Identification of individual root-knot nematodes using low coverage long-read sequencing.

Root-knot nematodes (RKN; genus Meloidogyne) are polyphagous plant pathogens of great economic importance to agriculturalists globally. These species are small, diverse, and can be challenging for accurate taxonomic identification. Many of the most important crop pests confound analysis with simple genetic marker loci as they are polyploids of likely hybrid origin. Here we take a low-coverage, long-read genome sequencing approach to characterisation of individual root-knot nematodes. We demonstrate library preparation for Oxford Nanopore Technologies Flongle sequencing of low input DNA from individual juveniles and immature females, multiplexing up to twelve samples per flow cell. Taxonomic identification with Kraken 2 (a k-mer-based taxonomic assignment tool) is shown to reliably identify individual nematodes to species level, even within the very closely related Meloidogyne incognita group. Our approach forms a robust, low-cost, and scalable method for accurate RKN species diagnostics.

Minc00344 and Mj-NULG1a effectors interact with GmHub10 protein to promote the soybean parasitism by Meloidogyne incognita and M. javanica.

Several economically important crops are susceptible to root-knot nematode (RKNs). Meloidogyne incognita and M. javanica are the two most reported species from the RKN complex, causing damage to several crops worldwide. The successful outcome of the Meloidogyne-plant interaction is associated with molecular factors secreted by the nematode to suppress the plant's immune response and promote nematode parasitism. In contrast, several plant factors are associated with defense against nematode infection. In this study, we identified and characterized the specific interaction of Minc00344 and Mj-NULG1a effectors with soybean GmHub10 (Glyma.19G008200) protein in vitro and in vivo. An Arabidopsis thaliana T-DNA mutant of AtHub10 (AT3G27960, an orthologous gene of GmHub10) showed higher susceptibility to M. incognita. Thus, since soybean and A. thaliana Hub10 proteins are involved in pollen tube growth and indirect activation of the defense response, our data suggest that effector-Hub10 interactions could be associated with an increase in plant susceptibility. These findings indicate the potential of these effector proteins to develop new biotechnological tools based on RNA interference and the overexpression of engineered Hub10 proteins for the efficient management of RKN in crops.

An improved draft genome assembly of Meloidogyne graminicola IARI strain using long-read sequencing.

The rice root-knot nematode Meloidogyne graminicola is a major biotic stress for the rice crop under upland, rain-fed lowland and irrigated cultivation conditions. Here, we present an improved draft genome assembly of M. graminicola IARI strain using the long-read sequencing approach (PacBio Sequel platform). The assembled genome size was 36.86 Mb with 514 contigs and N50 value of 105 kb. BUSCO estimated the genome to be 88.6% complete. Meloidogyne graminicola genome contained 17.83% repeat elements and showed 14,062 protein-coding gene models, 4,974 conserved orthologous genes, 561 putative secreted proteins, 49 RNAi pathway genes, 1,853 proteins involved in pathogen-host interactions, 1,575 carbohydrate-active enzymes, and 32,138 microsatellites. Five of the carbohydrate-active enzymes were found only in M. graminicola genome and were not present in any other analysed root-knot nematode genome. Together with the previous two genome assemblies, this improved genome assembly would facilitate comparative and functional genomics for M. graminicola.

Development of Real-Time and Conventional PCR Assays for Identifying a Newly Named Species of Root-Lesion Nematode (Pratylenchus dakotaensis) on Soybean.

A rapid and accurate PCR-based method was developed in this study for detecting and identifying a new species of root-lesion nematode (Pratylenchus dakotaensis) recently discovered in a soybean field in North Dakota, USA. Species-specific primers, targeting the internal transcribed spacer region of ribosomal DNA, were designed to be used in both conventional and quantitative real-time PCR assays for identification of P.dakotaensis. The specificity of the primers was evaluated in silico analysis and laboratory PCR experiments. Results showed that only P.dakotaensis DNA was exclusively amplified in conventional and real-time PCR assays but none of the DNA from other control species were amplified. Detection sensitivity analysis revealed that the conventional PCR was able to detect an equivalent to 1/8 of the DNA of a single nematode whereas real-time PCR detected an equivalent to 1/32 of the DNA of a single nematode. According to the generated standard curve the amplification efficiency of the primers in real-time PCR was 94% with a R2 value of 0.95 between quantification cycle number and log number of P.dakotaensis. To validate the assays to distinguish P.dakotaensis from other Pratylenchus spp. commonly detected in North Dakota soybean fields, 20 soil samples collected from seven counties were tested. The PCR assays amplified the DNA of P.dakotaensis and discriminated it from other Pratylenchus spp. present in North Dakota soybean fields. This is the first report of a species-specific and rapid PCR detection method suitable for use in diagnostic and research laboratories for the detection of P.dakotaensis.

