First report of barley root-knot nematode, Meloidogyne naasi from turfgrass in Idaho, with multigene molecular characterization.

Barley root-knot nematode, Meloidogyne naasi Franklin, 1965, is one of the most important pest nematodes infecting monocots (Franklin, 1965). Two-inch core soil samples collected from a golf course in Ada County, Idaho were submitted for identification in November of 2019. A high number of Meloidogyne sp. juveniles were recovered from both soil samples using sieving and decantation followed by the sugar centrifugal flotation method. They were examined by light microscopy, morphometric measurements, and multiple molecular markers, including the ribosomal 28S D2-D3 and intergenic spacer 2 (IGS-2) regions, mitochondrial markers cytochrome oxidase I (COI) and the interval from COII to 16S, and the protein-coding gene Hsp90. Morphometrics as well as BlastN comparisons with other root-knot nematode sequences from GenBank were consistent with identification as M. naasi. Phylogenetic trees inferred from 28S, IGS-2, COI, or Hsp90 alignments each separated the Idaho population into a strongly supported clade with other populations of M. naasi, while the COII-16S interval could not resolve M. naasi from M. minor. This report represents the first morphological and molecular characterization of Meloidogyne naasi from turfgrass in Idaho.

Molecular and Morphological Characterization of Tylenchus Zeae n. Sp. (Nematoda: Tylenchida) from Corn (Zea Mays) in South Carolina.

Specimens of a tylenchid nematode were recovered in 2019 from soil samples collected from a corn field, located in Pickens County, South Carolina, USA. A moderate number of Tylenchus sp. adults (females and males) were recovered. Extracted nematodes were examined morphologically and molecularly for species identification, which indicated that the specimens of the tylenchid adults were a new species, described herein as Tylenchus zeae n. sp. Morphological examination and the morphometric details of the specimens were very close to the original descriptions of Tylenchus sherianus and T. rex. However, females of the new species can be differentiated from these species by body shape and length, shape of excretory duct, distance between anterior end and esophageal intestinal valve, and a few other characteristics given in the diagnosis. Males of the new species can be differentiated from the two closely related species by tail, spicules, and gubernaculum length. Cryo-scanning electron microscopy confirmed head bearing five or six annules; four to six cephalic sensilla represented by small pits at the rounded corners of the labial plate; a small, round oral plate; and a large, pit-like amphidial opening confined to the labial plate and extending three to four annules beyond it. Phylogenetic analysis of 18S rRNA gene sequences placed Tylenchus zeae n. sp. in a clade with Tylenchus arcuatus and several Filenchus spp., and the mitochondrial cytochrome oxidase c subunit 1 (COI) gene region separated the new species from T. arcuatus and other tylenchid species. In the 28S tree, T. zeae n. sp. showed a high level of sequence divergence and was positioned outside of the main Tylenchus-Filenchus clade.

Morphological Characterization of Fresh and 20-Yr-Old Fixed Nematode Specimens of Sauertylenchus Maximus (Allen, 1955) Siddiqi, 2000 Deposited in the USDA Nematode Collection from Arlington National Cemetery, VA, USA.

Sauertylenchus maximus was discovered during a survey conducted at the Arlington National Cemetery, Virginia, for the type specimens of Hoplolaimus galeatus. Besides the fresh material, the fixed specimens of S. maximus were also studied by molecular and morphological means. The morphological and morphometric characteristics of the recovered fresh material were consistent with the original and other description(s) of this species. The fixed specimens used in this study were preserved in a 3% formaldehyde and 2% glycerin solution for over 20 yr. Molecular analyses of the fresh and fixed specimens were performed using internal transcribed spacer, D2-D2 expansion segments of 28S large subunits, and 18S small subunit ribosomal DNA sequences. To our knowledge, this represents the first report of S. maximus from Virginia and the first report of a successful DNA extraction from fixed nematode specimens.

First report of Seville root-knot nematode, Meloidogyne hispanica (Nematoda: Meloidogynidae) in the USA and North America.

