Depth distribution of plant-parasitic nematodes on bentgrass golf greens in Missouri and Indiana.

Control of plant-parasitic nematodes (PPNs) on golf putting greens with nematicides is dependent on the seasonal occurrence and depth distribution of target PPN populations. This study aimed to determine if plant-parasitic nematode populations on golf course putting greens in Missouri and Indiana peaked at a targetable depth at a specific time in the year, focusing primarily on lance (Hoplolaimus spp.) and root-knot (Meloidogyne spp.) nematodes. To elucidate species diversity in the region, rDNA from a subset of lance and root-knot nematodes was sequenced and analyzed, with additional micromorphology of a lance nematode assessed in scanning electron micrographs (SEM). Soil samples were taken to a depth of 25 cm and stratified into 5 cm increments during April, June, August and October at seven sites across Missouri, three in the Kansas City metro of Kansas in 2021 and in ten sites across Indiana in 2022. Samples were stratified in five-centimeter increments and aggregated for a total of 100 cm3 of soil at each depth for each sampling. Samples were processed using a semi-automatic elutriator followed by the sucrose-flotation method, and populations were counted using a hemocytometer and recorded. For molecular characterization, rDNA was extracted and analyzed from 31 individual lance nematodes from one site in Missouri and eight sites in Indiana, and 13 root-knot nematodes from nine sites across Indiana. A significant interaction occurred between sampling month and depth for lance and ring nematodes Missouri/KS, with both PPN populations peaking at the 0-5 cm depth during October, which is well after most targeted nematicide applications are applied. Ring nematodes in Indiana did not follow this trend and were most abundant in August at a depth of 0-5 cm. No significant interaction between depth and month occurred for lance or root-knot nematodes in Indiana, or root-knot nematodes in Missouri/KS. Hoplolaimus stephanus and H. magnistylus were the lance species identified on golf greens, and Meloidogyne naasi, M. graminicola and M. marylandi were the root-knot species identified. Scanning-electron micrographs confirmed morphological characteristics unique to H. stephanus.

Characterization of Hoplolaimus seinhorsti and Hoplolaimus pararobustus (Tylenchina: Hoplolaimidae) from banana, with phylogeny and species delineation in the genus Hoplolaimus.

The morphological and molecular characterisations of two lance nematode species isolated from the rhizosphere of banana, Hoplolaimus seinhorsti and H. pararobustus, are provided based on an integrative study that includes light and scanning electron microscopy, phylogenetic analysis and two tree-based molecular species delimitation methods (GMYC and bPTP). Nineteen new sequences were obtained, including 5 partial 18S rRNA, 6 D2-D3 of 28S rRNA, 1 ITS rRNA and 7 COI mtDNA (the first COI sequences of H. seinhorsti and H. pararobustus), and an updated morphological character comparison of 37 Hoplolaimus species is presented. The tree-based molecular species-delimitation approaches employed gave markedly differing results, and also showed remarkable discrepancies among the investigated genes, although the bPTP output was found to agree well with established morphological species delimitations. Both species-delimitation approaches did, however, provide the same output for the COI mtDNA sequences, and the COI mtDNA gene sequence was also found to correspond better to established morphological species. It is therefore recommended by this paper as representing the most suitable barcode marker for Hoplolaimus species identification. This integrative study also resulted in the corrective reassignment of 17 gene sequences that were previously unidentified or incorrectly classified, as well as concluding that H. pararobustus consists of two cryptic species.

First Genomic Resource of the Columbia Lance Nematode Hoplolaimus columbus.

The Columbia lance nematode Hoplolaimus columbus has been reported frequently from North America due to its negative impact on agricultural production. In this study, for the first time, we sequenced the whole genome of a female specimen by using whole-genome amplification and Illumina MiSeq. Data were de novo assembled to form scaffolds of 205.75 Mbp consisting of 118,374 contigs. The largest scaffold was 636,881 bp. Benchmarking Universal Single-Copy Orthologs completeness was 66.6% (eukaryotic dataset), and >8,000 unique genes were predicted by GeneMark-ES. In total, 61,855 protein sequences were predicted by AUGUSTUS, and 10,085 of them were annotated by PANNZER2 with at least one function. These data will provide valuable resources for studies focusing on pathogenicity and phylogenomics of plant-parasitic nematodes.

