Quantification of Pythium populations in ginseng soils - Mount Saint ...

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448applied soil ecology 40 (2008) 447–455Pythium populations in soils and plant roots have beenassessed using a variety of techniques. The simplest method isbased on the number of CFU formed after dilution plating onselective media (Jeffers and Martin, 1986). However, thismethod is labour intensive, does not allow for discriminationamong morphologically similar species, and can be hinderedby competition from other soil organisms. More sophisticatedimmunological methods, such as ELISA, have also beenemployed for Pythium quantification (Yuen et al., 1998), butthese methods are still culture dependent.More recently, real-time PCR (qPCR) has been used for thefast, accurate and culture independent quantification of avariety of pathogens (including Pythium spp.) from planttissues (Schaad and Frederick, 2002; Schena et al., 2004) andsoils (Okubara et al., 2005; Schroeder et al., 2006; Lievens et al.,2006; Kernaghan et al., 2007). Although a variety of chemistrieshave been developed for quantitative real-time PCR (Ginzinger,2002), SYBR green intercalating dyes represent the mostflexible approach to the analysis of species assemblages.Taxon specific PCR primers have been designed for a varietyof Pythium species, for use in both conventional (Kageyamaet al., 1997; Wang et al., 2003) and real-time PCR reactions(Schroeder et al., 2006). In the present study, we designed PCRprimers specific to P. ultimum Trow and P. irregulare Buismansensu stricto, the two most destructive Pythium species in southwesternOntario ginseng plantations (Reeleder and Brammall,1994). P. ultimum is fairly well circumscribed both morphologicallyand genetically (Van der Plaats-Niterink, 1981; Kageyamaet al., 2007). However, the species concept of P. irregulare iscurrently under revision (Matsumoto et al., 2000; Garzón etal.,2005, 2007) and the occurrence of morphologically similar, butgenetically divergent isolates (cryptic species) makes quantificationdifficult.Although real-time PCR quantification methods are moresensitive and specific than culture based methods (Atkinset al., 2003; Ippolito et al., 2004; Kernaghan et al., 2007), platingon selective media still gives valuable and complementarydata on soil pathogen populations. For example, relationshipsamong DNA concentration, microbial biomass and infectivitymay not be equivalent across species and pathogen DNA levelsmay not necessarily be a direct indication of inoculumpotential. The relationship between DNA concentration andinoculum potential should therefore be determined empiricallyfor each pathogen species.As the ability to accurately quantify populations of Pythiumspecies directly from soils prior to ginseng planting wouldallow for more accurate assessments of disease risk and areduction in the need for fumigation, we developed qPCRassays for P. irregulare and P. ultimum and used them in bothartificially infested soil and naturally infested ginsengcultivatedsoils. We also conducted concurrent estimates ofinoculum potential using counts of colony forming units onsemi-selective agar media.2. Materials and methods2.1. Conventional PCR, sequencing of isolates andquantification of reference DNAIsolates of target and related species from the Southern CropProtection and Food Research Centre (SCPFRC), London, ON,and the Canadian Collection of Fungal Cultures (CCFC),Ottawa, ON (Table 1) were cultured on V8 broth. DNA wasextracted using the DNeasy Plant Mini Kit (Qiagen, Mississauga,ON) and the ITS 1 and 2 regions amplified on anTable 1 – Species used in this study and the results of conventional PCR (using primers ITS1/ITS4) and testing of taxonspecific primersSpecies Accession # Source Conventional PCR and presence of restriction sites qPCRPiF/PiRPuF/PuRIsolates used for Pythium primer design and/or testingPythium heterothallicum 491 CCFC +Pythium irregulare 419 CCFC + F +P. irregulare 598 CCFC + F +P. irregulare (Group II) PID008 SCPFRC + F +Pythium paroecandrum 568 CCFC +P. irregulare (Group IV) PID018* SCPFRC +Pythium intermedium PID091* SCPFRC +Pythium splendens 435a CCFC +P. slpendens 225914 CCFC +Pythium sylvaticum 259 CCFC +Pythium ultimum 144 CCFC + B +P. ultimum 418 CCFC + B +P. ultimum PID095* SCPFRC + B +Pythium violae 400 CCFC +Isolates used for Pythium primer design onlyP. irregulare (Group II) PID003* SCPFRC + FP. irregulare (Group II) PID096* SCPFRC + FP. intermedium PID306* SCPFRC ++: Amplification of product with predicted size and melting temperature; : no amplification of target; *: isolates sequenced; F: presence of FspIrestriction site; B: presence of BccI restriction site.
