Recombinase Polymerase Amplification assay for detection of the British root-knot nematode, Meloidogyne artiellia.

Recombinase polymerase amplification (RPA) is an isothermal in vitro nucleic acid amplification technique that has been adopted for simple, robust, rapid, reliable diagnostics of nematodes. In this study, the real-time RPA assay and RPA assay combined with lateral flow dipsticks (LF-RPA) have been developed targeting the ITS rRNA gene of the British root-knot nematode, Meloidogyne artiellia. The assay provided specific and rapid detection of this root-knot nematode species from crude nematode extracts without a DNA extraction step with a sensitivity of 0.125 second-stage juvenile (J2) specimen per a reaction tube for real-time RPA during 11 min and a sensitivity of 0.5 J2 specimens per a reaction tube for LF-RPA during 25 min. The RPA assays were validated with a wide range of non-target root-knot nematodes. The LF-RPA assay has great potential for nematode diagnostics in the laboratory having minimal available equipment.

Morphological and Molecular Analysis of Two Mycophagous Nematodes, Aphelenchoides bicaudatus and A. rutgersi (Nematoda: Aphelenchoididae) from Florida Strawberry.

From 2016 to 2021, nematode surveys in Florida strawberry fields revealed several species of foliar nematodes (Aphelenchoides spp.). Aphelenchoides besseyi sensu stricto was detected only in 2016 and 2017 on photosynthetic strawberry leaves/buds, but other not well characterized populations of Aphelenchoides sp. were found on declining/dessicated leaves. Morphological analyses showed that these samples of Aphelenchoides sp. consisted of A. bicaudatus, a species detected in Florida for the first time, and A. rutgersi, a species previously reported in Florida from the citrus rhizosphere. These two species differed from A. besseyi in the shape of their tail terminus: bifurcate in A. bicaudatus; mucronate with a ventral thin mucro in A. rutgersi; and stellate in A. besseyi. One population each of these species was used for morphological and molecular analyses after being reared on Monilinia fructicola. Body and tail length differences were observed among Florida A. bicaudatus and other populations from the Far East and South Africa. Phylogenetic analyses of the rRNA gene sequences showed that Florida A. bicaudatus grouped with those of species from South Korea, Taiwan, and the Netherlands and several other populations listed as Aphelenchoides sp. from Brazil, Costa Rica, and Japan, which were considered as representatives of A. bicaudatus in this study. Similarly, sequences of Florida A. rutgersi grouped with those from environmental samples in Japan and North Carolina, which were listed as Aphelenchoides sp. and were considered as representatives of A. rutgersi in this study. Photosynthetic strawberry leaf samples were free from both A. bicaudatus and A. rutgersi, indicating that these two species did not damage strawberry. They were associated with desiccated leaves and/or propagative stolons, usually infected by fungi, confirming that they are mycetophagous under field conditions in this study. Results of soybean leaf inoculation on moist filter paper containing A. bicaudatus specimens showed that this species could become phytophagous under artificial conditions. Nematodes penetrated the leaf epidermis and migrated into the mesophyll causing leaf tissue discoloration/necrosis, which remained localized within the infested area. Soybean leaf damage was almost negligible, and no nematode reproduction was observed in the inoculated soybean areas.

Phytopathogenic Curtobacterium flaccumfaciens Strains Circulating on Leguminous Plants, Alternative Hosts and Weeds in Russia.

Many bacterial plant pathogens have a broad host range important for their life cycle. Alternate hosts from plant families other than the main (primary) host support the survival and dissemination of the pathogen population even in absence of main host plants. Metabolic peculiarities of main and alternative host plants can affect genetic diversity within and between the pathogen populations isolated from those plants. Strains of Gram-positive bacterium Curtobacterium flaccumfaciens were identified as being causal agents of bacterial spot and wilt diseases on leguminous plants, and other crop and weed plants, collected in different regions of Russia. Their biochemical properties and susceptibility to copper compounds have been found to be relatively uniform. According to conventional PCR assays, all of the isolates studied were categorised as pathovar Curtobacterim flaccumfaciens pv. flaccumfaciens, a pathogen of legumes. However, the strains demonstrated a substantial diversity in terms of virulence on several tested host plants and different phylogenetic relationships were revealed by BOX-PCR and alanine synthase gene (alaS) sequencing.