Morphological and molecular characterization of Hoplolaimus pararobustus (Schuurmans Stekhoven and Teunissen, 1938) Sher 1963 with its first report on Zea mays roots in Namibia.

In the summer of 2018, specimens of a Hoplolaimus population were extracted from a maize root sample collected near Stampriet, Namibia. This population was identified as Hoplolaimus pararobustus and is described and illustrated based on its morphological, morphometric, and molecular characteristics. To our knowledge, this is the first report H. pararobustus from maize roots. Females of the population had a mean body and stylet length of 1,100 µm and 36 µm, respectively. Esophagus with three nuclei in three pharyngeal glands. Lateral field reduced, ranging from a very faint line to just breaks in striae. The males were shorter than the females with a mean body length of 925 µm and the stylet slightly shorter, with a mean length of 34 µm. Phylogenetic analyses using partial sequences of 18 S and the expansion fragment D2-D3 of 28 S rDNA genes showed the close relation of this species and H. columbus. This Namibian population of H. pararobustus is the first Hoplolaimus species from Africa to be molecularly characterized.

Site specific nematode management-development and success in cotton production in the United States.

Variability in edaphic factors such as clay content, organic matter, and nutrient availability within individual fields is a major obstacle confronting cotton producers. Adaptation of geospatial technologies such global positioning systems (GPS), yield monitors, autosteering, and the automated on-and-off technology required for site-specific nematicide application has provided growers with additional tools for managing nematodes. Multiple trials in several states were conducted to evaluate this technology in cotton. In a field infested with Meloidogyne spp., both shallow (0 to 0.3 m) and deep (0 to 0.91 m) apparent electrical conductivity (ECa) readings were highly correlated with sand content. Populations of Meloidogyne spp. were present when shallow and deep EC values were less than 30 and 90 mS/m, respectively. Across three years of trials in production fields in which verification strips (adjacent nematicide treated and untreated rows across all soil zones) were established to evaluate crop response to nematicide application, deep EC values from 27.4-m wide transects of verification strips were more predictive of yield response to application of 1,3-dichloropropene than were shallow EC values in one location and both ECa values equally effective at predicting responses at the second location. In 2006, yields from entire verification strips across three soil zones in four production fields showed that nematicide response was greatest in areas with the lowest EC values indicating highest content of sand. In 2008 in Ashley and Mississippi Counties, AR, nematicide treatment by soil zone resulted in 36% and 42% reductions in the amount of nematicide applied relative to whole-field application. In 2007 in Bamberg County, SC, there was a strong positive correlation between increasing population densities of Meloidogyne incognita and increasing sand content. Trials conducted during 2007 and 2009 in South Carolina against Hoplolaimus columbus showed a stepwise response to increasing rates of aldicarb in zone 1 but not in zones 2 and 3. Site-specific application of nematicides has been shown to be a viable option for producers as a potential management tool against several nematode pathogens of cotton.

Plant-parasitic Nematode Communities and Their Associations with Soil Factors in Organically Farmed Fields in Minnesota.

