First report of the barley root-knot nematode, Meloidogyne naasi Franklin, 1965 from Pennsylvania, with new host report.

Meloidogyne naasi Franklin, 1965, the barley root-knot nematode, was originally found in field crops such as cereals, grasses, and sugar beet (Beta vulgaris L.) in England and Wales, (Franklin,1965). This nematode is one of the most significant root-knot nematodes impacting grains in European countries (Santos et al. 2020). Among root-knot nematode species, M. naasi, exhibits a distinct preference for grasses, with documented impacts on turfgrasses leading to reduced growth and vigor (Skantar et al., 2023; Cook and Yeates, 1993). In September 2022, root-knot nematode females and second-stage juveniles (J2) were recovered from roots of fowl manna grass, Glyceria striata (Lam.) Hitchc., during a nematode survey on natural vegetation at the Allegheny National Forest (41°30'13.8"N 79°09'46.2"W). Second-stage juvenile specimens were recovered from soil using sugar centrifugal flotation (Jenkins, 1964). Small galls with egg masses were dissected from fowl manna grass roots originally collected from the surveyed areas. In parallel, five plants of non-infected fowl manna grass were placed in a pot in the greenhouse using naturally nematode-infested soil collected from the same forested area. Small galls and female specimens recovered from these plants were dissected and processed for further analyses. Female and J2 were fixed in 3% formaldehyde solution and processed to glycerin (Golden, 1990; Hooper, 1970). The specimens were examined by light microscopy, morphometric measurements, and molecular markers, which included the D2-D3 region of the large ribosomal subunit 28S, and the rDNA internal transcribed spacer region (ITS). The perennial pattern of five females analyzed morphologically were consistent to the patterns observed for M. naasi. The perennial patterns had coarse ridges on the cuticle in dorsal region forming broken irregular lines around anal and phasmid area. We also noted a prominent fold that covered some of the anus and showed a curved line between vulval slit and phasmids, typical of M. naasi. The area around the vulval area had a few or no striae except for a few lines radiating from the vulval slit as in the original description. Measurements of ten J2 had a body length ranged between 380 and 410 µm, stylet 11-13 µm, tail 50-70 µm long with a hyaline tail terminus between 12-22 µm in length, 4 lines in the lateral field, a and c ratio between 29.23-35.91 and 5.79-7.9 fitting the original description by Franklin, 1965 and others populations found in the USA (Skantar et al., 2023). The matrix codes for the female specimes are A32, B324, C3, D3 and for J2's A2, B21, C123, D1, E3, F12 (Subbotin et al., 2021). The amplified DNA fragments were sequenced, resulting in an 726 bp fragment flanked by the D2-D3 primers (PP097762), while for the ITS primers an 634 bp fragment was obtained (PP092043). Both generated sequences for the specimens collected in Pennsylvania revealed >99% similarity to M. naasi sequences deposited at GenBank, and therefore, validating the morphological analyses. Based on both morphological and molecular analyses the specimens collected in the state of Pennsylvania were identified as M.naasi. To our knowledge, this is the first report of this species from this state and being associated with naturally infected fowl manna grass.

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.

First report of the beech leaf disease nematode Litylenchus crenatae mccannii (Nematoda: Anguinidae) in Michigan.