A high number of second stage juveniles of the root-knot nematode were recovered from soil samples collected from a corn field, located in Pickens County, South Carolina, USA in 2019. Extracted nematodes were examined morphologically and molecularly for species identification which indicated that the specimens of root knot juveniles were Meloidogyne hispanica. The morphological examination and morphometric details from second-stage juveniles were consistent with the original description and redescriptions of this species. The ITS rRNA, D2-D3 expansion segments of 28S rRNA, intergenic COII-16S region, nad5 and COI gene sequences were obtained from the South Carolina population of M. hispanica. Phylogenetic analysis of the intergenic COII-16S region of mtDNA gene sequence alignment using statistical parsimony showed that the South Carolina population clustered with Meloidogyne hispanica from Portugal and Australia. To our best knowledge, this finding represents the first report of Meloidogyne hispanica in the USA and North America.

Molecular and morphological characterization of a first report of Cactodera torreyanae Cid del Prado Vera & Subbotin, 2014 (Nematoda: Heteroderidae) from Minnesota, the United States of America.

Cactodera torreyanae Cid del Prado Vera & Subbotin, 2014 cysts were discovered during a Pale Potato Cyst Nematode (PCN) survey conducted by Minnesota Department of Agriculture as part of the Animal and Plant Health Inspection Service (APHIS) efforts to survey states for the presence of PCN. The soil samples were collected from a potato field, located in Karlstad, Kittson County, Minnesota, USA. Two out of 175 vials submitted for identification to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL) contained few cysts and juveniles of C. torreyanae. Cysts were dark brown in color, lemon-shaped to elongated with distinct vulval cone. Vulva with denticles present around fenestra, cyst length to width ratio between 1.6 and 2.3 and anus distinct. The juveniles had rounded stylet knobs, some sloping slightly posteriorly. The molecular analysis included sequence and phylogenetic analysis of ITS rRNA, D2-D3 expansion segments of 28S rRNA and COI of mtDNA genes. The nematode species was identified by both morphological and molecular means as Cactodera torreyanae. To the best of our knowledge this represents the first report of Cactodera torreyanae from the United States and first report of this cyst nematode species from potato fields. Definite host plant for this nematode remains unknown.

First report of Cactodera milleri Graney and Bird, 1990 from Colorado and Minnesota.

In 2019, Cactodera milleri cysts were discovered from soil samples collected from a Chenopodium quinoa field, located in Mosca, Alamosa county, Colorado, USA. Approximately 200 lemon shaped cysts and several hundred juveniles were recovered from the affected quinoa plants. The same species was also identified from several counties in Minnesota from samples submitted over the years by the Minnesota Department of Agriculture as part of the Animal and Plant Health Inspection Service (APHIS) efforts to survey states for the presence of Pale Potato Cyst Nematode. The cysts and juveniles (J2) were recovered from soil samples through sieving and Baermann funnel extraction. The nematode species was identified by both morphological and molecular means as Cactodera milleri (Graney and Bird, 1990). To our knowledge this represents the first report of Cactodera milleri from Colorado and Minnesota.

Molecular and morphological characterization of the amaryllis lesion nematode, Pratylenchus hippeastri (Inserra et al., 2007), from California.