A survey was conducted to determine the assemblage and abundance of plant-parasitic nematodes and their associations with soil factors in organically farmed fields in Minnesota. A total of 31 soil samples were collected from southeast (SE), 26 samples from southwest (SW), 28 from west-central (WC), and 23 from northwest (NW) Minnesota. The assemblage and abundance of plant-parasitic nematodes varied among the four regions. The soybean cyst nematode, Heterodera glycines, the most destructive pathogen of soybean, was detected in 45.2, 88.5, 10.7, and 0% of organically farmed fields with relative prominence (RP) values of 10.3, 26.5, 0.6, and 0 in the SE, SW, WC, and NW regions, respectively. Across the four regions, other common genera of plant-parasitic nematodes were Helicotylenchus (42.6, RP value, same below), Pratylenchus (26.9), Tylenchorhynchus and related genera (9.4), Xiphinema (5.6), and Paratylenchus (5.3). Aphelenchoides, Meloidogyne, Hoplolaimus, Mesocriconema, and Trichodorus were also detected at low frequencies and/or low population densities. The similarity index of plant-parasitic nematodes between two regions ranged from 0.44 to 0.71 and the similarity increased with decreasing distance between regions. The densities of most plant-parasitic nematodes did not correlate with measured soil factors (organic matter, pH, texture). However, the densities of Pratylenchus correlated negatively with % sand, and Xiphinema was correlated negatively with soil pH.

The complete mitochondrial genome of the Columbia lance nematode, Hoplolaimus columbus, a major agricultural pathogen in North America.

BACKGROUND: The plant-parasitic nematode Hoplolaimus columbus is a pathogen that uses a wide range of hosts and causes substantial yield loss in agricultural fields in North America. This study describes, for the first time, the complete mitochondrial genome of H. columbus from South Carolina, USA. METHODS: The mitogenome of H. columbus was assembled from Illumina 300 bp pair-end reads. It was annotated and compared to other published mitogenomes of plant-parasitic nematodes in the superfamily Tylenchoidea. The phylogenetic relationships between H. columbus and other 6 genera of plant-parasitic nematodes were examined using protein-coding genes (PCGs). RESULTS: The mitogenome of H. columbus is a circular AT-rich DNA molecule 25,228 bp in length. The annotation result comprises 12 PCGs, 2 ribosomal RNA genes, and 19 transfer RNA genes. No atp8 gene was found in the mitogenome of H. columbus but long non-coding regions were observed in agreement to that reported for other plant-parasitic nematodes. The mitogenomic phylogeny of plant-parasitic nematodes in the superfamily Tylenchoidea agreed with previous molecular phylogenies. Mitochondrial gene synteny in H. columbus was unique but similar to that reported for other closely related species. CONCLUSIONS: The mitogenome of H. columbus is unique within the superfamily Tylenchoidea but exhibits similarities in both gene content and synteny to other closely related nematodes. Among others, this new resource will facilitate population genomic studies in lance nematodes from North America and beyond.

Genetic variation of Hoplolaimus columbus populations in the United States using PCR-RFLP analysis of nuclear rDNA ITS regions.

Hoplolaimus columbus is an important nematode pest which causes economic loss of crops including corn, cotton, and soybean in the Southeastern United States. DNA sequences of the ITS1-5.8S-ITS2 region of ribosomal DNA from H. columbus were aligned and analyzed to characterize intraspecific genetic variation between eleven populations collected from Georgia, Louisiana, North Carolina, and South Carolina. In comparative sequence analysis with clones from either one or two individuals obtained from the eleven populations, we found variability existed among clones from an individual and that clonal diversity observed from within individuals was verified by PCR-RFLP. PCR-RFLP analysis with Rsa I and Msp I restriction enzymes yielded several fragments on 3.0% agarose gel that corresponded to different haplotypes in all populations and the sum of digested products exceeded the length of undigested PCR products, which revealed that ITS heterogeneity existed in a genome of H. columbus. This indicates that heterogeneity may play a role in the evolution of this parthenogenetic species.

Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA.

DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae.

Molecular Analysis of the Lance Nematode, Hoplolaimus spp., Using the First Internal Transcribed Spacer and the D1-D3 Expansion Segments of 28S Ribosomal DNA1.

