Research

Detection and Discrimination of neglectus and P. t ho rn ei in DNA Extracts from Soil

Guiping Yan and Richard W. Smiley, Oregon State University, Columbia Basin Agricultural Research Center, Pen- dleton 97801; Patricia A. Okubara, United States Department of Agriculture–Agricultural Research Service (USDA-ARS), Root Disease and Biological Control Research Unit, Pullman, WA 99164; Andrea Skantar, USDA- ARS, Nematology Laboratory, Beltsville, MD 20705; and Sandra A. Easley, Jason G. Sheedy, and Alison L. Thompson, Oregon State University, Columbia Basin Agricultural Research Center, Pendleton

ing three key diagnostic features (lip an- ABSTRACT nules, tail shape, and vulva position). For Yan, G. P., Smiley, R. W., Okubara, P. A., Skantar, A., Easley, S. A., Sheedy, J. G., and Thomp- example, vulval position has an overlap- son, A. L. 2008. Detection and discrimination of and P. thornei in DNA ping range for these species, requiring extracts from soil. Plant Dis. 92:1480-1487. measurements of multiple specimens to determine a mean for the ratio of vulval A species-specific polymerase chain reaction (PCR) method was developed to detect and iden- position to body length (12,13). In addi- tify the root-lesion Pratylenchus neglectus and P. thornei from soil. A primer set was tion, morphological identification is lim- Pratylenchus r designed from 28S rRNA gene sequences of the D3 expansion domain. Prime ited to examination of mature adult fe- specificity was confirmed with 23 isolates of 15 species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal males of these parthenogenic species. The species commonly associated with root rot. DNA obtained using a commercially available largest commercial nematode diagnostic kit and a method developed in our laboratory gave comparable amplification. PCR conditions laboratory in the Pacific Northwest quanti- were optimized and the two species were differentiated by PCR products of 144 bp for P. neglec- fies Pratylenchus spp. at the genus but not tus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, species level due to issues relating to reli- inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to ability and expense. However, the lab is a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method equipped and staffed for PCR diagnostic is rapid, efficient, and reliable, does not require expertise in nematode and morphol- services using plant and soil DNA. A ogy, and could be used as a rapid diagnostic tool for commercial and research applications for quick, reliable, and inexpensive molecular disease forecasting and management. method that is amenable to high- throughput diagnostic labs is needed for Additional keywords: detection sensitivity, DNA extraction, DNA purification distinguishing P. neglectus and P. thornei. A commercial testing program in South Australia provides DNA-based diagnostic services; however, the protocols are cur- Root-lesion nematodes, Pratylenchus that are both resistant and tolerant rently proprietary (21). A publically avail- spp., are widely distributed and economi- (28,34,36). Individual wheat cultivars dif- able procedure is needed to increase the cally important migratory endoparasites of fer in tolerance to these nematodes; culti- level of service and diagnostic efficiency many plant species (8). Recently, Praty- vars that exhibit tolerance to P. neglectus in laboratories elsewhere in the world, and lenchus neglectus and P. thornei were are not necessarily tolerant to P. thornei, particularly to facilitate more precise sur- shown to be widespread in wheat fields in and vice versa. Mechanisms for resistance veys of species distribution in the Pacific Oregon and Washington (27), and Idaho to these species are also under different Northwest where P. neglectus and P. (31). High populations of these species in species-specific genetic controls (36). thornei are widespread and damaging. commercial fields have reduced yields of Therefore, optimal cultivar selection re- Polymerase chain reaction (PCR)-based intolerant wheat cultivars by as much as quires that the lesion nematode species techniques have been reported for identify- 60% (29,30). Pratylenchus spp. populations present in each field or region be accu- ing species of Pratylenchus (22,32,37). exceeding the threshold for economic dam- rately identified. Identification of these The combination of PCR and restriction age are now thought to occur in up to 60% species will become even more important fragment-length polymorphism (RFLP) of wheat fields in the Pacific Northwest. P. if current biofuels initiatives result in addi- has been used to discriminate Pratylenchus neglectus is more widely distributed than P. tional production of biofuel crops. For spp. (32,37), but the RFLP portion of the thornei, but mixed populations have been example, increasing canola acreage has the test requires an additional step and time found within the same field (27). potential to significantly affect the level of that is not amenable to commercial, high- The best approach to control damage damage to subsequent wheat crops, de- throughput applications. PCR amplifica- from lesion nematodes is to grow cultivars pending on the Pratylenchus spp. present. tion with species-specific primers followed Canola may increase populations of P. by gel electrophoresis has been used effec- neglectus but is less likely to increase tively for discrimination of some plant- Corresponding author: G. P. Yan populations of P. thornei (36). parasitic nematodes (1,3,4,6,18,35,40). Al- E-mail: [email protected] Distinction between P. neglectus and P. Banna et al. (1) distinguished P. neglectus thornei based on morphological character- and P. thornei along with four other Praty- Accepted for publication 3 August 2008. istics requires detailed microscopic meas- lenchus spp. using PCR and species- urements by an experienced nematologist. specific primers derived from the internal doi:10.1094/ PDIS-92-11-1480 Morphological diagnosis may be further variable portion of the D3 expansion re- © 2008 The American Phytopathological Society complicated by difficulties in distinguish- gion of the 28S rDNA. Carrasco-

