Andrea M. Skantar

Detection and discrimination of Pratylenchus neglectus and P. thornei in DNA extracts from soil

A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei from soil. A primer set was designed from Pratylenchus 28S rRNA gene sequences of the D3 expansion domain. Primer specificity was confirmed with 23 isolates of 15 nematode species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and a method developed in our laboratory gave comparable amplification. PCR conditions were optimized and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy and morphology, and could be used as a rapid diagnostic tool for commercial and research applications for disease forecasting and management.

Developing a Real-Time PCR Assay for Detection and Quantification of Pratylenchus neglectus in Soil

Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R2 = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R2 = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.

Root-Knot Nematodes in Golf Course Greens of the Western United States

A survey of 238 golf courses in 10 states of the western United States found root-knot nematodes (Meloidogyne spp.) in 60% of the putting greens sampled. Sequence and phylogenetic analyses of 18S rRNA, D2-D3 of 28S rRNA, internal transcribed spacer-rRNA, and mitochondrial DNA gene sequences were used to identify specimens from 110 golf courses. The most common species, Meloidogyne naasi, was found in 58 golf courses distributed from Southern California to Washington in the coastal or cooler areas of those states. In the warmer regions of the Southwest, M. marylandi was recovered from 38 golf courses and M. graminis from 11 golf courses. This constitutes the first report of M. marylandi in Arizona, California, Hawaii, Nevada, and Utah, and the first report of M. graminis in Arizona, Hawaii, and Nevada. Two golf courses in Washington were infested with M. minor, the first record of this nematode in the Western Hemisphere. Columbia root-knot nematode, M. chitwoodi, was found in a single golf course in California. Polymerase chain reaction restriction fragment length polymorphism of the intergenic region between the cytochrome oxidase and 16S rRNA genes in the mitochondrial genome with restriction enzyme SspI was able to distinguish populations of M. graminis from M. marylandi, providing a fast and inexpensive method for future diagnosis of these nematodes from turf.

Morphological and molecular characterisation of Pratylenchus arlingtoni n. sp., P. convallariae and P. fallax (Nematoda: Pratylenchidae)

Pratylenchus arlingtoni n. sp. from the rhizosphere of grasses Poa pratensis and Festuca arundinacea at Arlington National Cemetery, VA, USA is characterised by six to eight lines in the lateral field, and pyriform to slightly overlapping pharyngeal glands. Morphological comparisons are made with lesion nematodes having similar morphometrics, six lateral lines, or crenate tail tips. Molecular sequences of the LS 28S rDNA were generated for the new species as well as P.fallax and P.convallariae. The new species differs by only 1% from identical sequences found in P.fallax and P.convallariae.

Multiplex Real-Time PCR Assays for the Identification of the Potato Cyst and Tobacco Cyst Nematodes

TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCNs) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time polymerase chain reaction (PCR). One tube contained a primer-probe set specific for G. pallida (pale potato cyst nematode) multiplexed with another primer-probe set specific for G. rostochiensis (golden potato cyst nematode). A second tube consisted of the G. pallida-specific primer-probe set multiplexed with a primer-probe set specific for G. tabacum (the morphologically similar tobacco cyst nematode). This internal transcribed spacer rDNA-based system was specific for the Globodera spp. of interest and successfully identified several populations of PCN. This rapid, sensitive, and specific quantitative PCR assay presents a useful tool for PCN regulatory response and management programs.

Species-Specific PCR Assays for Differentiating Heterodera filipjevi and H. avenae

Heterodera avenae and H. filipjevi are economically important cyst nematodes that restrict production of cereal crops in the Pacific Northwest United States and elsewhere in the world. Identification of these two species is critical for recommending and implementing effective management practices. Primers were designed from the internal transcribed spacer (ITS) regions of H. avenae and H. filipjevi ribosomal DNA. The primers were highly specific when examined on target isolates but did not amplify DNA from nontarget Heterodera, Globodera, Meloidogyne, Pratylenchus, and other nematode species tested. Polymerase chain reaction (PCR) and amplification conditions were established, and H. avenae and H. filipjevi were clearly distinguished by PCR fragments of 242 and 170 bp, respectively. Robust PCR amplification was achieved with DNA extracted from a single egg or second-stage juvenile (J2) using a laboratory-made worm lysis buffer, and DNA from 0.5 egg or J2 using a commercial kit. The PCR assays were successfully employed for differentiation of H. filipjevi and H. avenae populations collected from eight locations in three Pacific Northwest states. This is the first report of a species-specific ITS PCR assay to detect and identify H. filipjevi. The assays for both species will enhance diagnosis of cereal cyst nematode species in infested fields.