Rathayibacter tanaceti sp. nov., a Novel Actinobacterium from Tanacetum vulgare Infested by Foliar Nematode Aphelenchoides sp.

Two novel yellow-pigmented, rod-shaped and non-motile coryneform actinobacteria, strains VKM Ac-2596T and VKM Ac-2761, were isolated from a plant Tanacetum vulgare (Asteraceae) infested by foliar nematode Aphelenchoides sp. The strains exhibited the highest 16S rRNA gene sequence similarities to Rathayibacter agropyri CA4T (99.71%), Rathayibacter rathayi DSM 7485T (99.65%) and Rathayibacter iranicus VKM Ac-1602T (99.65%). The pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between VKM Ac-2596T and VKM Ac-2671 towards the type strains of Rathayibacter species did not exceed 85.24% and 29.40%, respectively, that are well below the thresholds for species delineation. The target strains had key chemotaxonomic properties typical of the genus Rathayibacter, namely, the DAB-based peptidoglycan, rhamnose and mannose as the predominant sugars and a rhamnomannan in the cell, the major menaquinone MK-10 and fatty acids of iso-anteiso type, with a large proportion of anteiso-15:0. The strains showed clear differences from the recognized Rathayibacter species in several phenotypic characteristics, including the difference in the composition of cell wall glycopolymers. Based on the results obtained in this study and the data published previously, we provide a description of a new species, Rathayibacter tanaceti sp. nov., with DL-642T (= VKM Ac-2596T = LMG 33114T) as the type strain.

Rapid Detection of the Strawberry Foliar Nematode Aphelenchoides fragariae Using Recombinase Polymerase Amplification Assay with Lateral Flow Dipsticks.

Rapid and reliable diagnostic methods for plant-parasitic nematodes are critical for facilitating the selection of effective control measures. A diagnostic recombinase polymerase amplification (RPA) assay for Aphelenchoides fragariae using a TwistAmp® Basic Kit (TwistDx, Cambridge, UK) and AmplifyRP® Acceler8® Discovery Kit (Agdia, Elkhart, IN, USA) combined with lateral flow dipsticks (LF) has been developed. In this study, a LF-RPA assay was designed that targets the ITS rRNA gene of A. fragariae. This assay enables the specific detection of A. fragariae from crude nematode extracts without a DNA extraction step, and from DNA extracts of plant tissues infected with this nematode species. The LF-RPA assay showed reliable detection within 18-25 min with a sensitivity of 0.03 nematode per reaction tube for crude nematode extracts or 0.3 nematode per reaction tube using plant DNA extracts from 0.1 g of fresh leaves. The LF-RPA assay was developed and validated with a wide range of nematode and plant samples. Aphelenchoides fragariae was identified from seed samples in California. The LF-RPA assay has great potential for nematode diagnostics in the laboratory with minimal available equipment.

Correction: Ryss, A.Y.; Subbotin, S.A. New Records of Wood- and Bark-Inhabiting Nematodes from Woody Plants with a Description of Bursaphelenchus zvyagintsevi sp. n. (Aphelenchoididae: Parasitaphelenchinae) from Russia. Plants 2023, 12, 382.

In the original publication [...].

Morphological and Molecular Diversity among Pin Nematodes of the Genus Paratylenchus (Nematoda: Paratylenchidae) from Florida and Other Localities and Molecular Phylogeny of the Genus.