The North American beech leaf disease (BLD) nematode, Litylenchus crenatae mccannii Handoo, Li, Kantor, Bauchan, McCann, Gabriel, Yu, Reed, Koch, Martin and Burke, 2020, is recognized as a newly emergent nematode species that causes BLD in beech trees (Fagus spp.) in North America (Carta et al. 2020; Kantor et al. 2022a). Since the first report of BLD on American beech (Fagus grandifolia Ehrh) within the Lake County, located at the north-eastern corner of the state of Ohio in 2012 (Carta et al. 2020), the disease has rapidly spread to other US states and a province in Canada (Erwing et al. 2018; Carta et al 2020; Marra and LaMondia 2020; Reed et al 2020; Kantor et al. 2022b). Currently, besides Ohio, this nematode has been reported in Pennsylvania, New York, Connecticut, Massachusetts, Maine, Rhode Island, New Jersey, West Virginia, and Virginia, as well as Ontario, Canada. Different life stages of L. crenatae mccannii were isolated from symptomatic American beech leaves from an isolated natural maple-beech stand in rural Saint Clair Cty., Michigan, US; presenting typical symptoms of beech leaf disease, i.e., swelling and darkening of interveinal leaf tissues. Samples were taken to the Forest Pathology Laboratory at Michigan State University where L. crenatae mccannii presence was confirmed in the leaves after which samples were sent to the Mycology and Nematology Genetic Diversity and Biology Laboratory (USDA-ARS) in Beltsville, Maryland for official confirmation. Nematodes were identified based on morphology and sequence analysis of the internal transcribed spacer (ITS), and the D2D3 region of the 28S large subunit ribosomal DNA. To validate the morphological identification two different ribosomal DNA loci were amplified, sequenced and the phylogenetic relationships were generated. The amplification yielded fragments of 784 and 741 bp flanked by the ITS (GenBank accession no. OP689654) and D2D3 (GenBank accession no. OP689710) primers, respectively. The sequences obtained for the specimens collected in Michigan revealed 100% similarity to L. crenatae mccannii sequences obtained from specimens collected from other geographical areas in the US, and therefore validating the morphological analyses as well. The ITS sequence shared a 99.75% similarity with the subspecies L. crenatae (GenBank accession no. LC383724.1), and 90.53% similarity to L. coprosma Zhao, Davies, Alexander and Riley, 2011 (GU727548.1). While the D2D3 sequences of both L. crenatae subspecies revealed a 100% similarity (versus LC383725.1), they revealed 95.35% similarity to L. coprosma (KY679564.1). Since the first confirmed detection of BLD in June 2022 in St. Clair Cty, BLD has been reported in Oakland and Wayne Ctys (7 reports total across the three counties), suggesting BLD spread in the SE of Michigan. BLD confirmation was based on either physical symptoms (leaf banding), and/or the presence of the beech leaf nematode by morphological or molecular confirmation. The presence of the beech leaf nematode in symptomatic leaves follow the results obtained by Carta et al. (2020) after inoculation of beech seedlings with L. crenatae mccannii. Based on both morphological and molecular analyses the specimens collected in the state of Michigan were identified as L. crenatae mccannii. To our knowledge, this is the first report of this species in conjunction with symptomatic F. grandifolia leaves in this state.

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.

Morphological and Molecular Characterization of Punctodera Stonei Brzeski, 1998 (Nematoda: Heteroderidae) from Virginia, USA.

In August of 2021, several cysts with juveniles and eggs were discovered during a vegetation survey conducted at the Arlington National Cemetery, Virginia. Eight soil samples were collected from the rhizosphere region of the common grass (Festuca arundinacea L.) and processed at the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL). Cysts were light to dark brown in color, and oval to pear-shaped without bullae in young cysts but present in older cysts and with prominent vulval cone. The juveniles had slightly concave stylet knobs projecting sometimes anteriorly, tail tapering gradually to a narrowly rounded terminus, and hyaline tail terminus conspicuous at least twice the length of stylet. The molecular analysis included the analysis of three gene sequence fragments: D2-D3 of 28S rRNA, ITS rRNA, and COI. The nematode species was identified by both morphological and molecular means as Stone's cyst nematode, Punctodera stonei. Detection of P. stonei in Virginia represents a new record of this species in the United States, and a second report after Canada in North America.

Genome Announcement: The Draft Genome of the Carrot Cyst Nematode Heterodera Carotae.

Heterodera carotae, the carrot cyst nematode, is a significant pest affecting carrot globally. Here we present the draft genome of H. carotae, which was generated from short read libraries from Illumina HiSeq technology, and the corresponding genome annotation.

Morphological and molecular characterization of Paratylenchus beltsvillensis n. sp. (Tylenchida: Paratylenchidae) from the rhizosphere of pine tree (Pinus virginiana Mill) in Maryland, USA.

