Morpho-Anatomy, In Vitro Culture, and Phylogenetic Studies of Two Helicotylenchus Species from Southern Alberta, Canada.

Spiral nematodes (Helicotylenchus spp.) are polyphagous parasitic species exhibiting a broad host range and geographical distribution. However, their diversity in the cultivated regions of southern Alberta remains understudied. Hence, we conducted a comprehensive survey of the region's arable lands for the presence of spiral nematodes and revealed two Helicotylenchus species, H. crassatus and H. oscephalus. H. crassatus consisted of two distinct morphotypes: one morphotype had a conoid tail with slight ventral projection on the distal end, whereas the other had a broadly rounded tail. This study presents the first documentation of H. crassatus and H. oscephalus from southern Alberta, Canada. Molecular characterization was based on the partial 18S rRNA, the D2-D3 of 28S rRNA, ITS rRNA, and COI gene sequences, complemented by detailed morphological studies using scanning electron microscopy. In this work, Helicotylenchus species were often co-detected with root lesion nematodes, which made the evaluation of their role in crop damage more difficult. To meet the requirements for threshold and pathogenicity assessments, we introduced both spiral nematode species to sterile carrot disks and evaluated the feasibility of their multiplication and mass production in vitro. The present findings expand the taxonomic records of Helicotylenchus spp. and improve diagnostics of these morphologically similar species. Furthermore, our in vitro culture technique will provide a reliable source of the initial inoculum for future plant-nematode interaction studies.

New Records of Ditylenchus Species from Southern Alberta, Canada.

The presence of plant-parasitic nematodes (PPNs) in cultivated areas is a limiting factor in achieving marketable crop yield. To control and alleviate the effects of these nematodes and determine appropriate management strategies, species-level identification is crucial. Therefore, we conducted a nematode diversity survey, which resulted in the detection of four Ditylenchus species in cultivated areas of southern Alberta, Canada. The recovered species had six lines in the lateral field, delicate stylets (>10 µm long), distinct postvulval uterine sacs, and pointed to rounded tail tips. The morphological and molecular characterization of these nematodes revealed their identity as D. anchilisposomus, D. clarus, D. tenuidens and D. valveus, all of which are members of the D. triformis group. All of the identified species were found to be new records in Canada except for D. valveus. Accurate Ditylenchus species identification is crucial because false-positive identification can result in the implementation of quarantine measures over the detected area. Our current study not only documented the presence of Ditylenchus species from southern Alberta, but also described their morpho-molecular characteristics and subsequent phylogenetic relationships with related species. The results of our study will aid in the decision on whether these species should become a part of nematode management programs since nontarget species can become pests due to changes in cropping patterns or climate.

Morpho-Molecular and Ultrastructural Characterization of Discocriconemella Parasinensis n. Sp. from Zhejiang Province, China.

During a recent inventory survey of the nematofauna of tea plantation at Zhejiang Province, China, Discocriconemella parasinensis n. sp. was detected in the rhizosphere of Camellia sinensis. The new species can be characterized by having the uninterrupted rounded labial disc, en face view showing rectangular-rounded labial plate without submedian lobes, R = 82.6 (80-86), Rex = 22 (21-23), stylet length of 68.3 (59-76) µm, excretory pore located 1-2 annuli posterior to the esophageal bulb, vulva open, postvulval body elongated conoid, and tail conoid with bilobed terminus. Morphologically, the species shares the same lip-type with D. discolabia, D. mauritiensis, D. mineira, D. perseae, and D. sinensis. Phylogenetic relationships of the new species based on D2-D3 expansion segments of 28S, ITS, and 18S rRNA genes revealed that D. parasinensis n. sp. formed a separated clade from other criconematid species, thereby supporting its status as a new species of the genus. The new species showed close phylogenetic relationships with Criconemoides geraerti.

Morphological and molecular characterization of Geocenamus persici n. sp. (Nematoda: Merliniinae) from China.

