Molecular Characterization of an Emerging Root-Knot Nematode Meloidogyne enterolobii in North Carolina, USA.

An emerging threat to agriculture, Meloidogyne enterolobii Yang & Eisenback, 1983, is a tropical species and considered to be the most damaging root-knot nematode (RKN) in the world because of its wide host range, aggressiveness, and ability to overcome resistance to RKN in many crops. It was first detected in the United States on ornamental plants in Florida in 2001 but has since been identified in North Carolina, South Carolina, and Louisiana. Several thousand RKN populations were collected from North Carolina field crops, ornamental plants, and turfgrasses for species identification in the Nematode Assay Laboratory in the North Carolina Department of Agriculture & Consumer Services. From 2006 to 2019, root systems showing galling symptoms were dissected under the microscope, and females were obtained for DNA analysis. When only soil samples were submitted, the second-stage juveniles or males were used instead. Molecular characterization was performed via polymerase chain reaction with species-specific primers and DNA sequencing on the ribosomal DNA 18S-ITS1-5.8S and 28S D2/D3 and mitochondrial DNA CoxII-16S. One hundred thirty-five representative RKN populations from North Carolina were characterized and identified as M. enterolobii. Six populations from China where the species was originally described were included in this study for identity confirmation and comparison. As of December 2019, M. enterolobii has been confirmed from a limited number of fields in 11 North Carolina counties: Columbus, Craven, Greene, Harnett, Johnston, Lenoir, Nash, Pitt, Sampson, Wayne, and Wilson. Currently, M. enterolobii is the most important emerging RKN species in the United States and causes severe damage to agronomic and horticultural crops, especially sweetpotato in North Carolina.

Morphological and molecular characteristics of Parasitodiplogaster religiosae n. sp. (Nematoda: Diplogastrina) associated with Ficus religiosa in China.

A new nematode species of the genus Parasitodiplogaster was recovered from syconia of Ficus religiosa at the Guangxiao Temple, Guangzhou, China. It is described herein as P. religiosae n. sp. and is characterised by possessing the longest and thinnest spicule of all currently described males in the genus, an elongated laterally "ε-shaped" and ventrally rhomboid-like gubernaculum, a stoma without teeth, consisting of a ring-like cheilostom with indistinct anteriolateral projections, a tube-like gymnostom and a funnel-like stegostom, monodelphic with a mean vulval position of 66%. There are three pre-cloacal and six post-cloacal male genital papillae with the arrangement P1, P2, P3, (C, P4), P5, P6d, P7, P8, P9d, Ph. This new species was easily differentiated from other members of the genus by DNA sequences of partial small subunit rRNA gene (SSU) and the D2-D3 expansion segments of the large subunit rRNA gene (LSU). Phylogenetic analysis also corroborated its reasonable placement within a well-supported monophyletic clade with other Parasitodiplogaster species and within the australis-group that includes P. australis and P. salicifoliae that are all associates of fig wasp pollinators (Platyscapa sp.) of figs of the subsection Urostigma.

Molecular Characterisation and Diagnosis of Root-Knot Nematodes (Meloidogyne spp.) from Turfgrasses in North Carolina, USA.

Root-knot nematodes (Meloidogyne spp.) are the most common and destructive plant-parasitic nematode group worldwide and adversely influence both crop quality and yield. In this study, a total of 51 root-knot nematode populations from turfgrasses were tested, of which 44 were from North Carolina, 6 from South Carolina and 1 from Virginia. Molecular characterisation was performed on these samples by DNA sequencing on the ribosomal DNA 18S, ITS and 28S D2/D3. Species-specific primers were developed to identify turfgrass root-knot nematode through simplex or duplex PCR. Four species were identified, including M. marylandi Jepson & Golden in Jepson, 1987, M. graminis (Sledge & Golden, 1964) Whitehead, 1968, M. incognita (Kofoid & White, 1919) Chitwood, 1949 and M. naasi Franklin, 1965 through a combined analysis of DNA sequencing and PCR by species-specific primers. M. marylandi has been reported from North Carolina and South Carolina for the first time. Molecular diagnosis using PCR by species-specific primers provides a rapid and cheap species identification approach for turfgrass root-knot nematodes.

Molecular Characterization and Phylogenetic Relationships of Plant-Parasitic Nematodes Associated with Turfgrasses in North Carolina and South Carolina, United States.

The near-full-length 18S ribosomal DNA (rDNA) gene and internal transcribed spacer 1 region were amplified and sequenced from 52 nematode populations belonging to 28 representative species in 13 families recovered from turfgrasses in North Carolina (38 populations) and South Carolina (14 populations). This study also included 13 nematode populations from eight other plant hosts from North Carolina for comparison. Nematodes were molecularly characterized and the phylogenetic relationships were explored based on 18S rDNA sequences. Phylogenetic analysis using Bayesian inference was performed using five groups of the plant-parasitic nematode populations Tylenchids, Criconematids, Longidorids, Xiphinematids, and Trichodorids. The 65 nematode populations were clustered correspondingly within appropriate positions of 13 families, including Belonolaimidae, Caloosiidae, Criconematidae, Dolichodoridae, Hemicycliophoridae, Hoplolaimidae, Heteroderidae, Longidoridae, Meloidogynidae, Paratylenchidae, Pratylenchidae, Telotylenchidae, and Trichodoridae. This study confirms previous morphological-based identification of the plant-parasitic nematode species found in turfgrasses and provides a framework for future studies of plant-parasitic nematodes associated with turfgrasses based upon DNA sequences and phylogenetic relationships.

