tonia solaniusing a repetitive DNA sequence clonedfrom a Sri Lankan isolate. Mycol Res 108:649653

White TJ, Brun T, Lee SB, Taylor J (1990) Amplification anddirect sequencing of fungal ribosomal RNA genes forphylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ,White TJ (eds) PCR Protocols. A guide to methods

and applications. Academic Press, San Diego, CA, pp315320

Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV(1990) DNA polymorphisms amplified by arbitraryprimers are useful as genetic markers. Nucleic AcidsRes 18:65316535

Williams RH, Ward E, McCartney HA (2001) Methods forintegrated air sampling and DNA analysis for detec-tion of airborne fungal spores. Appl EnvironMicrobiol67:24532459

Wittwer CT, Herrmann MG, Gundry CN, Elenitoba-Johnson KS (2001) Real-time multiplex PCR assays.Methods 25:430442

Wong ML, Medrano JF (2005) Real-time PCR for mRNA

quantitation. Biotechniques 39:7585Wu Z, Blomquist G, Westermark SO, WangXR (2002) Appli-

cation of PCR and probe hybridization techniques indetection of airborne fungal spores in environmentalsamples. J Environ Monit 4:673678

Wu Z, Tsumura Y, Blomquist G, Wang XR (2003) 18S rRNAgene variation among common airborne fungi, anddevelopment of specific oligonucleotide probes for thedetection of fungal isolates. Appl Environ Microbiol69:53895397

Zeng QY, Wang XR, Blomquist G (2003) Developmentof mitochondrial SSU rDNA-based oligonucleotideprobes for specific detection of common airbornefungi. Mol Cell Probes 17:281288

Zezza F, Pascale M, Mule G, Visconti A (2006) Detection ofFusarium culmorum in wheat by a surface plasmonresonance-based DNA sensor. J Microbiol Methods66:529537

Zhang Z, Zhang J, Wang Y, Zheng X (2005) Molecular de-tection ofFusarium oxysporumf. sp.niveumandMy-cosphaerella melonisin infected plant tissues and soil.FEMS Microbiol Lett 249:3947

Zhu ML, Mo MH, Xia ZY, Li YH, Yang SJ, Li TF, Zhang KQ(2006) Detection of two fungal biocontrol agentsagainst root-knot nematodes by RAPD markers.Mycopathologia 161:307316

Documents

Molecular Approaches for Studying Fungi in the Environment