The pin nematode, Paratylechus beltsvillensis n. sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George's County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 µm, stylet from 70 to 75 µm long with anchor shaped knobs, vulva located at 70-73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17-20 µm and gubernaculum = 5.0-5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.

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.

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.

Chia: Host Status for Meloidogyne incognita and Activity of Plant Extracts.

Chia (Salvia hispanica L.) seeds are used for food, drinks, oil, and animal feed, and all plant parts are employed in traditional medicine. The growing demand for the seed has created a need for improved disease management. Plant-parasitic nematodes have been found on other Salvia spp., but none have been reported from S. hispanica. Chia has also not been tested for production of compounds active against these nematodes. Therefore, aqueous extracts from shoots and roots of six chia lines, Brad's Organic, Cono, E2, G3, G5, and W13.1, were tested in laboratory assays. Some concentrations of all extracts were nematotoxic, killing about one-third of Meloidogyne incognita (Kofoid & White) Chitwood second-stage juveniles (J2s) in shoot extracts and up to nearly half of J2s in root extracts. Hatch was generally not affected by the extracts. In greenhouse trials, all six chia lines were hosts of M. incognita. Chia line G3 had approximately two times or more eggs per gram of root than Brad's Organic or Cono. When cucumber seedlings were transplanted into soil amended with chopped chia shoots (2.3 or 2.5% weight of fresh shoots/weight of dry soil), galling and egg production on cucumber roots were not suppressed. To our knowledge, this is the first report that chia is a host to M. incognita (or any phytoparasitic nematode) and that chia shoots and roots produce compounds active against a nematode.

On the molecular identity of Paratylenchus nanus (Nematoda: Tylenchida).

In this study, molecular characterization of Paratylenchus nanus collected from the type locality in Four Mile Run, Fall Church, Virginia using COI, D2-D3 of 28 S rRNA and ITS rRNA gene sequences was provided. We molecularly also characterized, Paratylenchus specimens collected from grasses in Devils Lake, Ramsey County, North Dakota indicated as the type locality in the original description of P. nanus by Cobb (1923). These nematodes were identified as representatives of the species P. projectus. Populations of P. nanus belonging to the molecular types A and B, and previously designated by Van den Berg et al. (2014) should be now identified as P. nanus and P. projectus, respectively.

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 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.

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.

DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis.

Specimens of Heterodera have been collected from alfalfa fields in Kearny County, Kansas & Carbon County, Montana. DNA barcoding with the COI mitochondrial gene indicate that the species is not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum likelihood phylogenetic trees show that the alfalfa specimens form a sister clade most closely related to H. glycines, with a 4.7% mean pairwise sequence divergence across the 862 nucleotides of the COI marker. Morphological analyses of juveniles and cysts conform to the measurements of H. medicaginis, the alfalfa cyst nematode originally described from the USSR in 1971. Initial host testing demonstrated that the nematode reproduced on alfalfa, but not on soybeans, tomato, or corn. Collectively, the evidence suggests that this finding represents the first record of H. medicaginis in North America. Definitive confirmation of this diagnosis would require COI sequence of eastern European isolates of this species.Specimens of Heterodera have been collected from alfalfa fields in Kearny County, Kansas & Carbon County, Montana. DNA barcoding with the COI mitochondrial gene indicate that the species is not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum likelihood phylogenetic trees show that the alfalfa specimens form a sister clade most closely related to H. glycines, with a 4.7% mean pairwise sequence divergence across the 862 nucleotides of the COI marker. Morphological analyses of juveniles and cysts conform to the measurements of H. medicaginis, the alfalfa cyst nematode originally described from the USSR in 1971. Initial host testing demonstrated that the nematode reproduced on alfalfa, but not on soybeans, tomato, or corn. Collectively, the evidence suggests that this finding represents the first record of H. medicaginis in North America. Definitive confirmation of this diagnosis would require COI sequence of eastern European isolates of this species.