Pin nematodes (Paratylenchus spp.) are root parasites found worldwide. They have different life cycles and feeding habits and can damage a wide range of plants. A remarkable diversity of pin nematode species was found in soil samples from Florida and other states of the USA, Canada, and Spain. Using integrative taxonomy, two new species (Paratylenchus hawaiiensis sp. n. and P. roboris sp. n.), six valid species (Paratylenchulus acti, P. aquaticus, P. goldeni, P. paralatescens, P. minutus (=P. shenzhenensis syn. n.), and P. straeleni), and two undescribed species were identified from Florida; P. goldeni, P. hamatus, P. hamicaudatus, P. holdemani, and P. pedrami were found in California, P. minutus in Hawaii, P. goldeni in Oregon and Washington, and one new species, Paratylenchus borealis sp. n., in Alaska. Outside the USA, Paratylenchus projectus was detected in samples from Canada and Spain as well as P. holdemani and Paratylenchus sp. from Spain. The pin nematode species from Belgium and Russia identified in former studies as Paratylenchus sp. F was herein described as a new species with the name of P. borealis sp. n., using a population from Alaska. Previously reported molecular type A of P. aquaticus from Hawaii was reclassified as P. hawaiiensis sp. n., using a population from Florida. Paratylenchus roboris sp. n. from Florida has obese sedentary females with a stylet 63-71 µm long. The results of the molecular analysis of P. shenzhenensis from Florida and China indicated that it was conspecific with P. minutus from Hawaii and considered here as its junior synonym. New 26 D2-D3 expansion segments of 28S rRNA, 17 ITS rRNA, and 20 COI gene sequences were obtained in this study. Phylogenetic relationships of Paratylenchus are reconstructed using the D2-D3 of 28S rRNA, ITS rRNA, and COI gene sequences. Congruence of molecular and morphological evolution and species identification problems are discussed. Obese females were found in two major clades of Paratylenchus. The problem of reference materials is discussed, and it is proposed to make more efforts to collect topotype materials of known Paratylenchus species for molecular study.

Draft Genome Sequences of 11 Rhodopsin Gene-Containing Actinobacteria (Geodermatophilaceae) from Saline Arid Habitats in the Central Asian Deserts.

Draft genome sequences of 11 strains of putative new species of Geodermatophilaceae were generated using Illumina technology. The genome sizes ranged from 4.19 to 4.99 Mb, with G+C contents of 73.5% to 74.6%, and contained genes for microbial rhodopsins. This study will contribute to our knowledge of the ecology and diversity of members of the family Geodermatophilaceae.

Molecular and Morphological Characterization of Tylenchus Zeae n. Sp. (Nematoda: Tylenchida) from Corn (Zea Mays) in South Carolina.

Specimens of a tylenchid nematode were recovered in 2019 from soil samples collected from a corn field, located in Pickens County, South Carolina, USA. A moderate number of Tylenchus sp. adults (females and males) were recovered. Extracted nematodes were examined morphologically and molecularly for species identification, which indicated that the specimens of the tylenchid adults were a new species, described herein as Tylenchus zeae n. sp. Morphological examination and the morphometric details of the specimens were very close to the original descriptions of Tylenchus sherianus and T. rex. However, females of the new species can be differentiated from these species by body shape and length, shape of excretory duct, distance between anterior end and esophageal intestinal valve, and a few other characteristics given in the diagnosis. Males of the new species can be differentiated from the two closely related species by tail, spicules, and gubernaculum length. Cryo-scanning electron microscopy confirmed head bearing five or six annules; four to six cephalic sensilla represented by small pits at the rounded corners of the labial plate; a small, round oral plate; and a large, pit-like amphidial opening confined to the labial plate and extending three to four annules beyond it. Phylogenetic analysis of 18S rRNA gene sequences placed Tylenchus zeae n. sp. in a clade with Tylenchus arcuatus and several Filenchus spp., and the mitochondrial cytochrome oxidase c subunit 1 (COI) gene region separated the new species from T. arcuatus and other tylenchid species. In the 28S tree, T. zeae n. sp. showed a high level of sequence divergence and was positioned outside of the main Tylenchus-Filenchus clade.

Distribution of Bacterial Endosymbionts of the Cardinium Clade in Plant-Parasitic Nematodes.