1480 Plant Disease / Vol. 92 No. 11 Ballesteros et al. (6) identified P. thornei pension containing nematode pieces were a MyCycler Thermal Cycler (Bio-Rad, from different life stages of the nematode pipetted into a 0.2-ml sterile Eppendorf Richmond, CA) as follows: 95°C for 3 min using PCR and specific sequence- tube with 8 µl of lysis buffer (500 mM followed by 35 cycles of 95°C for 30 s, characterized amplified region (SCAR) KCl; 100 mM Tris-Cl, pH 8.3; 15 mM 60°C (PNEG-F1/D3B5) or 62°C primers derived from a randomly amplified MgCl2; 10 mM dithiothreitol [DTT]; 4.5% (PTHO/D3B) for 30 s, and 72°C for 30 s, DNA fragment. Tween 20; and 0.1% gelatin). The tube with a final extension at 72°C for 7 min. Although the PCR-based methods de- contents were frozen at –20°C for at least PCR products were separated in 2% stan- scribed above distinguished P. neglectus 20 min, then thawed, and 2 µl of proteinase dard agarose gels stained with ethidium from P. thornei, they were designed to use K at 600 µg/ml was added. The tubes were bromide. Molecular size was estimated by DNA extracted from nematode cultures or incubated at 65°C in a water bath for 1 h, a 100-bp DNA ladder (Roche). Band pat- isolated individuals. There is a clear need and consecutively at 95°C for 10 min to tern was photographed under UV light for quick diagnostic methods that do not inactivate proteinase K. The nematode using a Polaroid digital camera, and ana- require time-consuming nematode extrac- lysis mix was centrifuged at 16,000 × g for lyzed by the Polaroid PhotoMAX Pro pro- tion (38), such as are currently available 5 min and the supernatant was transferred gram (Polaroid, Bedford, MA). This ex- for detecting plant-pathogenic bacteria and to a new 0.2-ml tube and stored in –20°C periment was performed four times. fungi in soil (9,11,15). There are few re- until used as the DNA template. To expand specificity evaluation to addi- ports on the use of molecular techniques Primer selection and evaluation for tional nontarget nematode species that for detecting and distinguishing nematodes specificity. The species-specific forward could not be obtained in culture or DNA species in soil. Atkins et al. (4) detected primer PTHO and the common reverse based on requests, an in silico analysis was false root-knot nematodes (Nacobbus spp.) primer D3B described by Al-Banna et al. conducted using GenBank D3 expansion from soil and tubers using PCR (1) were used to identify P. thornei (Table sequences from four Pratylenchus spp. and with species-specific primers and DNA 1). The species-specific forward primer four nematode genera and the computer extracted with a soil DNA extraction kit. PNEG-F1 and the common reverse primer software PrimerSelect 5.00 (DNASTAR, Iwahori et al. (14) detected root-knot D3B5 were designed to identify P. neglec- Inc, Madison, WI). Primer specificity was nematodes (Meloidogyne spp.) from soil tus. PNEG-F1 was designed based on the determined by the primer-template duplex using PCR-RFLP with DNA isolated with variable region in the alignment of the 28S stability values (ΔG) as described by a modified commercial soil DNA kit. rRNA D3 expansion domains obtained Schroeder et al. (26) and Okubara et al. The objectives of this study were to de- from GenBank. D3B5 was selected from (20). The Pratylenchus spp. were P. vulnus velop a quick, sensitive, reliable, and non- the conserved region of the same D3 ex- (U47547 and AJ545020), P. hexincisus proprietary diagnostic method for identify- pansion domain in order to produce a (U47554 and AF303949), P. brachyurus ing P. neglectus and P. thornei directly PCR fragment different in size from that (U47553), and P. coffeae (U47552, from soil, and to determine whether the produced with DNA from P. thornei. AF170428, and AF170436). The nematode method was applicable to a wide range of These primers were analyzed for anneal- species in other genera were Meloidogyne soils inhabited by these nematodes at a ing temperature, GC content, dimers, and incognita (AY355417), Globodera ta- range of population densities. hairpin loops using GeneRunner (version bacum solanacearum (AF393846), G. 3.05; Hastings Software, Inc., Hudson, pallida (AF393843), Paratrichodorus MATERIALS AND METHODS NY), and synthesized by Invitrogen pachydermus (AM180727), P. anemones DNA extraction from nematodes. (Carlsbad, CA). (AJ781505), and H. glycines (DQ328692). Nematodes were extracted from soil sam- Six isolates of P. neglectus and two iso- DNA extraction from soil and DNA ples using the Whitehead tray method (39). lates of P. thornei from Oregon, Washing- purification by polyvinylpolypyrroli- This method relies principally on the ac- ton, Montana, and Idaho (Table 2) were done powder column. Various extraction tive movement of migratory nematodes used to examine the specificity of the Pra- methods were evaluated by examining and from the moist soil sample into the sur- tylenchus primers. Isolates of five other comparing band intensity of PCR amplifi- rounding water. Adult females were mor- Pratylenchus spp. and three Meloidogyne cation on agarose gel. Total genomic DNA phologically identified (12,13) as either P. spp. (Table 2) were tested for primer speci- was extracted from soil using two com- neglectus (two lip annules, pointed but still ficity. Five plant-parasitic nematode spe- mercial soil DNA extraction kits, FastDNA round tail, and vulva position ratio on body cies ( avenae, H. filipjevi, Ty- SPIN Kit for Soil (Bio101, La Jolla, CA) at 76 to 87%) or P. thornei (three lip an- lenchorhynchus sp., Merlinius brevidens, and PowerSoil DNA Isolation Kit (MoBio, nules, slightly truncated tail on end, and and Paratylenchus sp.), three nematode Carlsbad, CA), according to the manufac- vulva ratio at 73 to 80%), and then placed communities frequently found in wheat turers’ recommendations. Four buffers onto surface-sterilized carrot disks to es- fields in eastern Oregon, and six fungal prepared in the laboratory also were com- tablish pure cultures. DNA was extracted species commonly associated with wheat pared. These were designated buffer A (pH from nematodes following the protocol root diseases were also used as controls 8.0, 30 mM Na2HPO4 and 90 mM described by Waeyenberge et al. (37), with (Table 2). PCR reactions of 25 µl con- NaH2PO4) (38), buffer B (200 mM Tris- some modifications. Ten nematodes of tained the DNA template (5 µl), 0.75 units HCl, pH 8.5; 250 mM NaCl; 25 mM mixed juvenile and adult stages were hand- of Taq polymerase (Roche, Mannheim, EDTA; 0.5% sodium dodecyl sulfate picked using a dental pick, put into 20 µl Germany), 200 µM dNTPs, 0.5 µM each [SDS]) (25), buffer C (500 mM KCl; 100 of sterilized nanopure water on a concave primer, 1× PCR buffer with 1.5 mM mM Tris-Cl, PH 8.3; 15 mM MgCl2; 10 glass slide, and cut into two pieces under a MgCl2, and 1× Cresol Red in 20% glyc- mM DTT; 4.5% Tween 20; 0.1% gelatin) dissecting microscope. Then, 10 µl of sus- erol. PCR amplification was performed in (37), and buffer D (equal amounts of

Table 1. Sequences of polymerase chain reaction (PCR) primers and the expected size of PCR products to discriminate Pratylenchus neglectus and P. thornei Species Primer namea Sequence (5′–3′) Band size (bp) Reference P. neglectus F: PNEG-F1 CGCAATGAAAGTGAACAATGTC 144 This study R: D3B5 AGTTCACCATCTTTCGGGTC … This study P. thornei F: PTHO GAAAGTGAAGGTATCCCTCG 288 1 R: D3B TCGGAAGGAACCAGCTACTA … 1,2,7 a F = forward primer and R = reverse primer.