DNA sequence analyses of the nuclear ribosomal ITS1 region of the ribosomal DNA and D1-D3 expansion segments of the 28S gene were conducted to characterize the genetic variation of six amphimictic Hoplolaimus species, including H. magnistylus, H. concaudajuvencus, H. galeatus, Hoplolaimus sp. 1, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, and two closely related parthenogenetic species, H. columbus and H. seinhorsti. PCR amplifications of the combined D1-D3 expansion segments and the ITS1 region each yielded one distinct amplicon. In the D1-D3 region, there was no nucleotide sequence variation between populations of H. columbus, H. magnistylus, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, whereas the ITS1 sequences had nucleotide variation among species. We detected conserved ITS1 regions located at the 3' and 5' end of ITS1 and also in the middle of the ITS1 among Hoplolaimus species. These regions were compared with sequences of distantly related Heterodera and Globedera. PCR-RFLP and sequence analysis of ITS1 and 28S PCR products revealed that several haplotypes existed in the same genome of H. columbus, H. magnistylus, H. seinhorsti, H. concaudajuvencus and Hoplolaimus sp. 1. Maximum likelihood and maximum parsimony analysis using the combined ITS1 and D1-D3 expansion segment sequences always produced trees with similar topology; H. columbus and H. seinhorsti grouped in one clade and the other six species (H. galeatus, H. concaudajuvencus, H. magnistylus, Hoplolaimus sp. 1, Hoplolaimus sp. 2, Hoplolaimus sp. 3) grouped in another. Molecular analysis supports morphological schemes for this genus to be divided into two groups based on several phenotypic traits derived from morphological evolution.

Saline Irrigation Affects Belonolaimus longicaudatus and Hoplolaimus galeatus on Seashore Paspalum.

Seashore paspalum (Paspalum vaginatum) has great potential for use in salt-affected turfgrass sites. Use of this grass on golf courses, athletic fields, and lawns in subtropical coastal areas may aid in conservation of freshwater resources. Belonolaimus longicaudatus and Hoplolaimus galeatus are considered among the most damaging root pathogens of turfgrasses in Florida. Glasshouse experiments were performed in 2002 and 2003 to examine the effects of increasing levels of irrigation salinity on B. longicaudatus and H. galeatus. Irrigation treatments were formulated by concentrating deionized water to six salinity levels (0, 5, 10, 15, 20, and 25 dS/m). Final population densities of H. galeatus followed a negative linear regression (r(2) = 0.92 and 0.83; P <= 0.01) with increasing salinity levels. Final population densities of B. longicaudatus were quadratically (r(2) = 0.72 and 0.78; P <= 0.01) related to increasing salinity levels from 0 to 25 dS/m. An increase in population densities of B. longicaudatus was observed at moderate salinity levels (10 and 15 dS/m) compared to 0 dS/m. Root-length comparisons revealed that B. longicaudatus caused root stunting at low salinity levels, 0 to 10 dS/m, but roots were not affected at 15 to 25 dS/m. These results indicate that the ability of B. longicaudatus to feed and stunt root growth was negatively affected at salinity levels of 15 dS/m and above.

Alternatives to fenamiphos for management of plant-parasitic nematodes on bermudagrass.

Plant-parasitic nematodes can be very damaging to turfgrasses. The projected cancellation of the registration for fenamiphos in the near future has generated a great deal of interest in identifying acceptable alternative nematode management tactics for use on turfgrasses. Two field experiments were conducted to evaluate the effectiveness of repeated applications of several commercially available nematicides and root biostimulants for reducing population densities of plant-parasitic nematodes and (or) promoting health of bermudagrass in nematode-infested soil. One experimental site was infested with Hoplolaimus galeatus and Trichodorus obtusus, the second with Belonolaimus longicaudatus. In both trials, none of the experimental treatments reduced population densities (P

High genetic diversity and geographic subdivision of three lance nematode species (Hoplolaimus spp.) in the United States.