applied soil ecology 40 (2008) 447–455 449Eppendorf Mastercycler (Brinkmann Instruments, Mississauga,ON) thermocycler in 50 mL reactions containing 1 UPlatinum 1 Taq DNA Polymerase, 1 PCR buffer, 200 mM dNTPmix (all from Invitrogen, Burlington, ON), 2.5 mM MgCl 2 , 0.8%BSA, 0.4 mM each of primers ITS 1 and ITS 4 (White et al., 1990)and 5 mL template DNA. Cycling parameters were 94 8C for2 min followed by 35 cycles of 94 8C for 1 min, 55 8C for 1 minand 72 8C for 2 min, followed by 72 8C for 10 min.PCR products generated from Pythium isolates from southwesternOntario ginseng soils (PID003, PID018, PID091,PID095, PID096, PID306) (Table 1) were purified with theMinElute PCR Purification Kit (Qiagen, Mississauga, ON) andsequenced on an ABI 377 Sequencer (Applied Biosystems,Foster City, CA) using BigDye 1 Terminator chemistry(Applied Biosystems); Genbank accession numbersDQ083527–DQ083532.Reference DNA was obtained from fresh mycelium of P.irregulare (PID 008) and P. ultimum (PID 095) (Table 1) byextracting with the DNeasy Plant Mini Kit (Qiagen) andquantifying with an Eppendorf BioPhotometer calibratedwith calf thymus DNA (Sigma) in Qiagen elution buffer(buffer AE).2.2. Design of taxon specific primers and identification ofspecific restriction sitesTaxon specific primer pairs targeting the internallytranscribed spacer regions of P. irregulare Groups I and II(sensu Matsumoto et al., 2000) (PiF 5 0 -GTAGCATGCGT-GTTTGCTTA-3 0 /PiR 5 0 -GCAAGCTGTGCATTCATTGC-3 0 ) andP. ultimum (PuF 5 0 -ATGATGGACTAGCTGATGAA-3 0 /PuR 5 0 -TTCCATTACACTTCATAGAA-3 0 )weredesignedonthebasisof an alignment of sequenced SCPFRC and CCFC Pythiumcultures and over 100 Pythium sequences from Genbankusing BioEdit ver. 7.0.0 (Hall, 1999). The predicted PCRproduct sizes were 508 and 407 bp, respectively. The extentof primer dimer formation was predicted using thePrimerSelect module of DNASTAR (Madison, WI). Specificityof real-time amplifications was tested on a Roche DiagnosticsLightCycler 1, using DNA extracted from a range ofrelated Pythium species, previously isolated from ginsengsoils (Table 1).In order to confirm target amplification from soil, thealignment of Pythium species used for taxon specific primerdesign was also used to identify restriction sites whichcould distinguish between the amplified DNA of our targetspecies and that of other Pythium species from naturallyinfested soils. Restriction digests of the ITS region withenzymes FspI and BccI were predicted to result in restrictiondigestion patterns characteristic of P. irregulare Groups I andII (sensu Matsumoto et al., 2000) andP. ultimum, respectively.To test for the presence of these restriction sites, digests ofPCR products generated from 17 Pythium isolates byconventional PCR (Table 1) were carried out by combining10 mL ofPCRproductwith5UofeitherFspIorBccI(NewEngland Biolabs, Pickering, Ontario), 2 mL of 10 reactionbuffer and 7 mL H 2 O. Reactions were incubated at 37 8C for3 h, separated electrophoretically on 2% agarose in TAEbuffer and analyzed with the Gel Doc EQ System (Bio-Rad,Hercules, CA).2.3. Collection and preparation of soilArtificially infested soil was prepared using Fox loamy sand(Brunosolic Gray Brown Luvisol; Typic Hapludalf; 0.9% OM)from the Agriculture and Agri-food Canada research farm atDelhi, Ontario (42847 0 N, 80838 0 W). Soil was steam pasteurizedat 74 8C for 30 min using a Lindig soil treatment system (LindigManufacturing, St. Paul, MN), air-dried, sieved through a 4 mmmesh, mixed and stored at room temperature until used. Soildilution (1 part soil in 4 parts 0.25% water agar) procedureswere carried out using P 5 ARP semi-selective media (Jeffers andMartin, 1986; Reeleder et al., 2006b) to confirm that pasteurizationhad eliminated viable propagules. P. irregulare (PID008)and P. ultimum (PID095) were grown for 2 weeks in clarified V8broth. Twenty mycelial mats were then washed with sterilewater and vacuum filtration, macerated using autoclavedmini-blenders and re-suspended in approx. 240 mL sterilewater. Macerates were mixed into pasteurized soil atapproximately 40 mL/kg soil in a large plastic bag and mixedby vigorously shaking, then allowing the mixed soil to air dry.The resulting inoculum was then further mixed withpasteurized field soil in different proportions. The resultingsoil dilutions were 100, 50, 10 and 1%. Each dilution was thendivided into six sub-samples, air dried in the greenhouse andstored at 4 8C until plating and DNA extraction.Samples of naturally infested soils (ranging from sands tosandy loams) were collected at seven sites in south-westernOntario. At each site, one 441 m 2 plot was constructed anddivided into 49.9 m 2 quadrats. Approx. 2 L of topsoil wasremoved from each quadrat. Sampling locations wererecorded by GPS. Soil samples from within each site werepooled, passively air dried at room temperature, mixed with acommercial cement mixer, sieved, and mixed again. Allequipment, including mixer and sieve were sterilized withhousehold bleach (diluted with water to 0.5% sodium hypochlorite)between soils. Soils were placed in plastic bags andstored at 4 8C until used. DNA was extracted and purified from5 g subsamples of each artificially and naturally infested soilas described in Kernaghan et al. (2007).2.4. qPCRqPCR was performed on extracts of samples of the artificiallyinfested soil using SYBR green chemistry on a RocheDiagnostics Light Cycler TM I. Twenty microlitres reactionmixtures were prepared in glass capillaries (Roche Diagnostics)and included 2 mL soil DNA extract, 9.8 mL Quanti-Tect SYBR Green PCR Master Mix (Qiagen), 0.1 U HotStarTaqDNA Polymerase (Qiagen) and 0.6 mM each of primers PiFand PiR or PuF and PuR. Reactions using PuF/PuR received1mM MgCl 2 in addition to the 2.5 mM present in theQuantiTect Master Mix.For each primer pair, PCR reactions were carried out onDNA extractions from each of the six subsamples taken fromeach soil dilution, as well as on a dilution series of referenceDNA (Quantified DNA from pure culture) and the negative(yeast) control extract. All reactions received a 15 min pretreatmentat 95 8C, then 60 cycles of 94 8C for 15 s, 64 8C for 15 s,72 8C for 25 s for the PiF/PiR primer set and 94 8C for 15 s, 54 8Cfor 30 s, 72 8C for 35 s for the PuF/PuR primer set. For all
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