Plant Disease / November 2008 1481 phosphate buffer [100 mM NaH2PO4, pH prepared as a substitute for spin filters tube. Then, 300 µl of cold NaOAc (3 M, 8.0] and SDS buffer [100 mM NaCl; 500 provided in the extraction kits. A hole (1 pH 5.2) was added and, following incuba- mM Tris, pH 8.0; 10% SDS]) (11). DNA mm) was made in the bottom of a 0.5-ml tion (5 min at –20°C), the suspension was extractions with these buffers were per- tube and was covered by small pieces cleared by centrifugation for 5 min at formed after mechanical lysis using lysing (0.005 g) of glass wool (Corning, NY) 16,000 × g. The supernatant (up to 650 µl) matrix tubes provided in the FastDNA using a forceps. The tube was inserted into was transferred to a clean tube. DNA was SPIN Kit, processed in a FastPrep FP120 a 1.5-ml tube with the lid removed. Dry subsequently precipitated with 600 µl of machine (Bio101, Savant) for 30 s at a PVPP powder (0.05 g; Sigma-Aldrich) was cold isopropanol for 20 min at room tem- speed of 5.5 m/s. Crude DNA extracts added to the 0.5-ml tube. The PVPP col- perature and collected by centrifugation at were then purified using High Pure spin umn was conditioned by the addition of 16,000 × g for 5 min. The resulting pellet filters (Roche) that contained water- 100 µl of sterilized nanopure water was rinsed with 70% ethanol and centri- insoluble polyvinylpolypyrrolidone (PVPP; (SNPH2O) twice, each followed by 3 min fuged for 5 min at 16,000 × g. The DNA Sigma-Aldrich, St. Louis) powder. of centrifugation at 400 × g. A final spin pellet was air dried and dissolved in 100 µl To replace the expensive lysing matrix for 30 s was performed just before use to of SNPH2O. The crude DNA solution was provided in FastDNA SPIN Kit, the effect remove residual water. added to the PVPP column described of glass beads was assessed, including 0.20 The optimized protocol developed in above, followed by centrifugation at 400 × g of 1.0-mm glass beads (Research Prod- this study is as follows. Soil was added to g for 1 min, incubation at room tempera- uct International Corp., Mt. Prospect, IL) a 2.0-ml screw-cap tube (Bio-Rad) with ture for 2 min, and a final centrifugation at with one ceramic sphere (6.4 mm; Fisher glass beads (0.53 g, 1 mm). Buffer D (300 400 × g for 5 min. The purified DNA in the Scientific, Pittsburgh), 0.80 g of 1.0-mm µl of each phosphate buffer and SDS tube was then used for PCR. glass beads, 0.53 g of 1.0-mm glass beads, buffer) was added and the tube was in- Optimization of PCR conditions with and 0.60 g of 1.0-mm glass beads com- verted several times. Chloroform:isoamyl DNA extracted from soil. To improve bined with 0.15 g of 0.1-mm glass beads. alcohol (400 µl, 24:1) was added to the PCR amplification for P. neglectus, the Extraction of DNA from soil often re- tube and shaken in a FastPrep FP120 ma- effect of bovine serum albumin (BSA) as a sults in co-extraction of humic substances chine for 30 s at a speed of 5.5 m/s. The PCR enhancer was assessed. A BSA solu- that inhibit DNA polymerase during PCR tube was centrifuged for 5 min at 16,000 × tion of 10 µg/µl was added to PCR reaction amplification. To remove humic substances g and the supernatant (less than 600 µl) mixtures to make final concentrations of 0, prior to PCR reaction, PVPP columns were transferred to a clean 1.5-ml centrifuge 0.1, 0.4, 0.8, and 1.0 µg/µl. Additional

Table 2. Isolates of Pratylenchus spp. and other nematode and fungal species used to examine the species-specific polymerase chain reaction primers for distinguishing P. neglectus and P. t ho r nei Speciesa N or Fb Isolate Origin Host Sourcec Pratylenchus neglectus N Pn1 La Grande, OR Wheat R. Smiley P. neglectus N Pn2 Lind, WA Wheat R. Smiley P. neglectus N Pn3 Moro, OR Wheat R. Smiley P. neglectus N Pn4 Heppner, OR Wheat R. Smiley P. neglectus N Pn5 Great Falls, MT Wheat A. Dyer P. neglectus N Pn6 Canyon County, ID Potato S. Hafez P. thornei N Pt1 Pendleton, OR Wheat R. Smiley P. thornei N Pt2 Pendleton, OR Wheat R. Smiley P. agilis N 031302 Wye, MD Corn L. Carta P. crenatus N 012204 Clarksville, MD Grass Z. Handoo P. zeae N 030204 North Carolina Corn L. Carta P. scribneri N 062805 Homestead, FL Tomato W. Klassen P. scribneri N 032102 Seneca County, OH Corn L. Carta P. penetrans N 030402 New York Corn L. Carta P. penetrans N 052704 Wisconsin Potato A. MacGuidwin Meloidogyne naasi N 110704 Linn County, OR Oat, wheat K. Merrifield M. chitwoodi N 110504 Parma, ID Potato S. Hafez M. hapla N 070808 Prosser, WA Grape E. Riga Heterodera avenae N Ha La Grande, OR Wheat R. Smiley H. filipjevi N Hf La Grande, OR Wheat R. Smiley Tylenchorhynchus sp. N Ty Pendleton, OR Wheat S. Easley Merlinius brevidens N Mb Pendleton, OR Wheat A. Thompson Paratylenchus sp. N Pa La Grande, OR Wheat G. Yan Nematode community 1 N Nc1 Pendleton, OR Wheat J. Sheedy Nematode community 2 N Nc2 Pendleton, OR Wheat J. Sheedy Nematode community 3 N Nc3 Pendleton, OR Wheat J. Sheedy Bipolaris sorokiniana F 103-18 Walla Walla, WA Wheat R. Smiley Fusarium culmorum F R5321 Chatham, Ontario, Canada Wheat R. Smiley F. pseudograminearum F 032-06 Moro, OR Wheat R. Smiley Gaeumannomyces graminis var. tritici F 99401 Dayton, WA Wheat R. Smiley Rhizoctonia oryzae F 2-3-2 Wenatchee, WA Wheat M. Mazzola R. solani AG-8 F 1727B Wenatchee, WA Wheat M. Mazzola a Nematode