A new stunt nematode species, Geocenamus persici n. sp. was recovered from the rhizosphere of peach (Prunus persica) in Zhejiang Province, China. This species is characterized by a dome-shaped labial region with six bulging sectors. The lip region is slightly offset from the rest of the body with five annuli; cuticle with over 30 longitudinal striae; stylet slender, 19 to 21 m long; excretory pore at the anterior region of the basal pharyngeal bulb; tail cylindrical, 55 to 79 m long with striated terminus. Males are common. Morphologically, the species is close to G. brevicaudatus, G. chengi, G. conicaudatus, G. quadrifer, G. rugosus, and G. tartuensis. Phylogenetic relationships of the new species placed G. persici n. sp. with other species of subfamily Merliniinae. However, it has a sister relationship with G. chengi. Since the new species is found in the peach plantations, our study will lay a foundation to initiate investigations on the occurrence of stunt nematodes in peach growing areas and will aid the researchers to determine whether this group of nematodes cause peach plantation diseases.

Morphological and Molecular Characterization of Quinisulcius curvus from China.

A stunt nematode species, Quinisulcius curvus, recovered from the rhizosphere of sea randa (Guettarda speciosa), is described and characterized herein based on integrative taxonomy. Morphometrics and distribution of all reported populations of Q. curvus are also discussed. The Chinese population of Q. curvus displayed slight variation in stylet length; however, the rest of the characters matches well with the original description. This is the first record of Q. curvus from Hainan, China, and the first molecular characterization for this species. Phylogenetic analysis based on partial 18S, 28S and ITS sequences placed Q. curvus with related stunt nematodes species, but clearly separated from them. The present study expanded the geographic record and provided molecular data on Q. curvus from China.

Overview of the Genus Boleodorus and First Reports of Boleodorus thylactus and B. volutus from Southern Alberta, Canada.

The present study provides the morphological and molecular characterization of Boleodorus thylactus and B. volutus populations, recovered from agricultural fields of southern Alberta. Despite a significant abundance of this group of nematodes, none of the Boleodorus species were previously reported in Canada. Therefore, representative adult specimens of each population were photographed and examined morphometrically. Phylogenetic trees were reconstructed using partial D2-D3 expansion segments of the 28S and 18S rDNA sequences to understand the relationships of Boleodorus species with Tylenchidae-related genera. Boleodorus species are relevant to soil ecological studies and therefore we summarized the important morphological and morphometric characters in tabular form for easy and efficient species identification. Moreover, we discuss the associated hosts and the distribution of all described Boleodorus species. This study will serve as a guide and basic framework for species diagnostics in the genus Boleodorus and will aid in filling the gaps in our knowledge of the species present in our cultivated lands.

Integrative taxonomy, distribution, and host associations of Geocenamus brevidens and Quinisulcius capitatus from southern Alberta, Canada.

Two stunt nematode species, Geocenamus brevidens and Quinisulcius capitatus, were recovered from the potato growing regions of southern Alberta, described and characterized based on integrative taxonomy. Morphometrics, distribution, and host associations of both species are discussed. The Canadian populations of both species displayed minor variations in morphometrical characteristics (viz., slightly longer bodies and tails) from the original descriptions. The populations of G. brevidens and Q. capitatus species examined in this study are proposed as standard and reference populations for each respective species until topotype specimens become available and molecularly characterized. Phylogenetic analyses, based on partial 18S, 28S, and ITS sequences, placed both species with related stunt nematode species. The present study updates the taxonomic records of G. brevidens and Q. capitatus from a new location, southern Alberta, Canada, and will aid in the decision whether these stunt nematodes should be included in nematode management programs.

Species Diversity of Pin Nematodes (Paratylenchus spp.) from Potato Growing Regions of Southern Alberta, Canada.

Pin nematodes ( Paratylenchus spp.) are polyphagous parasitic species with a wide host range and geographical distribution; their diversity is unknown in the potato growing region of Alberta, Canada. The present study aims to provide morphological and molecular characterization of three pin nematode species, namely P. neoprojectus , P. tateae , and a new species, Paratylenchus enigmaticus sp. nov. All of them were recovered from the potato growing region of southern Alberta. The nematodes were isolated using the sieving and flotation-centrifugation method, and their morphology was assessed by light microscopy. Molecular characterization was performed using partial 18S, D2-D3 expansion domains of the 28S and ITS ribosomal genes. This study is the first report of molecular characterization of P. tateaeand P. neoprojectus , being new records from southern Alberta, and two Spanish populations of P. tateae comprising the first report of this species in Europe. The phylogenetic analysis of the 18S, D2-D3 expansion domains of the 28S and ITS ribosomal DNA regions underscores the importance of using molecular data for accurate species identification and clarifies the status of P. nanus type B and P. sheri . Moreover, our findings will be useful to determine the impact of pin nematodes on potato production in future field research.