Lance nematodes (Hoplolaimus spp.) feed on the roots of a wide range of plants, some of which are agronomic crops. Morphometric values of amphimictic lance nematode species overlap considerably, and useful morphological characters for their discrimination require high magnification and significant diagnostic time. Given their morphological similarity, these Hoplolaimus species provide an interesting model to investigate hidden diversity in crop agroecosystems. In this scenario, H. galeatus may have been over-reported and the related species that are morphologically similar could be more widespread in the United States that has been recognized thus far. The main objectives of this study were to delimit Hoplolaimus galeatus and morphologically similar species using morphology, phylogeny, and a barcoding approach, and to estimate the genetic diversity and population structure of the species found. Molecular analyses were performed using sequences of the cytochrome c oxidase subunit 1 (Cox1) and the internal transcribed spacer (ITS1) on 23 populations. Four morphospecies were identified: H. galeatus, H. magnistylus, H. concaudajuvencus, and H. stephanus, along with a currently undescribed species. Pronounced genetic structure correlated with geographic origin was found for all species, except for H. galeatus. Hoplolaimus galeatus also exhibited low genetic diversity and the shortest genetic distances among populations. In contrast, H. stephanus, the species with the fewest reports from agricultural soils, was the most common and diverse species found. Results of this project may lead to better delimitation of lance nematode species in the United States by contributing to the understanding the diversity within this group.

Description of Hoplolaimus bachlongviensis sp. n. (Nematoda: Hoplolaimidae) from banana soil in Vietnam.

BACKGROUND: The genus Hoplolaimus Daday, 1905 belongs to the subfamily Hoplolaimine Filipiev, 1934 of family Hoplolaimidae Filipiev, 1934 (Krall 1990). Daday established this genus on a single female of H. tylenchiformis recovered from a mud hole on Banco Island, Paraguay in 1905 (Sher 1963​, Krall 1990). Hoplolaimus species are distributed worldwide and cause damage on numerous agricultural crops (Luc et al. 1990 Robbins et al. 1998​). In 1992, Handoo and Golden reviewed 29 valid species of genus Hoplolaimus Dayday, 1905 (Handoo and Golden 1992). Siddiqi (2000) recognised three subgenera in Hoplolaimus: Hoplolaimus (Hoplolaimus) with ten species, is characterized by lateral field distinct, with four incisures, excretory pore behind hemizonid; Hoplolaimus (Basirolaimus) with 18 species, is characterized by lateral field with one to three incisures, obliterated, excretory pore anterior to hemizonid, dorsal oesophageal gland quadrinucleate; and Hoplolaimus (Ethiolaimus) with four species is characterized by lateral field with one to three incisures, obliterated; excretory pore anterior to hemizonid, dorsal oesophageal gland uninucleate (Siddiqi 2000). Since then, Hoplolaimus puriensis Ali, Shaheen & Pervez, 2009 has been described (​Ali et al. 2009). Up to now, there have been two species of genus Hoplolaimus reported in Vietnam, viz H. seinhorsti and H. chambus (Nguyen and Nguyen 2000). NEW INFORMATION: Hoplolaimus bachlongviensis sp. n. was isolated from banana soil in Bach Long Vi Island, Vietnam. The female of this species is described and illustrated below. Some diagnostic characters of this species include body slightly curved ventrally, offset lip region exhibiting three to four annules, lateral field reduced, pharyngeal glands with six nuclei, excretory pore anterior to hemizonid, epiptygma absent, intestine not overlapping rectum and male was not found.

Control of the Lance Nematode, Hoplolaimus galeatus, on 'Tifdwarf' Bermudagrass by Chemical Dips.

Dipping of bare-rooted 'Tifdwarf' bermudagrass sprigs in 1000 ppm of the 2,4-dichlorophenyl ester of methanesulfonic acid plus 2150 ppm 1,2-dibromo-3-chloropropane or in 1000 ppm ethyl 4-(methylthio)-m-tolyl isopropyl phosphoramidate for 30 min eliminated Hoplolaimus galeatus (Cobb) Thorne. No phytotoxic effect was observed and bermudagrass growth was improved.

Occurrence, Parasitism, and Pathogenicity of Nematodes Associated with Sycamore (Platanus occidentalis L.).