communities 1, 2, and 3 were extracted from soils that were not infested with any P. neglectus or P. thornei but were infested with many other plant-parasitic and non-plant-parasitic nematodes; the Whitehead tray method was used for nematode extraction. b N = nematodes and F = fungi. c Isolates were obtained from R. Smiley, S. Easley, G. Yan, A. Thompson, and J. Sheedy, Oregon State University, Columbia Basin Agricultural Research Center, Pendleton; A. Dyer, Montana State University, Bozeman; S. Hafez, University of Idaho, Southwest Idaho Research and Extension Center, Parma; L. Carta and Z. Handoo, United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Nematology Laboratory, Beltsville, MD; W. Klassen, University of Florida, Tropical Research and Education Center, Homestead; A. MacGuidwin, University of Wisconsin, Department of Plant Pa- thology, Madison; K. Merrifield, Oregon State University, Department of Botany and Plant Pathology, Corvallis; E. Riga, Washington State University, Irrigated Agriculture Research and Extension Center, Prosser; and M. Mazzola, USDA-ARS, Wenatchee, WA.

1482 Plant Disease / Vol. 92 No. 11 adjustments made to the previously de- tions. A no-DNA template was used as a Pn4, Pn5, and Pn6 when combined with scribed PCR protocol included decreasing negative control and DNA from pure nema- primer D3B5 but did not produce an am- the DNA template from 5 to 1 µl for P. tode cultures was used as positive controls. plicon with DNA from P. thornei isolates neglectus and from 5 to 0.2 µl for P. Pt1 and Pt2 (Fig. 1A). As expected, the thornei. The number of PCR cycles was RESULTS primer pair PTHO/D3B amplified a spe- increased from 35 to 40 for both species. Primer specificity. The primer PNEG- cific band (288 bp) from P. thornei cul- Detection sensitivity of PCR amplifi- F1 designed for P. neglectus amplified a tures Pt1 and Pt2 but did not generate an cation. Soil was inoculated with P. neglec- unique PCR product (144 bp) with DNA amplicon with DNA from P. neglectus tus or P. thornei to evaluate the sensitivity from P. neglectus isolates Pn1, Pn2, Pn3, cultures Pn1, Pn2, Pn3, Pn4, Pn5, and Pn6 of PCR detection. A Walla Walla silt loam that was not infested with P. neglectus and Table 3. Numbers of Pratylenchus neglectus, P. t h o r n e i , and other plant-parasitic and non-plant- P. thornei was collected from Pendleton, parasitic nematodes extracted from naturally infested soils using the Whitehead tray and morphologi- OR and was sieved through a 3-mm sieve. cal identification procedures and polymerase chain reaction (PCR) assays Thereafter, the soil was autoclaved (121°C, a b 115 kPa) two times for 45 min each to Nematodes/kg of soil PCR assay completely kill all living organisms. Juve- Soilc Year collected Pn Pt OP NP Pn Pt niles of P. neglectus and P. thornei were S1 2007 87 0 1,136 3,843 – – added separately to 1 g of autoclaved soil S2 2007 93 0 0 3,988 – – using a dental pick with the aid of a micro- S3 2007 113 0 0 10,251 – – scope. Concentrations of 0, 1, 2, 3, and 5 S4 2007 343 0 2,286 5,373 + – juveniles/g of soil were used. Nematode S5 2007 815 0 0 4,158 + –

DNA was extracted from the soils using S6 2007 1,237 0 0 4,948 + – S7 2007 2,697 0 0 4,238 + – the method developed in this study and S8 2007 3,609 570 190 30,388 + + DNA was quantified using the NanoDrop S9 2006 4,185 0 135 3,240 + – ND-1000 Spectrophotometer (Wilmington, S10 2007 4,689 0 408 2,854 + – DE). The DNA extraction procedure was S11 2006 6,740 0 124 3,463 + – conducted from eight different samples for S12 2007 7,774 0 2,418 11,056 + – each level of inoculation. PCR reactions S13 2007 11,118 0 139 2,085 + – S14 2007 13,704 98 196 25,059 + – were performed in duplicate for each inde- pendent DNA extraction under the opti- S15 2007 16,836 324 324 8,256 + + S16 2007 17,959 0 770 12,828 + – mum conditions for the soil samples as S17 2007 0 0 2,575 3,277 – – described above with BSA at a final con- S18 2007 0 0 1,033 2,195 – – centration of 0.8 µg/µl. The sensitivity for S19 2007 0 0 898 2,437 – – PCR amplification was determined by the S20 2007 0 117 0 1,756 – – ability to detect a minimum number of S21 2007 0 126 631 1,767 – + juveniles/g of soil. S22 2006 0 315 473 630 – +

Validation of species identification in S23 2007 0 635 741 4,340 – + S24 2007 0 1,936 129 1,678 – + soil. Sixteen soil samples were collected S25 2007 0 3,277 0 2,622 – + from various fields between Heppner and S26 2007 0 4,903 0 2,790 – + Condon in Morrow County, OR during S27 2007 0 7,198 0 1,400 – + 2006 and 2007. This location was known S28 2007 0 9,430 0 982 – + to be significantly infested with P. neglec- S29 2006 0 13,721 0 1,083 – + tus and minimally infested with P. thornei. S30 2007 0 15,998 0 4,081 – + Fourteen soil samples were taken from a Pn = P. neglectus, Pt = P. thornei, OP = other plant-parasitic nematodes, and NP = non-plant-parasitic fields adjacent to the Columbia Basin Ag- nematodes. ricultural Research Center near Pendleton b Presence (+) or absence (–) of Pn (144 bp) or Pt (288 bp) were detected by PCR using PNEG- in Umatilla County, OR during 2006 and F1/D3B5 or PTHO/D3B, respectively. 