Root-lesion nematodes (Pratylenchus spp.) are among the most important nematode pests on grapevine along with root-knot, dagger, and ring nematodes. In 2019, two samples of both soil and roots were collected from a vineyard in Delano, Kern County, California and submitted to the United States Department of Agriculture, ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory, Beltsville, MD, for identification purposes. Females and juveniles of Pratylenchus sp. were recovered from the root and soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Both morphological observations and molecular analysis of internal transcribed spacer (ITS), 28S large subunit ribosomal DNA, and mitochondrial cytochrome oxidase (COI) sequences indicated that the specimens recovered from the soil and roots were Pratylenchus hippeastri. To the best of the authors' knowledge, this is the first report of P. hippeastri from California including the first record of this species on grapevine and the second state record in North America. Damages caused by nematodes cannot be over-emphasized, although economic importance of P. hippeastri has never been established. Hence, there is an urgent need to investigate the economic impact of this nematode in vineyards in California State in order to develop sustainable management strategies.Root-lesion nematodes (Pratylenchus spp.) are among the most important nematode pests on grapevine along with root-knot, dagger, and ring nematodes. In 2019, two samples of both soil and roots were collected from a vineyard in Delano, Kern County, California and submitted to the United States Department of Agriculture, ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory, Beltsville, MD, for identification purposes. Females and juveniles of Pratylenchus sp. were recovered from the root and soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Both morphological observations and molecular analysis of internal transcribed spacer (ITS), 28S large subunit ribosomal DNA, and mitochondrial cytochrome oxidase (COI) sequences indicated that the specimens recovered from the soil and roots were Pratylenchus hippeastri. To the best of the authors' knowledge, this is the first report of P. hippeastri from California including the first record of this species on grapevine and the second state record in North America. Damages caused by nematodes cannot be over-emphasized, although economic importance of P. hippeastri has never been established. Hence, there is an urgent need to investigate the economic impact of this nematode in vineyards in California State in order to develop sustainable management strategies.

Characterization of Vittatidera zeaphila (Nematoda: Heteroderidae) from Indiana with molecular phylogenetic analysis of the genus.

In the summer of 2016, a field of corn (Zea mays) in Spencer County, Indiana was observed with heavily stunted plants, and from the affected roots a large number of cysts were recovered. Soil samples were submitted to one of us (JF), who extracted the nematode cysts and sent them to the USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), Beltsville, MD for morphological and molecular identification. Cysts and the recovered second-stage juveniles (J2) that were examined morphologically conformed to the measurements of Vittatidera zeaphila, the goose cyst nematode originally described from Tennessee, USA in 2010. The molecular analysis of J2 showed the sample from Spencer County matched exactly with V. zeaphila according to ribosomal DNA markers ITS, 28S, and 18S, and with mitochondrial cytochrome oxidase I (COI). The nuclear marker heat shock protein 90 (Hsp90) was also analyzed for the first time from the Indiana population of V. zeaphila. Similarities to existing cyst nematode sequences are reported herein. Geographically, although the county is across the Ohio River from Kentucky, the previously reported Hickman County, Kentucky location and Indiana detection are approximately 200 miles apart. To the best of our knowledge, this is the first report of V. zeaphila in Indiana.In the summer of 2016, a field of corn (Zea mays) in Spencer County, Indiana was observed with heavily stunted plants, and from the affected roots a large number of cysts were recovered. Soil samples were submitted to one of us (JF), who extracted the nematode cysts and sent them to the USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), Beltsville, MD for morphological and molecular identification. Cysts and the recovered second-stage juveniles (J2) that were examined morphologically conformed to the measurements of Vittatidera zeaphila, the goose cyst nematode originally described from Tennessee, USA in 2010. The molecular analysis of J2 showed the sample from Spencer County matched exactly with V. zeaphila according to ribosomal DNA markers ITS, 28S, and 18S, and with mitochondrial cytochrome oxidase I (COI). The nuclear marker heat shock protein 90 (Hsp90) was also analyzed for the first time from the Indiana population of V. zeaphila. Similarities to existing cyst nematode sequences are reported herein. Geographically, although the county is across the Ohio River from Kentucky, the previously reported Hickman County, Kentucky location and Indiana detection are approximately 200 miles apart. To the best of our knowledge, this is the first report of V. zeaphila in Indiana.

Molecular and morphological characterization of the alfalfa cyst nematode, Heterodera medicaginis, from Utah.