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.

GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes.

An important assessment prior to genome assembly and related analyses is genome profiling, where the k-mer frequencies within raw sequencing reads are analyzed to estimate major genome characteristics such as size, heterozygosity, and repetitiveness. Here we introduce GenomeScope 2.0 (https://github.com/tbenavi1/genomescope2.0), which applies combinatorial theory to establish a detailed mathematical model of how k-mer frequencies are distributed in heterozygous and polyploid genomes. We describe and evaluate a practical implementation of the polyploid-aware mixture model that quickly and accurately infers genome properties across thousands of simulated and several real datasets spanning a broad range of complexity. We also present a method called Smudgeplot (https://github.com/KamilSJaron/smudgeplot) to visualize and estimate the ploidy and genome structure of a genome by analyzing heterozygous k-mer pairs. We successfully apply the approach to systems of known variable ploidy levels in the Meloidogyne genus and the extreme case of octoploid Fragaria × ananassa.

A Single, Shared Triploidy in Three Species of Parasitic Nematodes.

The root-knot nematodes of the genus Meloidogyne are important and damaging parasites capable of infecting most flowering plants. Within this genus, several species of the Meloidogyne incognita group show evidence of paleopolyploidy in their genomes. We used our software tool POInT, the Polyploidy Orthology Inference Tool, to phylogenetically model the gene losses that followed that polyploidy. These models, and simulations based on them, show that three of these species (M. incognita, M. arenaria and M. javanica) descend from a single common hybridization event that yielded triplicated genomes with three distinguishable subgenomes. While one of the three subgenomes shows elevated gene loss rates relative to the other two, this subgenome does not show elevated sequence divergence. In all three species, ancestral loci where two of the three gene copies have been lost are less likely to have orthologs in Caenorhabditis elegans that are lethal when knocked down than are ancestral loci with surviving duplicate copies.

Molecular characterization of root-knot nematodes (Meloidogyne spp.) from Arkansas, USA.

Root-knot nematodes (Meloidogyne spp.) are the most common major pathogens of many crops throughout the world, impacting both the quantity and quality of marketable yields. In this study, a total of 244 root-knot nematode populations from various hosts from 39 counties in Arkansas were tested to determine the species diversity. Molecular characterization was performed on these populations by DNA sequencing of the ribosomal DNA 18S-ITS-5.8S, 28S D2/D3 and a mitochondrial DNA fragment flanking cytochrome oxidase gene subunit II - the intergenic spacer. Five species were identified, including M. incognita (Kofoid & White, 1919) Chitwood, 1949 from soybean, cotton, corn and various vegetables (232 samples); M. hapla Chitwood, 1949 from rose (1 sample); M. haplanaria Eisenback, Bernard, Starr, Lee & Tomaszewski, 2003 from okra, tomato, peanut, Indian hawthorn, ash, willow and elm trees (7 samples); M. marylandi Jepson & Golden in Jepson, 1987 from grasses (3 samples); and M. partityla Kleynhans, 1986 from pecan (1 sample) through a combined analysis of DNA sequencing and PCR by species-specific primers. Meloidogyne incognita is the most abundant species that was identified in 95% samples and was the only species in field crops including soybean and cotton, except for one population of M. haplanaria from soybean in Logan County (TK201). Species-specific primers were used to verify M. incognita through PCR by species-specific primers. Unlike historical data, M. arenaria, M. javanica and M. graminis were not detected from any of the samples collected during this study. This result is essential for effective and sustainable management strategies against root-knot nematodes in Arkansas.

Multigene phylogeny of root-knot nematodes and molecular characterization of Meloidogyne nataliei Golden, Rose & Bird, 1981 (Nematoda: Tylenchida).