2007. This location was known to be c Soils were collected in Morrow (S1-S16) and Umatilla (S17-S30) Counties of Oregon. highly infested with P. thornei but mini- mally infested with P. neglectus. Nema- todes were isolated from approximately 200 g of fresh moist soil using the White- head tray method. Nematodes in 1 ml of extracted suspension were identified and quantified on a nematode-counting slide under a microscope and converted to the number per kg of soil. Nematodes were identified as P. neglectus and P. thornei (13), other plant-parasitic nematode genera (17), and non-plant-parasitic nematodes; the numbers for each group are listed in Table 3. DNA was extracted from 0.5 g of moist soil using both the PowerSoil DNA Isolation Kit and our in-house method. DNA extractions were conducted four times for each sample and PCR was per- Fig. 1. Specific polymerase chain reaction amplification for Pratylenchus neglectus (144 bp) and P. formed under the optimum conditions for thornei (288 bp) from pure cultures using species-specific primers. A, Amplified with P. neglectus- soil samples, as described above. The iden- specific primer set PNEG-F1/D3B5. B, Amplified with P. thornei-specific primer pair PTHO/D3B. tifications determined by PCR assay were DNA templates from the isolates Pn1, Pn2, Pn3, Pn4, Pn5, Pn6, Pt1, and Pt2 (Table 2) were used. Nc: compared with morphological identifica- negative control without DNA template; M: 100-bp DNA molecular weight ladder.

Plant Disease / November 2008 1483 (Fig. 1B). The sizes of the specific PCR DNA extraction from soil. Both com- the MoBio PowerSoil DNA Kit (Fig. 2B, fragments were consistent with those in- mercial kits and all extraction buffers lane 3). ferred from the nucleotide sequences. Fur- tested in this study allowed the detection All four treatments using glass beads thermore, no specific PCR product was of P. thornei and P. neglectus in soil (Fig. yielded DNA and allowed the detection of observed when tested with DNA extracted 2). MoBio PowerSoil DNA Kit (lane 3) P. thornei with the FastPrep FP120 instru- from five other nontarget Pratylenchus generated a slightly brighter PCR band ment and buffer D. The brightest bands spp. (a total of seven isolates) and three than that by FastDNA SPIN Kit (lane 4). were achieved with 0.53 g of 1.0-mm glass Meloidogyne spp. (Table 2). Similarly, no Among the four buffers, buffer D (lane 8) beads as well as 0.20 g of 1.0-mm glass specific PCR product was produced with produced the brightest amplification beads combined with one ceramic sphere. DNA from five other plant-parasitic nema- band. Buffer C that was used for DNA Minimal amplification resulted from 0.60 tode species (H. avenae, H. filipjevi, Tylen- extraction from nematode cultures (lane g of 1-mm glass beads combined with 0.15 chorhynchus sp., Merlinius brevidens, and 7) was least effective, with relatively faint g of 0.1-mm glass beads. Intermediate Paratylenchus sp.), three nematode com- amplification (Fig. 2A). There was no amplification was obtained with 0.80 g of munities extracted from local wheat field difference in the intensity of bands ob- 1.0-mm glass beads. A similar effect was soils, and six fungal species (Table 2). In served when using the buffer A (lane 5) observed for detection of P. neglectus from silico analysis with four other related Pra- and buffer B (lane 6). Therefore, we used soil. tylenchus spp. and six species in four buffer D in further experiments. Notably, Removal of PCR inhibitors in soil. No nematode genera showed that the new P. t ho rne i DNA extracted with buffer D PCR amplification product was produced species-specific primer PNEG-F1 does not (lane 8) produced an amplification frag- with crude DNA extracts obtained with form hybrids with the sequences of these ment as bright as that with the MoBio each of the four extraction buffers. To rule nontarget organisms because the stability PowerSoil DNA Kit (Fig. 2A, lane 3). out PCR inhibitors co-extracted from soil, values (ΔG) obtained for these nontarget Similarly, P. neglectus DNA extracted PVPP columns were used to replace spin hybrids were below the default stability from soil with buffer D (lane 8) also pro- filters provided in commercial kits. After cut-off (–33 kcal/mol). duced a band almost as bright as that with centrifugation at 400 × g, DNA extracts from all samples produced visible ampli- cons without further dilution of the DNA extracts. Higher speed (720 × g) centrifu- gation for 5 min resulted in DNA extracts that could not be amplified with the same PCR conditions. PCR amplification of nematode DNA from soil. In spite of the special PVPP purification step, PCR amplification from soil extracts with the P. neglectus-specific primer set was relatively weak compared with that from nematode pure cultures. To improve the amplification, BSA was added to the PCR reaction mixtures at different concentrations (0.1, 0.4, 0.8, and 1.0 µg/µl). No marked difference was observed for the band intensity between different concentrations of BSA; however, the pres- ence of BSA enhanced PCR amplification over control samples. The effect of BSA on Fig. 2. Effect of DNA extraction methods on polymerase chain reaction (PCR) detection. A, DNA from the soil sample S29 was used for PCR amplification with -specific primer PCR using DNA extracted with our in- pair PTHO/D3B: lane 1, positive control using DNA from hand-picked P. thornei. B, DNA from the house method was more evident than on soil sample S11 was