List of Type Specimens Deposited Since 1998 in the United States Department of Agriculture Nematode Collection, Beltsville, Maryland.

The United States Department of Agriculture Nematode Collection (USDANC) is one of the largest and most valuable in existence and includes millions of specimens housed in over 39,800 permanent slides and 9,300 vials. This collection preserves type specimens of nematodes to serve as a reference for identifications and future taxonomic revisions. Also, the collection provides useful information on nematode hosts, occurrence, and distribution. The present list includes type specimens added to the USDANC since 1998. Since that time, the collection has expanded, with 474 type species mounted and preserved on 2,564 glass slides and 180 vials. We encourage nematologists throughout the world to deposit type specimens in the USDANC for use by future generations.

First Report of the Spiral Nematode Helicotylenchus microlobus Infecting Soybean in North Dakota.

Spiral nematodes (Helicotylenchus spp.) are common plant-parasitic nematodes in fields of many crops. In June 2015, two soil samples were collected from a soybean field in Richland County, ND. Nematodes were extracted from soil using the sugar centrifugal flotation method (Jenkins, 1964). Plant-parasitic nematodes were identified to genus based on morphological features and counted. Both samples contained spiral nematodes from 1,500 to 3,300 per kilogram of soil. In June and August 2016, 10 soil samples were collected from the same field. Nematodes were extracted, and nine of the samples had spiral nematodes ranging from 125 to 3,065 per kilogram of soil. One soil sample with 1,500 spiral nematodes per kilogram was used to inoculate two soybean cultivars Sheyenne and Barnes each in four replicates. After 15 wk of growth at 22°C in a greenhouse room, the population of spiral nematodes was found to have increased greatly. The final density was 9,300 ± 1,701 spiral nematodes per kilogram of soil for Sheyenne and 9,451 ± 2,751 for Barnes. The reproductive factor in Sheyenne and Barnes was 6.2 and 6.3, respectively, indicating that this spiral nematode infects and reproduces well on these two soybean cultivars. Infected soybean roots had small brown lesions on the surface. Individual spiral nematodes were handpicked and examined morphologically and molecularly for species identification. Morphological measurements of adult females (n = 15) included body length (mean = 708.5 µm, range = 600.0-812.0 µm), stylet (27.6, 26.0-29.0), body width (28.3, 25.0-33.0), lip region end to posterior end of pharyngeal glands (142.5, 130.0-152.0), anal body width (15.8, 14.0-17.5), tail length (20.3, 15.0-25.0), tail annules (11.6, 10.0-14.0), a (25.0, 21.4-27.1), b (5.0, 4.4-5.7), c (35.4, 30.2-41.7), c' (1.3, 1.0-1.6), and V (61.8%, 60.0-63.7). The spiral nematode was identified as Helicotylenchus microlobus according to morphological and morphometric characteristics (Subbotin et al., 2015). DNA was extracted from single nematodes (n = 8) using the Proteinase K method (Kumari and Subbotin, 2012). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers rDNA2/rDNA1.58S (Cherry et al., 1997). The PCR products were then purified and sequenced. The consensus ITS rDNA sequence (accession no. KY271078, 822 bp) that was deposited into the GenBank shared 99% identity with two isolates of H. microlobus from California (KM506860.1 and KM506859.1) and one isolate of H. microlobus from Spain (KM506862.1) (Subbotin et al., 2015). It had only 91% sequence identity with seven isolates of H. pseudorobustus (KM506875.1, KM506880.1, KM506876.1, KM506874.1, KM506872.1, KM506879.1, and KM506878.1) from California, Switzerland, and New Zealand, a spiral nematode species very closely related to H. microlobus in morphology. The molecular tests confirmed the identity of this spiral nematode as H. microlobus. The H. microlobus nematode was reported as one of the most commonly observed spiral nematodes in soil samples in the state of Minnesota, and all 13 soybean cultivars tested except Hawkeye were rated as hosts (Taylor, 1960). To our knowledge, this is the first report of H. microlobus in North Dakota.