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 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.

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.

Occurrence of Meloidogyne fallax in North America, and Molecular Characterization of M. fallax and M. minor from U.S. Golf Course Greens.

Several species of root-knot nematodes (Meloidogyne spp.) are known to have significant presence on turfgrass in golf course greens, particularly in the western United States. Nematodes isolated from a golf course in King County, WA were identified as Meloidogyne minor based on analysis of the large ribosomal subunit (LSU 28S D2-D3 expansion segment), the internal transcribed spacers 1 and 2 (ITS rDNA), the intergenic spacer region 2 (IGS2), and the nuclear protein-coding gene Hsp90. Sequence-characterized amplified region (SCAR) primers that were originally designed to be specific for M. fallax were found to cross-react with M. minor. A population from California was determined to be M. fallax based on juvenile tail morphology and analysis of the ribosomal markers and Hsp90, comprising the first report of this species in North America. Using trees based on Hsp90 genomic alignments, the phylogenetic relationships of these populations and known root-knot nematode species were congruent with previous trees based on ribosomal genes. Resolution of M. fallax and M. chitwoodi using Hsp90 was equivalent to species separation obtained with 28S or 18S rDNA alignments. The strengths and weaknesses of ribosomal and Hsp90 markers, and the use of SCAR polymerase chain reaction as diagnostic tools are discussed.