In the spring of 2019, a cyst nematode was discovered from soil samples collected from an alfalfa field in Millard County, Utah. The soil samples were submitted to one of us (SH), who extracted the nematode cysts and sent them to the USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), Beltsville, MD for morphological and molecular identification. Cysts and living nematode juveniles (J2) recovered from the cysts were examined morphologically and molecularly for species identification which indicated that the specimens were Heterodera medicaginis. This represents the first record of H. medicaginis in Utah and the second report of this nematode in North America.In the spring of 2019, a cyst nematode was discovered from soil samples collected from an alfalfa field in Millard County, Utah. The soil samples were submitted to one of us (SH), who extracted the nematode cysts and sent them to the USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), Beltsville, MD for morphological and molecular identification. Cysts and living nematode juveniles (J2) recovered from the cysts were examined morphologically and molecularly for species identification which indicated that the specimens were Heterodera medicaginis. This represents the first record of H. medicaginis in Utah and the second report of this nematode in North America.

First report of the cactus cyst nematode, Cactodera cacti, from a cactus garden in Idaho.

In April 2018, a cyst nematode was discovered from soil samples collected from a cactus garden collection in Meridian, Ada County, Idaho, USA. The cactus garden collection field reported was observed with localized areas of heavily stunted plants. Roots from affected plants displayed moderate numbers of nematode cysts. Living nematode juveniles (J2) recovered from the cysts were examined morphologically and molecularly for species identification which indicated that the specimens were Cactodera cacti. This is the first report of the cactus cyst nematode, C. cacti in Idaho.In April 2018, a cyst nematode was discovered from soil samples collected from a cactus garden collection in Meridian, Ada County, Idaho, USA. The cactus garden collection field reported was observed with localized areas of heavily stunted plants. Roots from affected plants displayed moderate numbers of nematode cysts. Living nematode juveniles (J2) recovered from the cysts were examined morphologically and molecularly for species identification which indicated that the specimens were Cactodera cacti. This is the first report of the cactus cyst nematode, C. cacti in Idaho.

Developing a Real-Time PCR Assay for Direct Identification and Quantification of Pratylenchus penetrans in Soil.

The root-lesion nematode Pratylenchus penetrans is a major pathogen of potato worldwide. Yield losses may be exacerbated by interaction with the fungus Verticillium dahliae in the potato early dying disease complex. Accurate identification and quantification of P. penetrans prior to planting are essential for developing effective integrated pest control measures. However, distinction between P. penetrans and other Pratylenchus spp. based on morphology is a tedious task. A SYBR Green I-based qPCR assay was developed to discriminate, identify, and quantify P. penetrans in field soil. P. penetrans-specific qPCR primers were designed from the D2-D3 region of the 28S rDNA. The specificity of the assay was evaluated using eight isolates of P. penetrans populations and 31 isolates of other nematode species. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. There was a high correlation between the P. penetrans numbers artificially added to soil or estimated from naturally infested field soils by conventional methods, and the numbers quantified using the qPCR assay. Grinding the field soil prior to DNA extraction improved P. penetrans detection from soil. The qPCR assay will not only be useful for differentiating P. penetrans from mixed populations of Pratylenchus spp., but also for efficient detection and quantification of P. penetrans from field soil. The assay requires no expertise in nematode taxonomy and morphology, and may serve as a useful diagnostic tool in research, diagnostic labs, and extension services for pest management.

Development of Real-Time and Conventional PCR Assays for Identifying Stubby Root Nematode Paratrichodorus allius.

Paratrichodorus allius is an important pest on many crops, particularly on potato due to its ability to transmit Tobacco rattle virus causing corky ringspot disease on tubers. Detection and identification of P. allius are important for effective disease management. In this study, a rapid and reliable molecular diagnosis of this nematode targeting internal transcribed spacer ribosomal DNA was established. The specificity of the designed primers was evaluated using 29 nematode species and results showed that a single amplicon was produced from DNA of P. allius only. Detection sensitivity analysis indicated that a 9.6 × 10-4 ng of DNA template could be detected by conventional PCR and 1.92 × 10-4 ng of DNA by real-time PCR. The PCR assays amplified DNA of stubby root nematodes isolated from 18 soil samples in North Dakota and Minnesota, which were confirmed as P. allius by sequencing. Both conventional PCR and real-time PCR assays amplified target nematodes from complex nematode communities, supporting the success of this molecular diagnosis of P. allius. This is the first report of P. allius identification using the real-time PCR method and from nematode communities with other nematodes using conventional PCR. The new PCR assays provide rapid species identification and are suitable for use in diagnostic laboratories and detection of field infestations with P. allius.