used for PCR amplification with P. neglectus-specific primer set PNEG-F1/D3B5: DNA extracted with the PowerSoil DNA lane 1, positive control using DNA from hand-picked P. neglectus; lane 2, negative control without Isolation Kit. The addition of BSA (0.8 DNA template; lane 3, PowerSoil DNA Isolation Kit; lane 4, FastDNA SPIN Kit for Soil; lane 5, buffer µg/µl) also allowed amplification at a A; lane 6, buffer B; lane 7, buffer C; lane 8, buffer D; lane M, 100-bp DNA molecular weight ladder. wider range of DNA concentrations, from 0.1 to 2.7 ng/µl. In contrast, detection of P. thornei from soil could be achieved with- out BSA. However, PCR amplification for P. thornei required a lower concentration of DNA from 0.1 to 0.5 ng/µl. Detection sensitivity of the PCR reac- tion in artificially inoculated soils. Vary- ing numbers of P. neglectus and P. thornei juveniles (n = 1, 2, 3, and 5) were inocu- lated into 1 g of sterile soils. Specific bands were amplified from all levels of the inoculation for both species even when one nematode was added (Fig. 3). No band was generated from the control soil (sterilized but not inoculated). The PCR test can de- tect one P. neglectus juvenile per gram of soil in seven of the eight independent DNA Fig. 3. Level of detection for the polymerase chain reaction amplification in artificially inoculated soils. A, Inoculation of Pratylenchus thornei: zero juvenile (J) (#0), one J (#1), two J (#2), three J (#3), extractions and one P. thornei juvenile per and five J (#5); B, Inoculation of P. neglectus: zero J (#0), one J (#1), two J (#2), three J (#3), and five gram of soil in 100% of the assays. This J (#5); M: 100-bp DNA ladder. detection sensitivity equates to half the

1484 Plant Disease / Vol. 92 No. 11 estimated economic threshold (2,000 detected and discriminated in a variety of cific amplicon with P. thornei DNA but did nematodes/kg of soil) for natural infesta- soil samples using a DNA extraction and not amplify DNA from other closely re- tions in dryland fields in the Pacific PCR amplification method developed in lated Pratylenchus spp., including P. ne- Northwest (30). The specific amplicons this study. This approach utilized mechani- glectus, P. brachyurus, P. penetrans, P. were produced in each of the eight repli- cal disruption of nematodes within the soil scribneri, and P. vulnus (1). For P. neglec- cates for the other inoculum levels. sample in the FastPrep homogenizer, ex- tus, the species-specific primer PNEG-F1 Discrimination of P. neglectus and P. tracted DNA from soil with or without the was designed from the variable region of thornei in naturally infested soils. The use of a commercial kit, and applied spe- D3 expansion domain of the 28S rRNA. developed protocol was validated for de- cies-specific primers to detect multicopy No cross reactivity was observed between tecting and discriminating P. neglectus and DNA of the target Pratylenchus spp. The the sequence of this primer and DNA from P. thornei in 30 soil samples harboring a assays were sensitive, reliably detecting other closely related Pratylenchus spp. range of population densities, from 0 to one juvenile of genomic DNA in 1 g of reported in the Pacific Northwest by in 17,959 P. neglectus/kg of soil and 0 to sterile, inoculated soil. The assays also silico analysis. Moreover, when challenged 15,998 P. thornei/kg of soil. In all, 2 of 16 proved highly specific when evaluated with five other nontarget Pratylenchus soil samples (Table 3, S4 and S5) from the against template DNA from a range of spp., eight nematode species in other gen- Morrow County field sites produced bands nematode and fungal species that are era, three nematode communities, and six with the same size as the P. neglectus- common in wheat fields in the Pacific fungal species, the specific amplicons were positive control (144 bp) in three of the Northwest. Although PCR assays to dis- not produced. DNA from three soil sam- four independent DNA extractions when criminate Pratylenchus spp. have been ples that did not contain P. neglectus and P. tested with PNEG-F1/D3B5. Eleven sam- previously reported (1,6,32,37), they were thornei but had other plant-parasitic and ples (S6 to S16) produced the expected not evaluated for detecting P. neglectus and non-plant-parasitic nematodes typical of amplicons in each replicate with PNEG- P. thornei directly from soil. dryland wheat fields in eastern Oregon F1/D3B5. The other three samples (S1 to For P. thornei, the species-specific also did not yield the specific amplicons, S3) did not produce the expected amplicon primer PTHO described by Al-Banna et al. demonstrating the specificity of the prim- in any of the replicates. The limit of PCR (1) was used. This primer produced a spe- ers in the soil community complex. Further detection for P. neglectus was 343 nema- todes/kg of soil, as determined by the Whitehead tray method. The specific am- plicon was not produced in any of the four replicates with the P. thornei-specific primer set PTHO/D3B for all except two samples. The amplicons were evident for these two samples (S8 and S15), which also contained 570 and 324 P. thornei/kg of soil, respectively, as determined by the Whitehead tray method. DNA from 2 of 14 samples (Table 3, S21 and S22) collected from Umatilla County sites generated the expected