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.

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.

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.

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.

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.

Molecular characterization of putative parasitism genes in the plant-parasitic nematode Meloidogyne hispanica.

Meloidogyne hispanica (Mhi) is a difficult-to-control polyphagous root-knot nematode (RKN) species of emerging importance for economically valuable crops. Nematode secretions are likely to be the first signals perceived by the plant and are thought to be involved in various aspects of the plant-nematode interaction. The aims of this work were to identify and characterize M. hispanica parasitism genes: cathepsin L cysteine protease (cpl-1), calreticulin (crt-1), ß-1,4-endoglucanase-1 (eng-1) and manganese superoxide dismutase (mnsod). As there are no genomic data available for M. hispanica, primers were designed from the conserved regions of the putative parasitism genes in M. incognita and M. hapla and used to amplify the genes in M. hispanica, which led to the successful amplification of these genes in M. hispanica. Partial gene sequences were also obtained for M. arenaria, M. hapla, M. hispanica, M. incognita and M. javanica cpl-1, crt-1, eng-1 and mnsod genes, and their phylogenetic relationship analysed. In order to determine whether these genes are differentially expressed during M. hispanica development, cDNA was amplified from mRNA isolated from eggs, second-stage juveniles (J2) and females. Amplification products were observed from cDNA of all developmental stages for the Mhi-cpl-1 and Mhi-crt-1 genes. However, the gene Mhi-crt-1 exhibited intense amplification bands in females, while the Mhi-eng-1 gene was equally amplified in eggs and J2 and the Mhi-mnsod gene was only expressed in eggs. In comparison to the other RKN species, the genes Mhi-eng-1 and Mhi-mnsod showed transcription in different nematode developmental stages.

Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism.

Tulipaline A: structure-activity aspects as a nematicide and V-ATPase inhibitor.

Carbonyl groups are known to form covalent adducts with endogenous proteins, but so far, their nematicidal mechanism of action of has been overlooked. The nematicidal activity of ten lactones was tested in vitro against the root knot nematodes Meloidogyne incognita and Meloidogynearenaria. In particular, the saturated lactones α-methylene-γ-butyrolactone or tulipaline A (1) and γ-butyrolactone (3) were active against M. incognita with an EC50/48h of 19.3±10.0 and 40.0±16.2mg/L respectively. Moreover the α, ß-unsaturated lactone 5,6-dihydro-2H-pyran-2-one (2) exhibited the strongest nematicidal activity against the two species with EC50/48h 14.5±5.3 and 21.2±9.7mg/L respectively. Here we propose that the toxic effects of lactones and aldehydes on M.incognita and M. arenaria might be a consequence of their vacuolar-type H(+)-ATPase (V-ATPase) inhibition activity; in fact α-methylene-γ-butyrolactone (1) and salicylaldehyde (12) produced an increased pH in lysosomal-like organelles on HeLa human cell line and this alteration was most likely related to a V-ATPase impairment.

An updated and annotated checklist of the Dolichodoridae (Nematoda: Tylenchoidea) of Iran.

The list of plant parasitic nematodes of the family Dolichodoridae, known from Iran, is updated. 81 species belonging to 13 genera and three subfamilies are included in the list. Data for 29 species are added, of which seven species viz. Neodolichorhynchus phaseoli, Pratylenchoides crenicauda, P. erzurumensis, P. utahensis, Scutylenchus paniculoides, Trophurus impar and Tylenchorhynchus variannus are new records for the Iranian nematofauna. The list of species, further information on their morphometrics, references, referring to full or partial descriptions, associated plants, geographical distribution and some taxonomic remarks are provided. More detailed studies on some doubtful populations are proposed. The information on the taxonomic position of species in different classification schemes, as well as, the tendency of the species to certain climatic condition or ecological niche are provided. Challenges on the reliable identification of this group of nematodes in Iran are discussed and finally, suggestions were proposed for future studies.

Genetic diversity of the golden potato cyst nematode Globodera rostochiensis and determination of the origin of populations in Quebec, Canada.

The golden cyst nematode (Globodera rostochiensis), native to South America, has been introduced in many parts of the world, including Europe and North America. Recently, it was found for the first time in the province of Quebec, Canada in the locality of St. Amable near Montreal. To date, very few studies have examined the population genetics of this pest. Consequently, there is a lack of knowledge about the genetic structure and evolution of this nematode. In this study, twelve new microsatellite markers were developed in order to explore these questions. These markers were used to genotype fifteen populations originating from different regions of the world, including five from Canada. Within populations, the highest genetic diversity was consistently observed in the populations from Bolivia, the postulated region of origin of the golden nematode, and the lowest in populations from British Columbia (Canada) and New York (USA). The two Quebec populations were very similar to each other and to the population found in Newfoundland, but surprisingly, they were significantly different from three other North American populations including those from New York and British Columbia. Based on our results, we conclude that the golden cyst nematode has been introduced in North America at least twice from distinct regions of the world.

Heterodera fengi n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China--a new cyst nematode in the Cyperi group.