Ten species of stylet-bearing nematodes were recovered in a survey of sycamore (Platanus occidentalis L. ) stands in Georgia. Helicotylenchus, Xiphinema, and Criconemoides were the genera found most frequently. Populations of Hoplolaimus galeatus, Scutellonema brachyurum, Helicotylenchus dihystera and H. pseudorobustus increased on greenhouse-grown sycamore, but Trichodorus christiei, Xiphinema americanum, Meloidogyne hapla, M. arenaria and M. incognita did not. Hoplolaimus galeatus and S. brachyurum are semi-endoparasites; H. dihystera and H. pseudorobustus are migratory endoparasites. Hoplolaimus galeatus caused extensive root necrosis and marked decrease of fresh weights of seedling roots and tops. Helicotylenchus dihystera and S. brachyurum produced only qualitatively different sparse and unhealthy root growth. Helicotylenchus pseudorobustus caused only a reduction in root surface area.

Description and Larval Heteromorphism of Hoplolaimus concaudajuvencus, n. sp. (Nematoda: Hoplolain-lidae).

Hoplolaimus concaudajuvencus n. sp., of the genus Hoplolaimus Daday, 1905, characterized by larval heteromorphism, is described and illustrated as recovered from ryegrass/bermudagrass golf green turf in Florida. Females and males are closely related to H. galeatus (Cobb, 1913) Thorne, 1935, but have longer stylets with more definitely tulip-shaped stylet knobs which anteriorly tend to close upon the stylet shaft more than in H. galeatus. First and second-stage larvae have a conically-pointed tail unlike any known species of the genus. Subsequent stages, including females, have rounded tails essentially similar to other species of the genus and males possess the typical hopolaimid tail and bursa. The first molt was found to occur within the egg.

Tolerance to Heterodera glycines in Soybean.

Fifty-four susceptible soybean, Glycine max, cultivars or plant introductions were evaluated for tolerance to H. glycines, the soybean cyst nematode (SCN). Seed yields of genotypes were compared in nematicide-treated (1,2-dibromo-3-chloropropane, 58 kg a.i./ha) and nontreated plots at two SCN-infested locations over 3 years. Distinct and consistent levels of tolerance to SCN were observed among soybean genotypes. PI 97100, an introduction from Korea, exhibited the highest level of tolerance with an average tolerance index ([yield in nontreated plot / yield in nematicide-treated plot] x 100) of 96 over 2 years. Coker 156 and Wright had moderate levels of tolerance (range in index values 68 to 95) compared to the intolerant cuhivars Bragg and Coker 237 (range in index values 33 to 68). Most of the soybean genotypes evaluated were intolerant to SCN. The rankings of five genotypes for tolerance to SCN and Hoplolaimus columbus were similar. Tolerance for seed yield was more consistently correlated with tolerance for plant height (r = 0.55 to 0.64) than for seed weight (r = 0.23 to 0.65) among genotypes.

Performance of Soybean Cultivars in Hoplolaimus columbus-infested Fields.

Yield performance and host suitability to Hoplolaimus columbus of 18 soybean cultivars in maturity groups V and VI and 21 cultivars in groups VII and VIII were evaluated in 10 experiments. No cultivar was highly resistant to H. columbus. Within individual experiments, few differences were detected in yield losses among cultivars; however, over all locations Braxton, Coker 485, and Leflore were intolerant to H. columbus. Braxton also exhibited pronounced chlorosis at all locations. Coker 368, Coker 488, Deltapine 506, Foster, Kirby, Ring Around 680, and Young sustained high yields.

A key and diagnostic compendium to the species of the genus hoplolaimus daday, 1905 (nematoda: hoplolaimidae).

An identification key to 29 valid species of Hoplolaimus is given. A compendium of the most important diagnostic characters for use in identification of species is included as a practical alternative and supplement to the key. Diagnosis of Hoplolaimus is emended and lists of species of the genus, their synonymies, species inquirendae, nomina nuda, and species transferred to other genera are given. Hoplolaimus sheri, H. chambus, H. casparus, and H. capensis are recognized as valid species.