P. thornei amplicon (288 bp) in three of the four replicates when amplified with PTHO/D3B. DNA from eight samples (S23 to S30) also generated the expected P. thornei amplicon in each replicate. Sample S20 did not produce the expected amplicon in any of the replicates. The limit of PCR detection for P. thornei was 126 nema- todes/kg of soil. The specific amplicon was not observed for the samples from S20 to S30 in any of the replicates with PNEG- F1/D3B5. Soil samples (S17, S18, and S19) that had no P. neglectus and P. thornei, according to the Whitehead tray method, also did not produce the specific amplicons in each replicate when tested with both primer pairs, confirming the specificity of the primers even with the presence of DNA from other plant-parasitic and nonparasitic nematodes in the samples (Table 3). The banding patterns for DNA extractions from nine soil samples obtained by our in-house DNA extraction assay and tested with both species-specific PCR primer sets is shown in Figure 4A. A similar banding pattern was observed for these Fig. 4. Identification of Pratylenchus thornei and P. neglectus in naturally infested soil samples. A, DNA extractions obtained by the PowerSoil Nematode DNA was extracted from soil without any components of a commercial kit. DNA was am- DNA isolation kit (Fig. 4B). plified with the P. t h o r n e i -specific primer set PTHO/D3B (top) and with the P. neglectus-specific primer set PNEG-F1/D3B5 (bottom). B, Nematode DNA was extracted from soil with the PowerSoil DNA Isolation Kit. DNA was amplified with the P. t h o r n e i -specific primer set PTHO/D3B (top) and DISCUSSION with the P. neglectus-specific primer set PNEG-F1/D3B5 (bottom). The soil samples S4, S8, S11, S12, Two plant-parasitic root-lesion nema- S15, S19, S21, S26, and S30 were shown in Table 3. Pc: positive control from nematode culture; Nc: todes, P. neglectus and P. thornei, were negative control without DNA template.

Plant Disease / November 2008 1485 testing of these primers with local species (phosphate and SDS buffer), 0.53 g of 1.0- S3 or the lack of P. thornei DNA detection may be necessary if these primers were mm disruption glass beads, glass wool, and for S20 by PCR is probably due to the used for PCR identification with DNA purification column with dry PVPP pow- uneven distribution of nematodes in soil extracts from soil in other areas. der yielded PCR-quality DNA that was (23) and the small amount of soil (≤1 g) Published species-specific primers (1) comparable with that yielded by the Pow- that can be processed in the FastPrep produced PCR fragments with very little erSoil DNA kit. The estimated cost for one FP120 homogenizer. However, the White- difference (2 bp) in length for the two reaction is $0.30, which greatly reduces head tray method requires a minimum of Pratylenchus spp. due to the use of a the cost of DNA extraction and purification 48 h to extract approximately 60% of the common reverse primer, D3B. Therefore, from soil. nematode population from silt loams, and we designed a reverse primer D3B5 from The effect of BSA on PCR amplification it may take as many as 6 days to extract all the conserved sequences of 28S rRNA D3 was different for the two target nematode nematodes (5). In contrast, DNA extraction expansion region. This resulted in PCR species at lower DNA concentrations (0.1 and PCR amplification, as used in this fragments that had larger size differences to 0.5 ng/µl). BSA greatly enhanced detec- study, require 5 to 6 h. that could be easily separated by agarose tion of P. neglectus in soil, whereas P. Pratylenchus spp.-infested soil repre- gel electrophoresis. Additionally, the for- thornei could be detected without the addi- sents a serious threat to intolerant wheat ward primer PNEG-F1 for P. neglectus tion of BSA. The effect of BSA on PCR cultivars. Therefore, the detection and proved to be more sensitive and robust for was not correlated with BSA concentra- discrimination of these nematodes in soil detection of DNA from soil extracts than tions. Similarly, Kageyama et al. (15) are very important in the Pacific North- the PNEG primer described by Al-Banna found that BSA was essential for detecting west. The molecular method developed in et al. (1) at the annealing temperature of V. dahliae and enhanced detection of Py- this study allows nematode DNA to be 60°C. thium ultimum but was not necessary for extracted directly from soil, facilitating the The DNA extraction method developed detecting Plasmodiophora brassicae in rapid and inexpensive detection and identi- in this study was based on the method of soil. BSA is thought to stabilize Taq poly- fication of P. neglectus and P. thornei with Damm and Fourie (11), which was devel- merase and neutralize inhibitory contami- equipment commonly available in research oped to extract DNA from the soilborne nants (11,16,19,24). and commercial laboratories. In commer- fungal pathogens Phaeomoniella chlamy- PCR detected one juvenile in 1 g of ster- cial labs, Pratylenchus spp. are often quan- dospora and Cylindrocarpon spp. With ile, inoculated soil, equating to 1,000 juve- tified at the genus level, which is now rec- some modifications, we used this protocol niles/kg of soil. It could detect even lower ognized as inadequate for serving wheat- to extract Pratylenchus neglectus and P. densities of nematodes in naturally in- based agricultural systems where Praty- thornei DNA from soil for PCR detection. fested soil: 126 Pratylenchus thornei lenchus spp.