Heteroderafengi n. sp. is described and illustrated from bamboo (Phyllostachys pubescens Mazel) based on morphology and molecular analyses of rRNA LSU D2D3 region and ITS. This new species belongs to the Cyperi group. Cysts are characterized by prominent vulval cone with ambifenestrate, bifurcate underbridge that is thicker in middle and a 47.0 (40.0-60.0) µm long vulval slit, but without bullae. The second-stage juveniles are characterized by a 23.2 (22.0-24.0) µm long stylet with slightly projected or anteriorly flattened knobs, three incisures in lateral field, a 70.2 (62.5-77.0) µm long tail with bluntly rounded terminus and hyaline portion ca 58.9 (50.0-62.5)% of the tail length. Males are characterized by a 25.1 (24.5-26.3) µm long stylet with rounded knobs sloping posteriorly, four incisures in lateral field, a 29.8 (27.5-31.3) µm long spicule with bifurcate tip. Phylogenetic analysis shows that the species has unique D2D3 and ITS rRNA sequences and RFLP-ITS-rRNA profiles. Heteroderafengi n. sp. is closest to H. elachista in dendrograms inferred from both DNA sequences.

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.

Morphological and molecular characterization of Globodera populations from Oregon and Idaho.

An unusual population of cyst nematode was found in soils collected from a Powell Butte, OR field with a cropping history including potato, wheat, other crops, and significant weed presence. These nematodes could not be placed with certainty into any known species and exhibited some unique morphological features in some specimens. Compared with Globodera pallida, the cyst body length was slightly longer and the second-stage juvenile stylet length was slightly shorter. In some individuals, the J2 stylet knob height was greater and the tail annules were more prominent than in G. pallida, and the tail abruptly narrowed, with a slight constriction near the posterior third of the hyaline terminus. Compared with G. rostochiensis, the hyaline tail terminus had a larger number of refractive bodies, and cysts of this population had a smaller Granek's ratio and fewer cuticular ridges between the anus and vulva. In some individuals, the tail termini of second-stage juveniles were more bluntly pointed, and the stylet knobs were more anteriorly directed with greater height. Unlike G. tabacum, the cyst wall often lacked a network-like pattern and, in some individuals, the juvenile tail terminus distinctly narrowed after a constriction. Molecularly, the population was distinct from G. pallida, G. rostochiensis, and G. tabacum. Multiplex polymerase chain reaction of the internal transcribed spacer (ITS) rDNA region gave results similar to G. tabacum; however, ITS restriction fragment length polymorphism patterns were observed to have individual bands in common with G. rostochiensis and G. pallida. Phylogenetic analysis based on ITS1 and -2 rDNA sequences showed greatest similarity to populations from Argentina and Chile; together, they form a moderately supported clade, distinct from G. rostochiensis, G. tabacum, G. "mexicana," European type G. pallida, and several G. pallida populations from South America.

Multiplex PCR for the simultaneous identification and detection of Meloidogyne incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls.

Meloidogyne incognita, M. enterolobii, and M. javanica are the most widespread species of root-knot nematodes in South China, affecting many economically important crops, ornamental plants, and fruit trees. In this study, one pair of Meloidogyne universal primers was designed and three pairs of species-specific primers were employed successfully to rapidly detect and identify M. incognita, M. enterolobii, and M. javanica by multiplex polymerase chain reaction (PCR) using DNA extracted from individual galls. Multiplex PCR from all M. incognita, M. enterolobii, and M. javanica isolates generated two fragments of ≈500 and 1,000, 500 and 200, and 500 and 700 bp, respectively. The 500-bp fragment is the internal positive control fragment of rDNA 28S D2/D3 resulting from the use of the universal primers. Other Meloidogyne spp. included in this study generated only one fragment of ≈500 bp in size. Using this approach, M. incognita, M. enterolobii, and M. javanica were identified and detected using DNA extracted directly from individual galls containing the Meloidogyne spp. at various stages of their life cycle. Moreover, the percentage of positive PCR amplification increased with nematode development and detection was usually easy after the late stage of the second-stage juvenile. The protocol was applied to galls from naturally infested roots and the results were found to be fast, sensitive, robust, and accurate. This present study is the first to provide a definitive diagnostic tool for M. incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls using a one-step multiplex PCR technique.

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.

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.

Support for the 'out-of-Southeast Asia' hypothesis for the origin of Australian populations of Radopholus similis (Cobb, 1893) (Nematoda: Pratylenchidae).

Radopholus similis is one of the world's ten most economically important plant-parasitic nematodes. It is especially a problem in banana cultivation, where the nematodes' feeding reduces yields and causes toppling disease. It has been suggested that the genus Radopholus Thorne, 1949 might have an Australian origin, but the native range of R. similis (Cobb, 1893) is not well known. Here we undertake a phylogeographical study of samples of R. similis from banana plantations down the eastern seaboard of Australia, with additional samples from Costa Rica and accessions from GenBank, to examine the origin of pest populations of R. similis. The lack of genetic diversity of R. similis within Australia, and its sharing of a worldwide pest haplotype, suggest that populations of R. similis in Australia were introduced from a single source population, most likely from the Southeast Asian region. This might not be the case in Africa, where extensive genetic diversity has been found.

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.