Multiple displacement amplification (MDA) of total genomic DNA from Meloidogyne spp. and comparison to crude DNA extracts in PCR of ITS1, 28S D2-D3 rDNA and Hsp90

Summary ‐ Because the quantity of nematode specimens available for molecular analysis is often limited, the number of analyses possible is constrained by the availability of DNA. Multiple displacement amplification (MDA) was assessed for whole genome amplification of crude DNA from several Meloidogyne species. MDA produced microgram quantities of template that resulted in successful amplification of the ribosomal internal transcribed spacer (ITS1) and 28S D2-D3 expansion regions, producing PCR results that were comparable to template generated by the single nematode smash method. MDA greatly improved degenerate primer PCR of single-copy Hsp90, a gene which is more sensitive than multi-copy ribosomal genes to limited DNA template. MDA should expand the number of molecular analyses possible for single nematodes. MDA will also be useful for archiving DNA from valuable specimens and provide a way for laboratories to share identical genetic material for nematode diagnosis.

Taxonomy, Morphology and Phylogenetics of Coffee-Associated Root-Lesion Nematodes, Pratylenchus spp

This review includes a synthesis of information on eight species of root-lesion nematodes (Pratylenchus spp.) that parasitize coffee or inhabit its rhizosphere. It includes a table of important morphological characters, a diagnostic key, photographs of anterior ends and tails of specimens from the USDA nematode collection, and a phylogenetic tree based on ribosomal DNA with drawings of scanning electron microscopic face-patterns. Information sources are evaluated and future research needs are outlined.

Supplemental description of Paraphelenchus acontioides (Tylenchida: Aphelenchidae, Paraphelenchinae), with ribosomal DNA trees and a morphometric compendium of female Paraphelenchus

Summary – Nematodes were isolated from surface-sterilised stems of cheatgrass, Bromus tectorum (Poaceae), in Colorado, grown on Fusarium (Hypocreaceae) fungus culture, and identified as Paraphelenchus acontioides. Morphometrics and micrographic morphology of this species are given to supplement the original description and expand the comparative species diagnosis. A tabular morphometric compendium of the females of the 23 species of Paraphelenchus is provided as the last diagnostic compilation was in 1984. Variations in the oviduct within the genus are reviewed to evaluate the taxonomic assignment of P. deckeri , a morphologically transitional species between Aphelenchus and Paraphelenchus. Sequences were generated for both 18S and 28S ribosomal DNA, representing the first identified species within Paraphelenchus so characterised. These sequences were incorporated into phylogenetic trees with related species of Aphelenchidae and Tylenchidae. Aphelenchus avenae isolates formed a well supported monophyletic sister group to Paraphelenchus. The ecology of Paraphelenchus, cheat grass and Fusarium is also discussed.

Real-Time PCR for Detection and Identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae

A number of seed, leaf, and stem gall nematodes are of significance to the forage and landscape grass and livestock industries. In North America, the bentgrass nematode, Anguina agrostis, reduces seed production on Agrostis tenuis and several other grass species. Anguina funesta is a seed-gall nematode that is most significant for its association with the toxigenic bacteria Rathayibacter toxicus. The wheat seed gall nematode A. tritici causes significant damage to wheat and other cereals; although it has been found in many countries worldwide, it has not been detected in the United States since 1975. Molecular methods based upon sequence variation in the ribosomal internal spacer region are useful for accurate identification of Anguina spp. Described herein are new species-specific primers and TaqMan probes for real-time polymerase chain reaction (PCR) identification of A. agrostis, A. funesta, A. tritici, and A. pacificae. Primer and probe combinations were each specific for the intended species and were sensitive enough to detect as few as 1.25 copies of nematode ribosomal DNA. PCR was also specific and sensitive in duplex assays that included genus-specific internal control primers as well as species-specific primers and probes. These standardized real-time PCR protocols should facilitate fast and accurate identification of Anguina spp. by diagnostic laboratories.