Use of Chemical Flocculation and Nested PCR for Heterodera glycines Detection in DNA Extracts from Field Soils with Low Population Densities.

The soybean cyst nematode (SCN) Heterodera glycines is a major pathogen of soybean worldwide. Distinction between SCN and other members of the H. schachtii sensu stricto group based on morphology is a tedious task. A molecular assay was developed to detect SCN in field soils with low population densities and to differentiate SCN from other species. Various numbers of SCN eggs or juveniles were inoculated into 10 g of sterilized soil from which soil DNA was extracted using the PowerSoil DNA Isolation Kit. A specific amplicon was amplified using published SCN-specific primers SCNF1/SCNR1. This primer set was evaluated for the first time to detect SCN directly in soil DNA extracts. The specificity of the primers was confirmed by testing 36 isolates of other nematode species. The PCR assay detected one SCN egg or juvenile added to 10 g of soil. The assay was validated using 35 field soil samples. Grinding the field soil coupled with PCR inhibitor removal by AlNH4(SO4)20.12H2O treatment of soil DNA extracts followed by nested PCR enabled SCN detection as low as 12 SCN eggs/200 g soil. The PCR assay not only provides a sensitive method for SCN detection at low densities but also provides a discrimination method for SCN from other closely related nematodes.

Molecular and Morphological Characterization of Xiphinema chambersi Population from Live Oak in Jekyll Island, Georgia, with Comments on Morphometric Variations.

A population of Xiphinema chambersi from the root zone around live oak (Quercus virginiana Mill.) trees on Jekyll Island, GA, is described using both morphological and molecular tools and compared with descriptions of type specimens. Initially, because of a few morphological differences, this nematode was thought to represent an undescribed species. However, on further examination, the morphometrics of the nematodes from live oak tend to agree with most of the morphometrics in the original description and redescription of X. chambersi except for few minor differences in V% relative to body length, slightly shorter stylet length, different c value, and the number of caudal pores. We consider these differences to be part of the normal variation within this species and accordingly image this new population of X. chambersi and redescribe the species. The new population is characterized by having females with a body length of 2.1 to 2.5 mm; lip region slightly rounded and set off from head; total stylet length 170 to 193 µm; vulva at 20.4% to 21.8% of body length; a monodelphic, posterior reproductive system; elongate, conoid tail with a blunt terminus and four pairs of caudal pores, of which two pairs are subdorsal and two subventral. Sequence data from the D2-D3 region of the 28S rRNA molecule subjected to GenBank sequence comparison using BLAST showed that the sequence had 96% and 99% similarity with X. chambersi from Alabama and Florida, respectively. Phylogenetic relationships of X. chambersi with other xiphinematids based on analysis of this DNA fragment are presented. This finding represents a new location of X. chambersi in Georgia on live oak for this species.

Morphological and Molecular Characterization of Two Aphelenchoides Endophytic in Poplar Leaves.

During a long-term, large network study of the ecology of plant endophytes in native habitats, various nematodes have been found. Two poplar species, Populus angustifolia (narrowleaf cottonwood) and Populus trichocarpa (black cottonwood), are important ecological and genomic models now used in ongoing plant-pathogen-endophyte interaction studies. In this study, two different aphelenchid nematodes within surface-sterilized healthy leaves of these two Populus spp. in northwestern North America were discovered. Nematodes were identified and characterized microscopically and molecularly with 28S ribosomal RNA (rRNA) and 18S rRNA molecular markers. From P. angustifolia, Aphelenchoides saprophilus was inferred to be closest to another population of A. saprophilus among sequenced taxa in the 18S tree. From P. trichocarpa, Laimaphelenchus heidelbergi had a 28S sequence only 1 bp different from that of a Portuguese population, and 1 bp different from the original Australian type population. The 28S and 18S rRNA trees of Aphelenchoides and Laimaphelenchus species indicated L. heidelbergi failed to cluster with three other Laimaphelenchus species, including the type species of the genus. Therefore, we support a conservative definition of the genus Laimaphelenchus, and consider these populations to belong to Aphelenchoides, amended as Aphelenchoides heidelbergi n. comb. This is the first report of these nematode species from within aboveground leaves. The presence of these fungal-feeding nematodes can affect the balance of endophytic fungi, which are important determinants of plant health.