Identification and Characterization of a Novel Protein Disulfide Isomerase Gene (MgPDI2) from Meloidogyne graminicola.

Protein disulfide isomerase (PDI) is a multifunctional enzyme that catalyzes rate-limiting reactions such as disulfide bond formation, isomerization, and reduction. There is some evidence that indicates that PDI is also involved in host-pathogen interactions in plants. In this study, we show that the rice root-knot nematode, Meloidogyne graminicola, has evolved a secreted effector, MgPDI2, which is expressed in the subventral esophageal glands and up-regulated during the early parasitic stage of M. graminicola. Purified recombinant MgPDI2 functions as an insulin disulfide reductase and protects plasmid DNA from nicking. As an effector, MgPDI2 contributes to nematode parasitism. Silencing of MgPDI2 by RNA interference in the pre-parasitic second-stage juveniles (J2s) reduced M. graminicola multiplication and also increased M. graminicola mortality under H2O2 stress. In addition, an Agrobacterium-mediated transient expression assay found that MgPDI2 caused noticeable cell death in Nicotiana benthamiana. An intact C-terminal region containing the first catalytic domain (a) with an active motif (Cys-Gly-His-Cys, CGHC) and the two non-active domains (b and b') is required for cell death induction in N. benthamiana. This research may provide a promising target for the development of new strategies to combat M. graminicola infections.

Delatylus andersoni n. gen., n. sp. (Nematoda: Neotylenchidae) Isolated from White Pine (Pinus monticola) Lumber from USA and Intercepted in Ningbo, China.

Three populations of neotylenchid nematodes were isolated in Ningbo, P. R. China, from white pine lumber (Pinus monticola) imported from the USA. The nematodes were morphologically intermediate between Hexatylus and Deladenus. The nematodes were molecularly characterized based on sequences of the rDNA small subunit 18S, large subunit 28S D2/D3, and internal transcribed spacer sequences. The phylogenetic inferences placed the nematodes with other neotylenchid nematodes, i.e., Fergusobia and Rubzovinema. Based on the morphology and phylogenetic analysis, this nematode is described herein as Delatylus andersoni n. gen., n. sp. The new genus/species is characterized by the female body habitus ranging from nonobese to semiobese and from straight to dorsally curved when heat relaxed, cephalic framework with six unequal sized lip sectors, lateral fields having 10 to 12 lines, 4 to 5 guide rings on the stylet, excretory pore posterior to the nerve ring, spermatheca diminished or absent, vulvar opening large, and relative proximity of vulva to the anus. Detailed morphological and molecular characterization of the new genus/species is presented along with the comparison of the related genera.

Description and Distribution of Three Criconematid Nematodes from Hangzhou, Zhejiang Province, China.

Populations of Criconemoides parvus , Discocriconemella hengsungica , and Discocriconemella limitanea , isolated in Hangzhou, China from the rhizosphere soil of woody perennials were characterized morphologically and molecularly. The morphometric data of the Chinese populations were compared with populations from other regions of the world. DNA barcoding with the mitochondrial COI gene confirmed conspecificity of Chinese and Costa Rican populations of D. limitanea . Phylogenetic assessment using a near full-length 18S ribosomal DNA sequence provided weak support for a grouping of Criconemoides parvus from China and C. annulatus from western North America. The phylogenetic position of D. hengsungica from China and an unknown species of Discocriconemella from Thailand relative to D. limitanea suggests that the genus Discocriconemella is not monophyletic. The study provides the first record of D. hengsungica in China and confirms the presence of C. parvus previously reported from China. Biogeographic implications of these nematode distributions are discussed.