Description of Schistonchus altissimus n. sp. (Nematoda: Aphelenchoididae), an associate of Ficus altissima in China.

A new nematode species was recovered from the syconia of Ficus altissima from the residential area of Huajingxincheng, Guangzhou, Guangdong Province, China during a survey of nematode diversity. Schistonchus altissimus n. sp. is characterised by having females with a short post-uterine sac, an ovoid spermatheca and a conoid tail with a mucron in the female, excretory pore located near the lip; and males with amoeboid sperm, a conoid tail without a mucron and three pairs of subventral papillae, no gubernaculum, and hook-shaped spicules with a cucullus and a thorn-shaped rostrum. Schistonchus altissimus n. sp. is typologically differentiated from all other described species in this genus, except for S. microcarpus, by having a spicule with cucullus on the male tail tip. Schistonchus altissimus n. sp. is easily differentiated from other sequenced species by the partial small subunit rRNA gene (SSU), D3 expansion segment of the large subunit rRNA gene (LSU) and mitochondrial DNA subunit I (mtCOI). Phylogenetic analysis with partial SSU sequences suggests that S. altissimus n. sp. is in a highly supported monophyletic clade with two Chinese species (S. microcarpus and S. centerae) and two neotropical species (S. aureus and Schistonchus sp. ex Ficus colubrinae Standl.). Based on inferences using LSU D3 sequence data, S. altissimus n. sp. has a closer relationship with four Chinese species (S. centerae, S. fistulosus, S. guangzhouensis and S. microcarpus) than with S. hirtus and S. superbus, also from China.

Diversity and Occurrence of Plant-parasitic Nematodes Associated with Golf Course Turfgrasses in North and South Carolina, USA.

One hundred and eleven golf courses from 39 counties in the Carolinas were surveyed for plant-parasitic nematodes. Species diversity within habitats was analyzed with five diversity indices including Diversity index (H'), Evenness (J'), Richness (SR), Dominance (λ) and Diversity (H2 ). The results revealed a remarkably high diversity of 24 nematode species belonging to 19 genera and 11 families. Of those, 23 species were found in SC, 19 species in NC, and 18 species were detected in both states. Helicotylenchus dihystera, Mesocriconema xenoplax, Hoplolaimus galeatus, Tylenchorhynchus claytoni, Belonolaimus longicaudatus, Meloidogyne graminis and Paratrichodorus minor were the most prevalent and abundant species in golf course turfgrasses in both states. Twelve species were new records of plant parasitic nematodes in turfgrasses in both NC and SC. The results also revealed effects of different habitats on diversity of nematode species in turfgrass ecosystem. H' and SR values were higher in SC than in NC. H', J' and H2 values were significantly higher in sandy than in clay soil in NC, but no significant differences between sand and clay soil were detected in SC or in pooled data from both states. There were no significant differences for all indices among the management zones (putting green, fairway and tee) in NC. However, in SC and pooled data, H', SR and H2 were significantly higher in putting greens than in fairways and tees. Significant differences from different grass species (bermudagrass, creeping bentgrass and zoysiagrass) were detected only in H', which was significantly higher in zoysiagrass than in bentgrass or bermudagrass in NC. In pooled data, H' was significantly higher in zoysiagrass samples than in creeping bentgrass samples but was not significantly different from bermudagrass samples.

Description of Hemicaloosia graminis n. sp. (Nematoda: Caloosiidae) Associated with Turfgrasses in North and South Carolina, USA.

A new nematode species was discovered during a diversity survey of plant-parasitic nematodes on turfgrass conducted in North and South Carolina in 2010 and 2011. It is described herein as Hemicaloosia graminis n. sp. and is characterized by two annuli in the lip region, one lateral line, body 610.0-805.0 µm long, stylet 65.0-74.6 µm long, vulva at 84.1% -85.8% of the body , 254-283 annuli, vulva at the 38-53(rd) annulus from tail terminus, 12-14 annuli between vulva and anus, tail elongate-pointed, 67.5-84.8 µm long in females and spicule straight, 31.0 µm long, caudal alae well developed, two lateral lines in males. The newly described species is morphologically closest to H. paradoxa, but has a longer stylet (65.0-74.6 vs 61.0-65.0 µm) and a higher V-value (84.1-85.8 vs 78.1-84.0%), less RV (38-53 vs 50-56), higher RVan (12-14 vs 10) in females, and a shorter tail (30.1 vs 36.7 µm) and more anteriorly located excretory pore (105.9 vs 140.0 µm) in the male. It was easily differentiated from other species based on near-full-length small subunit rRNA gene (SSU) and ITS1 sequences. Phylogenetic analysis from SSU supports placement in a monophyletic clade with the genus Caloosia. An identification key and a table of distinguishing characteristics are presented for all seven species of Hemicaloosia.