The root-knot nematodes of the genus Meloidogyne are highly adapted, obligate plant parasites, consisting of nearly one hundred valid species, and are considered the most economically important group of plant-parasitic nematodes. Six Meloidogyne species: M. arenaria, M. hapla, M. incognita, M. microtyla, M. naasi and M. nataliei were previously reported in Michigan, USA. For this study, Meloidogyne nataliei was isolated from the grapevine Vitis labrusca from the type locality in Michigan, USA, and was characterized using isozyme analysis and ribosomal and mitochondrial gene sequences. No malate dehydrogenase activity was detected using macerate of one, five, six, seven or ten females of M. nataliei per well. However, one strong band (EST = S1; Rm: 27.4) of esterase activity was detected when using homogenates of ten egg-laying females per well. Phylogenetic analyses of sequences of the partial 18S ribosomal RNA, D2-D3 of 28S rRNA, internal transcribed spacer of rRNA, mitochondrial cytochrome oxidase subunit I genes and the cytochrome oxidase subunit II-16S rRNA intergeneric fragment from fifty-five valid Meloidogyne species and M. nataliei were conducted using Bayesian inference and maximum likelihood methods. From these results, we infer 11 distinct clades among studied species, with M. nataliei and M. indica composing a basal lineage. Seventy five percent of these species belong to seven clades within the Meloidogyne superclade. Characterization of these clades is provided and evolutionary trends within the root-knot nematodes are discussed.

Microsatellite markers reveal two genetic groups in European populations of the carrot cyst nematode Heterodera carotae.

The cyst nematode Heterodera carotae, which parasitizes carrot roots, has been recorded in many countries in Europe (Italy, The Netherlands, Switzerland, France, Denmark, …), in South Africa and in North America (Canada, USA). To date, there is a lack of knowledge about the genetic structure of the populations of this economically important nematode. The aim of this work was to study the structuration of the genetic diversity of the carrot cyst nematode at the European scale. We have developed a set of thirteen polymorphic microsatellite markers and used it to genotype seventeen European populations of H. carotae coming from France, Switzerland, Italy, Denmark and one non-European population from Canada. As previously showed for other cyst nematode species, the H. carotae populations were characterised by a strong heterozygote deficit. A Bayesian clustering analysis revealed two distinct genetic clusters, with one group located in the north of Europe and a second one located in the south of Europe. Moreover, our results highlighted rather limited gene flow at small spatial scale and some events of long distance migration. This first investigation of the genetic diversity of H. carotae populations would be useful to develop sustainable control strategies.

On the Close Relatedness of Two Rice-Parasitic Root-Knot Nematode Species and the Recent Expansion of Meloidogyne graminicola in Southeast Asia.

Meloidogyne graminicola is a facultative meiotic parthenogenetic root-knot nematode (RKN) that seriously threatens agriculture worldwide. We have little understanding of its origin, genomic structure, and intraspecific diversity. Such information would offer better knowledge of how this nematode successfully damages rice in many different environments. Previous studies on nuclear ribosomal DNA (nrDNA) suggested a close phylogenetic relationship between M. graminicola and Meloidogyne oryzae, despite their different modes of reproduction and geographical distribution. In order to clarify the evolutionary history of these two species and explore their molecular intraspecific diversity, we sequenced the genome of 12 M. graminicola isolates, representing populations of worldwide origins, and two South American isolates of M. oryzae. k-mer analysis of their nuclear genome and the detection of divergent homologous genomic sequences indicate that both species show a high proportion of heterozygous sites (ca. 1⁻2%), which had never been previously reported in facultative meiotic parthenogenetic RKNs. These analyses also point to a distinct ploidy level in each species, compatible with a diploid M. graminicola and a triploid M. oryzae. Phylogenetic analyses of mitochondrial genomes and three nuclear genomic sequences confirm close relationships between these two species, with M. graminicola being a putative parent of M. oryzae. In addition, comparative mitogenomics of those 12 M. graminicola isolates with a Chinese published isolate reveal only 15 polymorphisms that are phylogenetically non-informative. Eight mitotypes are distinguished, the most common one being shared by distant populations from Asia and America. This low intraspecific diversity, coupled with a lack of phylogeographic signal, suggests a recent worldwide expansion of M. graminicola.

Technical and economic efficiency of methods for extracting genomic DNA from Meloidogyne javanica.

Plant parasitic nematodes reduce the production of agricultural crops. Species diagnosis is essential to predict losses, determine economic damage levels and develop integrated pest management programs. DNA extraction techniques need to be improved for precise and rapid molecular diagnosis of nematodes. The objective of the present study was to evaluate the efficiency of DNA extraction and amplification by PCR, cost and execution time by Chelex, Worm Lysis Buffer Method (WLB), Holterman Lysis Buffer Method (HLB) and FastDNA methods for nematodes of the Meloidogyne genus. The qualitative and quantitative efficiency of DNA extraction varied between methods. The band size of the amplified PCR product with WLB, Chelex and HLB methods was 590 bp. Extraction with the FastDNA is not recommended for DNA extraction from nematodes because it results in a low DNA concentration without bands in PCR amplification, besides presenting high cost. The efficiency of the WLB method to extracting DNA from Meloidogyne javanica was greater, ensuring a higher concentration and purity of the extracted material and guaranteeing lower costs and greater ease of PCR amplification.