Assessment of DAPG-producing Pseudomonas fluorescens for Management of Meloidogyne incognita and Fusarium oxysporum on Watermelon.

Pseudomonas fluorescens isolates Clinto 1R, Wayne 1R, and Wood 1R, which produce the antibiotic 2,4-diacetylphloroglucinol (DAPG), can suppress soilborne diseases and promote plant growth. Consequently, these beneficial bacterial isolates were tested on watermelon plants for suppression of Meloidogyne incognita (root-knot nematode: RKN) and Fusarium oxysporum f. sp. niveum (Fon). In a greenhouse trial, Wayne 1R root dip suppressed numbers of RKN eggs per gram root on 'Charleston Gray' watermelon by 28.9%. However, in studies focused on 'Sugar Baby' watermelon, which is commercially grown in Maryland, a Wayne 1R root dip did not inhibit RKN reproduction or plant death caused by Fon. When all three isolates were applied as seed coats, plant stand in the greenhouse was reduced up to 60% in treatments that included Fon ± P. fluorescens, and eggs per gram root did not differ among treatments. In a microplot trial with Clinto 1R and Wayne 1R root dips, inoculation with P. fluorescens and/or Fon resulted in shorter vine lengths than treatment with either P. fluorescens isolate plus RKN. Root weights, galling indices, eggs per gram root, and second-stage juvenile (J2) numbers in soil were similar among all RKN-inoculated treatments, and fruit production was not affected by treatment. Plant death was high in all treatments. These studies demonstrated that the tested P. fluorescens isolates resulted in some inhibition of vine growth in the field, and were not effective for enhancing plant vigor or suppressing RKN or Fon on watermelon.

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.

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.

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.

Description of Globodera ellingtonae n. sp. (Nematoda: Heteroderidae) from Oregon.

A new species of cyst nematode, Globodera ellingtonae, is described from soil collected from a field in Oregon. Second-stage juveniles (J2) of the species are characterized by body length of 365-515 µm, stylet length of 19-22.5 µm, basal knobs rounded posteriorly and pointed anteriorly, tail 39-55 µm, hyaline tail terminus 20-32.5 µm, and tail tapering uniformly but abruptly narrowing and constricted near the posterior third of the hyaline portion, ending with a peg-like, finely rounded to pointed terminus. Cysts are spherical to sub-spherical, dark to light brown and circumfenestrate and cyst wall pattern is ridge-like with heavy punctations. Males have a stylet length of 21-25 µm and spicule length of 30-37 µm with a pointed thorn-like tip. Females have a stylet length of 20-22.5 µm, one head annule and labial disc, heavy punctations on the cuticle, and short vulval slit 7.5-8 µm long. Morphologically this new, round-cyst species differs from the related species G. pallida, G. rostochiensis, G. tabacum complex and G. mexicana by its distinctive J2 tail, and by one or another of the following: shorter mean stylet length in J2, females and males; number of refractive bodies in the hyaline tail terminus of J2; cyst morphology including Granek's ratio; number of cuticular ridges between the anus and vulva; and in the shape and length of spicules in males. Its relationship to these closely related species are discussed. Based upon analysis of ribosomal internal transcribed spacer (ITS) sequences, G. ellingtonae n. sp. is distinct from G. pallida, G. rostochiensis, G. tabacum and G. mexicana. Bayesian and Maximum Parsimony analysis of cloned ITS rRNA gene sequences indicated three clades, with intraspecific variability as high as 2.8%. In silico analysis revealed ITS restriction fragment length polymorphisms for enzymes Bsh 1236I, Hinf I, and Rsa I that overlap patterns for other Globodera species.