-specific tolerance and resis- Adding 0.53 g of 1.0-mm glass beads or nematodes/kg of soil and 343 P. neglectus tance are being developed. This PCR- 0.20 g of 1.0-mm glass beads combined nematodes/kg of soil. The discrepancy based diagnostic method has the potential with one ceramic sphere to the extraction between artificially inoculated soil and for implementation in commercial as well buffer increased the amplification inten- naturally infested soil may be due to dif- as research applications. Protocols re- sity. Considering the relative cost of a ferences in sample size and nematode ported here are being adapted for use in ceramic sphere, 0.53 g of 1.0-mm glass counting methods used. One juvenile was real-time PCR applications, thus enabling beads were selected in place of the lysing the minimum number of nematodes that labs to avoid physical separation, micro- matrix provided in FastDNA SPIN Kit. can be added into 1 g of sterile soil, scopic identification, and counting of P. Glass beads aided in the disruption or whereas the number of target nematodes neglectus and P. thornei from soil. shearing of the nematode cuticle, allowing from natural soil was obtained using the release of DNA. Cullen and Hirsch (9) Whitehead tray method by counting the ACKNOWLEDGMENTS number of nematodes per milliliter of sus- This research was supported by the Oregon State reported that the use of bead-beating in the University, Agricultural Research Foundation cell lysis step increased the DNA yield and pension extracted from approximately 200 project ARF 7117, and a contract with the USDA- also decreased the amount of humic com- g of soil and converting to the number in 1 ARS (SCA 58-5348-9-100, “Control of Root Dis- pounds when monitoring a Rhizobium kg of soil. More importantly, the detection eases of Wheat and Barley”), and USDA-ARS leguminosarum bv. viciae strain RSM2004 sensitivity was much lower than the eco- Project No. 5248-22000-012-00D (P.A.O.). We nomic threshold level (2,000 nematodes/kg thank S. H. Hulbert and C. Yin at Washington State in Rothamsted field soils. Kageyama et al. University for use of the NanoDrop ND-1000 (15) reported that adding 0.2 g of 1-mm of soil) in the Pacific Northwest (30), indi- Spectrophotometer; A. Dyer, S. Hafez, L. Carta, Z. glass beads to the extraction buffer was cating that this method could be useful for Handoo, W. Klassen, A. MacGuidwin, K. Merri- necessary for detecting Verticillium dahl- disease forecasting and management. This field, E. Riga, and M. Mazzola for providing iso- iae in soil. sensitivity was higher than recent reports lates of control nematodes and fungi; and C. Wat- Purification of DNA from soil was es- for Meloidogyne and Nacobbus spp., son for technical support. sential for detecting the two target nema- where the level of detection was 5,000 LITERATURE CITED tode species. Two merits of the DNA puri- juveniles/kg of soil (14) and 30,000 juve- 1. Al-Banna, L., Ploeg, A. T., Williamson, V. M., fication method developed in this study are niles/kg of soil (4), respectively. and Kaloshian, I. 2004. Discrimination of six that (i) it does not require the expensive During the present study, the presence Pratylenchus species using PCR and species- of P. neglectus and P. thornei was success- specific primers. J. Nematol. 36:142-146. Micro Bio-Spin column or Sephadex G-75 2. Al-Banna, L., Williamson, V., and Gardner, S. used by Cullen et al. (10) and (ii) it is easy fully detected in most of the soil samples L. 1997. Phylogenetic analysis of nematodes to prepare the PVPP column. Extraction of taken from Morrow County or Umatilla of the genus Pratylenchus using nuclear 26 S DNA from soil frequently results in co- County, Oregon. Each pathogen was spe- rDNA. Mol. Phylogenet. Evol. 7:94-102. extraction of humic substances that inter- cifically detected in naturally infested field 3. Amiri, S., Subbotin, S. A., and Moens, M. soils that contained a higher number of 2002. Identification of the beet cyst nematode fere with PCR amplification (9,11,33). Heterodera schachtii by PCR. Eur. J. Plant PVPP is an inexpensive chemical that can nematodes than the respective detection Pathol. 108:497-506. be utilized for eliminating the majority of limits. Particularly, two soil samples were 4. Atkins, S. D., Manzanilla-López, R. H., humic and fulvic acids because it binds infested with both P. neglectus and P. Franco, J., Peteira, B., and Kerry, B. R. 2005. phenolic compounds (9–11). Commercial thornei and, accordingly, the two species A molecular diagnostic method for detecting were detected by PCR. Compared with the Nacobbus in soil and in potato tubers. Nema- soil DNA extraction kits are expensive tology 7:193-202. (e.g., the cost of the MOBio PowerSoil Whitehead tray method and subsequent 5. Bell, N. L., and Watson, R. N. 2001. Optimis- DNA kit is approximately $4.16 per reac- microscopic identification, the lack of P. ing the Whitehead and Hemming tray method tion). Our in-house assay, using buffer D neglectus DNA detection for S1, S2, and to extract plant parasitic and other nematodes

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