A High-Throughput Molecular Pipeline Reveals the Diversity in Prevalence and Abundance of Pratylenchus and Meloidogyne Species in Coffee Plantations.

Coffee yields are adversely affected by plant-parasitic nematodes and the pathogens are largely underreported because a simple and reliable identification method is not available. We describe a polymerase chain reaction-based approach to rapidly detect and quantify the major Pratylenchus and Meloidogyne nematode species that are capable of parasitizing coffee. The procedure was applied to soil samples obtained from a number of coffee farms in Brazil, Vietnam, and Indonesia to assess the prevalence of these species associated both with coffee (Coffea arabica and C. canephora) and its intercropped species Musa acuminata (banana) and Piper nigrum (black pepper). Pratylenchus coffeae and P. brachyurus were associated with coffee in all three countries but there were distinct profiles of Meloidogyne spp. Meloidogyne incognita, M. exigua, and M. paranaensis were identified in samples from Brazil and M. incognita and M. hapla were detected around the roots of coffee in Vietnam. No Meloidogyne spp. were detected in samples from Indonesia. There was a high abundance of Meloidogyne spp. in soil samples in which Pratylenchus spp. were low or not detected, suggesting that the success of one genus may deter another. Meloidogyne spp. in Vietnam and Pratylenchus spp. in Indonesia were more numerous around intercropped plants than in association with coffee. The data suggest a widespread but differential nematode problem associated with coffee production across the regions studied. The issue is compounded by the current choice of intercrops that support large nematode populations. Wider application of the approach would elucidate the true global scale of the nematode problem and the cost to coffee production. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Morphological and molecular characterization of Trophurus wuhuensis n. sp. (Nematoda: Telotylenchinae) from soil associated with Cinnamomum camphora in China.

A new plant nematode species, Trophurus wuhuensis n. sp., was collected from the soil associated with Cinnamomum camphora in Wuhu, Anhui Province, China. The new species is characterized by having a female with a slender body 660.5-801.5 µm in length, stylet 12-14 µm long, knobs directed laterad, lateral field marked by short and scattered grooves, post-vulval uterine sac shorter than vulval body diameter, post-rectal intestinal sac absent, tail cylindroid, terminus with deep wrinkles; and male with a pointed tail terminus and spicules 16-18 µm long. The internal transcribed spacer sequences of ribosomal DNA (ITS rDNA) and partial 18S ribosomal DNA (18S rDNA) from T. wuhuensis n. sp. were amplified and sequenced. A phylogenetic analysis based on sequences of 18S rDNA fragments is given in this study.

Molecular characterization and species delimiting of plant-parasitic nematodes of the genus Pratylenchus from the penetrans group (Nematoda: Pratylenchidae).

Root-lesion nematodes of the genus Pratylenchus are an important pest parasitizing a wide range of vascular plants including several economically important crops. However, morphological diagnosis of the more than 100 species is problematic due to the low number of diagnostic features, high morphological plasticity and incomplete taxonomic descriptions. In order to employ barcoding based diagnostics, a link between morphology and species specific sequences has to be established. In this study, we reconstructed a multi-gene phylogeny of the Penetrans group using nuclear ribosomal and mitochondrial gene sequences. A combination of this phylogenetic framework with molecular species delineation analysis, population genetics, morphometric information and sequences from type location material allowed us to establish the species boundaries within the Penetrans group and as such clarify long-standing controversies about the taxonomic status of P. penetrans, P. fallax and P. convallariae. Our study also reveals a remarkable amount of cryptic biodiversity within the genus Pratylenchus confirming that identification on morphology alone can be inconclusive in this taxonomically confusing genus.

Cloning and characterization of the first serine carboxypeptidase from a plant parasitic nematode, Radopholus similis.

Radopholus similis is an important parasitic nematode of plants. Serine carboxypeptidases (SCPs) are peptidases that hydrolyse peptides and proteins and play critical roles in the development, invasion, and pathogenesis of certain parasitic nematodes and other animal pathogens. In this study, we obtained the full-length sequence of the SCP gene from R. similis (Rs-scp-1), which is 1665 bp long and includes a 1461-bp open reading frames encoding 486 amino acids with an 18-aa signal peptide. This gene is a double-copy gene in R. similis. Rs-scp-1 was expressed in the procorpus, esophageal glands and intestines of females and in the esophageal glands and intestines of juveniles. Rs-scp-1 expression levels were highest in females, followed by juveniles and males, and lowest in eggs. Rs-scp-1 expression levels were significantly suppressed after R. similis was soaked in Rs-scp-1 dsRNA for 12 h. Nematodes were then inoculated into Anthurium andraeanum after RNAi treatment. Compared with water treatment, R. similis treated with RNAi were reduced in number and pathogenicity. In summary, we obtained the first SCP gene from a plant parasitic nematode and confirmed its role in the parasitic process.