Bacteria of the genus "Candidatus Cardinium" and related organisms composing the Cardinium clade are intracellular endosymbionts frequently occurring in several arthropod groups, freshwater mussels and plant-parasitic nematodes. Phylogenetic analyses based on two gene sequences (16S rRNA and gyrB) showed that the Cardinium clade comprised at least five groups: A, B, C, D and E. In this study, a screening of 142 samples of plant-parasitic nematodes belonging to 93 species from 12 families and two orders using PCR with specific primers and sequencing, revealed bacteria of Cardinium clade in 14 nematode samples belonging to 12 species of cyst nematodes of the family Heteroderidae. Furthermore, in this study, the genome of the Cardinium cHhum from the hop cyst nematode, Heterodera humuli, was also amplified, sequenced and analyzed. The comparisons of the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values for the strain Cardinium cHhum with regard to related organisms with available genomes, combined with the data on 16S rRNA and gyrB gene sequence identities, showed that this strain represents a new candidate species within the genus "Candidatus Paenicardinium". The phylogenetic position of endosymbionts of the Cardinium clade detected in nematode hosts was also compared to known representatives of this clade from other metazoans. Phylogenetic reconstructions based on analysis of 16S rRNA, gyrB, sufB, gloEL, fusA, infB genes and genomes and estimates of genetic distances both indicate that the endosymbiont of the root-lesion nematode Pratylenchus penetrans represented a separate lineage and is designated herein as a new group F. The phylogenetic analysis also confirmed that endosymbionts of ostracods represent the novel group G. Evolutionary relationships of bacterial endosymbionts of the Cardinium clade within invertebrates are presented and discussed.

New Records of Wood- and Bark-Inhabiting Nematodes from Woody Plants with a Description of Bursaphelenchus zvyagintsevi sp. n. (Aphelenchoididae: Parasitaphelenchinae) from Russia.

Wood- and bark-inhabiting parasitic nematodes are of great economic importance. Nematodes can cause wilt diseases in conifers and deciduous trees. In 2014-2022, during nematology surveys conducted in different regions of Russia and Belarus, adults and dauer juveniles of nematodes were collected from wood, bark and beetle vectors. Using traditional morphological taxonomic characters integrated with molecular criteria, we identified in the studied samples the following nematode species: Aphelenchoides heidelbergi, Bursaphelenchus eremus, B. fraudulentus, B. michalskii, B. mucronatus, B. willibaldi, Deladenus posteroporus, Diplogasteroides nix and Laimaphelenchus hyrcanus, several unidentified species: Aphelenchoides sp.1 and sp.2, Cryptaphelenchus sp.1, sp.2 and sp.3, Laimaphelenchus sp.1, Micoletzkya sp.1, Parasitaphelenchus sp.1, Parasitorhabditis sp.1, three unidentified tylenchid nematodes and a new species, Bursaphelenchus zvyagintsevi sp.n. Morphological descriptions and molecular characterization are provided for B. zvyagintsevi sp. n. belonging to the Abietinus group and B. michalskii belonging to the Eggersi group. Findings of Aphelenchoides heidelbergi, Bursaphelenchus eremus, B. michalskii, Deladenus posteroporus, Diplogasteroides nix and Laimaphelenchus hyrcanus are new records for Russia. Phylogenetic positions of studied species were reconstructed using D2-D3 expansion segments of 28S rRNA gene sequence analysis. The data obtained in this study may help to detect the refugia of opportunistic plant pests and find possible native biocontrol nematode agents of insect vectors causing diseases.

Draft Genome Sequences of 9 Actinobacteria from the Family Microbacteriaceae Associated with Insect- and Nematode-Damaged Plants.

Draft genome sequences of 9 strains of known and putative new species of Microbacteriaceae isolated from insect- and nematode-damaged plants were generated using Illumina technology. The data obtained will contribute to the development of the genome-based prokaryote taxonomy and the knowledge on the biology of the microbial group investigated.

Morphological and Molecular Characterization of Punctodera Stonei Brzeski, 1998 (Nematoda: Heteroderidae) from Virginia, USA.

In August of 2021, several cysts with juveniles and eggs were discovered during a vegetation survey conducted at the Arlington National Cemetery, Virginia. Eight soil samples were collected from the rhizosphere region of the common grass (Festuca arundinacea L.) and processed at the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL). Cysts were light to dark brown in color, and oval to pear-shaped without bullae in young cysts but present in older cysts and with prominent vulval cone. The juveniles had slightly concave stylet knobs projecting sometimes anteriorly, tail tapering gradually to a narrowly rounded terminus, and hyaline tail terminus conspicuous at least twice the length of stylet. The molecular analysis included the analysis of three gene sequence fragments: D2-D3 of 28S rRNA, ITS rRNA, and COI. The nematode species was identified by both morphological and molecular means as Stone's cyst nematode, Punctodera stonei. Detection of P. stonei in Virginia represents a new record of this species in the United States, and a second report after Canada in North America.

Expanding the RNA virome of nematodes and other soil-inhabiting organisms.

In recent years, several newly discovered viruses infecting free-living nematodes, sedentary plant-parasitic nematodes, and migratory root lesion nematodes have been described. However, to the best of our knowledge, no comprehensive research focusing exclusively on metagenomic analysis of the soil nematode community virome has thus far been carried out. In this work, we have attempted to bridge this gap by investigating viral communities that are associated with soil-inhabiting organisms, particularly nematodes. This study demonstrates a remarkable diversity of RNA viruses in the natural soil environment. Over 150 viruses were identified in different soil-inhabiting hosts, of which more than 139 are potentially new virus species. Many of these viruses belong to the nematode virome, thereby enriching our understanding of the diversity and evolution of this complex part of the natural ecosystem.

First report of Seville root-knot nematode, Meloidogyne hispanica (Nematoda: Meloidogynidae) in the USA and North America.

A high number of second stage juveniles of the root-knot nematode were recovered from soil samples collected from a corn field, located in Pickens County, South Carolina, USA in 2019. Extracted nematodes were examined morphologically and molecularly for species identification which indicated that the specimens of root knot juveniles were Meloidogyne hispanica. The morphological examination and morphometric details from second-stage juveniles were consistent with the original description and redescriptions of this species. The ITS rRNA, D2-D3 expansion segments of 28S rRNA, intergenic COII-16S region, nad5 and COI gene sequences were obtained from the South Carolina population of M. hispanica. Phylogenetic analysis of the intergenic COII-16S region of mtDNA gene sequence alignment using statistical parsimony showed that the South Carolina population clustered with Meloidogyne hispanica from Portugal and Australia. To our best knowledge, this finding represents the first report of Meloidogyne hispanica in the USA and North America.

Molecular and morphological characterization of a first report of Cactodera torreyanae Cid del Prado Vera & Subbotin, 2014 (Nematoda: Heteroderidae) from Minnesota, the United States of America.

Cactodera torreyanae Cid del Prado Vera & Subbotin, 2014 cysts were discovered during a Pale Potato Cyst Nematode (PCN) survey conducted by Minnesota Department of Agriculture as part of the Animal and Plant Health Inspection Service (APHIS) efforts to survey states for the presence of PCN. The soil samples were collected from a potato field, located in Karlstad, Kittson County, Minnesota, USA. Two out of 175 vials submitted for identification to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL) contained few cysts and juveniles of C. torreyanae. Cysts were dark brown in color, lemon-shaped to elongated with distinct vulval cone. Vulva with denticles present around fenestra, cyst length to width ratio between 1.6 and 2.3 and anus distinct. The juveniles had rounded stylet knobs, some sloping slightly posteriorly. The molecular analysis included sequence and phylogenetic analysis of ITS rRNA, D2-D3 expansion segments of 28S rRNA and COI of mtDNA genes. The nematode species was identified by both morphological and molecular means as Cactodera torreyanae. To the best of our knowledge this represents the first report of Cactodera torreyanae from the United States and first report of this cyst nematode species from potato fields. Definite host plant for this nematode remains unknown.

Morphological and molecular characterization of Paratylenchus beltsvillensis n. sp. (Tylenchida: Paratylenchidae) from the rhizosphere of pine tree (Pinus virginiana Mill) in Maryland, USA.