Expression and evolutionary analyses of three acetylcholinesterase genes (Mi-ace-1, Mi-ace-2, Mi-ace-3) in the root-knot nematode Meloidogyne incognita.

The full cDNA of Mi-ace-3 encoding an acetylcholinesterase (AChE) in Meloidogyne incognita was cloned and characterized. Mi-ace-3 had an open reading frame of 1875 bp encoding 624 amino acid residues. Key residues essential to AChE structure and function were conserved. The deduced Mi-ACE-3 protein sequence had 72% amino acid similarity with that of Ditylenchus destructor Dd-AChE-3. Phylogenetic analyses using 41 AChEs from 24 species showed that Mi-ACE-3 formed a cluster with 4 other nematode AChEs. Our results revealed that the Mi-ace-3 cloned in this study, which is orthologous to Caenorhabditis elegans AChE, belongs to the nematode ACE-3/4 subgroup. There was a significant reduction in the number of galls in transgenic tobacco roots when Mi-ace-1, Mi-ace-2, and Mi-ace-3 were knocked down simultaneously, whereas little or no effect were observed when only one or two of these genes were knocked down. This is an indication that the functions of these three genes are redundant.

Morphological characterisation of a new and two known species of Ditylenchus Filipjev, 1936 (Nematoda: Anguinidae) from Iran.

Ditylenchus sturhani sp. n. from the rhizosphere of grasses in East Azarbaijan province, Northwestern Iran, is described and illustrated. The new species is characterised by a straight to slightly arcuate body 656-865 µm in length, presence of four incisures in each lateral field, slender stylet 10-12 µm in length, pharyngeal basal bulb offset or slightly overlapping the intestine, post-uterine sac 1.0-1.9 vulval body diameter long and conical tail with finely rounded terminus. Differences with closely related species including D. solani, D. ausafi, D. angustus, D. dipsaci, D. drepanocercus, D. obesus and D. gallaeformans are discussed. In addition, Iranian populations of two known species, D. valveus and D. virtudesae, are morphologically characterised.

Reported populations of Meloidogyne ethiopica in Europe identified as Meloidogyne luci.

The tropical group of root-knot nematodes (RKN) including Meloidogyne ethiopica and M. luci is a highly polyphagus and damaging group of agricultural pests. M. ethiopica has been detected in several European countries (Slovenia, Italy, Greece) and also in Turkey. However, a description of a new sister species M. luci calls for reclassification of all European and Turkish M. ethiopica populations reported up to date as M. luci. Accurate identification can be accomplished through analysis of the esterase isozyme pattern, which is the most distinguishing character between the two otherwise very similar species. Both species display a three banded esterase pattern where the upper band is slightly shifted between the two species. In addition, molecular characterization of M. ethiopica and M. luci populations revealed that the ITS, SSU, and LSU of the rDNA regions are not appropriate markers for studying relationships among the tropical group of RKNs. However, the COII/lRNA region on mtDNA proved to be very useful for analyzing the phylogenetic relationship of these very closely related species/populations. Mitochondrial sequences with low levels of heteroplasmy allowed clustering of all M. luci populations in a monophyletic clade with a clear separation of this recently described species from M. ethiopica. At the same time, a very close relationship between M. ethiopica and M. luci was confirmed again.

Description of Basiria birjandiensis n. sp (Nematoda: Tylenchidae) from South Khorasan province with a checklist of the family Tylenchidae Örley 1880 from Iran.

In order to identify plant-parasitic nematodes (family Tylenchidae Örley 1880) associated with Ziziphus zizyphus in Iran, 360 soil and root samples were collected from South Khorasan province during 2012-2014. Herein, a new species of Basiria and several known members of the family Tylenchidae are reported. B. birjandiensis n. sp. is characterized by short body length (584-748 µm [660.6±72.3]), lip region with flat apex, stylet 11-12 µm (11.3±0.5), excretory pore position varying from isthmus level to the middle of the basal bulb (78-91 µm from the anterior end of the body), post-vulval uterine sac 8-14 µm (10.7±1.9) long, filiform tail (151-181 µm, c= 3.7-4.2, c´= 14.3-17.2) and body annuli 0.5-1 µm (0.6±0.1) wide. A checklist of Tylenchidae species from Iran is also presented.