The pin nematode, Paratylechus beltsvillensis n. sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George's County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 µm, stylet from 70 to 75 µm long with anchor shaped knobs, vulva located at 70-73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17-20 µm and gubernaculum = 5.0-5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.

Report of the Texas peanut root-knot nematode, Meloidogyne haplanaria (Tylenchida: Meloidogynidae) from American pitcher plants (Sarracenia sp.) in California.

During the winter and spring of 2021, the root-knot nematodes were extracted from samples of galled roots of potted American pitcher plants (Sarracenia sp.). Samples were collected from a botanical garden nursery in Los Angeles County, California. The root-knot nematode was identified by molecular methods as Meloidogyne haplanaria. In the USA, M. haplanaria was initially found in Texas, and subsequently reported from Arkansas and Florida. Molecular characterization of the Californian M. haplanaria isolate was done using the analysis of the D2-D3 of 28S rRNA, ITS rRNA, mitochondrial l-rRNA, COI, and nad5 gene sequences. Some rRNA gene clusters of M. haplanaria were similar with those of M. arenaria. Possible hybridization events within mitotic parthenogenetic root-knot nematodes are discussed. This study confirmed that reliable diagnostics of M. haplanaria should be based on mtDNA sequence analysis. This is a first report of M. haplanaria from Sarracenia sp. and California. Consequently, this nematode was considered to be eradicated from this botanical garden nursery and the State of California.

'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov., an endosymbiotic bacterium associated with nematode species of the genus Xiphinema (Nematoda, Longidoridae).

An intracellular bacterium, strain IAST, was observed to infect several species of the plant-parasitic nematode genus Xiphinema (Xiphinema astaregiense, Xiphinema incertum, Xiphinema madeirense, Xiphinema pachtaicum, Xiphinema parapachydermum and Xiphinema vallense). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAST was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EBT (92.9 % sequence identity) and 'Candidatus Glomeribacter gigasporarum' BEG34 (89.8 % identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes (atpD, lepA and recA) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8-1.2 µm wide and 2.5-6.0 µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing 'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov.

Morphology, development stages, and phylogeny of the Rhabditolaimus ulmi (Nematoda: Diplogastridae), a phoront of the bark beetle Scolytus multistriatus from the elm Ulmus glabra Huds. in Northwest Russia.

The nematode Rhabditolaimus ulmi was found in galleries, adults, and larvae of Scolytus multistriatus, the vector of the Dutch elm disease, in St. Petersburg parks. This nematode co-occurred with Bursaphelenchus ulmophilus, which is another phoretic partner of S. multistriatus. Nematodes were cultured on the fungus Botryotinia fuckeliana in potato sugar agar (PA) and used for morphological analyses of adults, juveniles, eggs, and dauers. Nematode females showed a didelphic female genital tract rather than a monoprodelphic gonad as reported in the original description. Male bursa peloderan, caudal papillae include three preanal pairs and one precloacal unpaired papillae; seven postanal papilla pairs, among which one is pore-like and possibly the phasmid homolog, one subdorsal, and a pair of three closely situated posteriorly at bursa alae. The juvenile stages differ in size and structure of their sexual primordia. Sex of juveniles may be identified from the third stage. The dauer juvenile is a phoretic third juvenile stage (DJ3), which enters and remains localized in the buccal cavity of beetle adults and last-instar larvae and also under the elytra and in the ovipositor's cavity of pupae and imagoes. The first molt J1-J2 occurred inside the eggshell. Adult females laid eggs in early stages of embryonic development or containing molted J2. The propagative non-phoretic J2 inside the egg and J3 have a long and well-developed median bulb. The phoretic dauer DJ3 has a small spherical bulb like the J1 juvenile within the egg. In a sterile fungal culture, the nematodes feed on both mycelium and their unidentified ecto-symbiotic bacteria, located on nematode surface coat and multiplying in PA. Diagnosis and tabular key to the Rhabditolaimus species are given. Phylogenetic analysis of the D2-D3 of 28S rRNA gene sequences resulted in the Bayesian consensus tree with the highly supported clade of the Rhabditolaimus species.