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The voltage-gated calcium channel (VGCC) β subunit (Ca v β) protein is a kind of cytosolic auxiliary subunit that plays an important role in regulating the surface expression and gating characteristics of high-voltage-activated (HVA) calcium channels. Ditylenchus destructor is an important plant-parasitic nematode. In the present study, the putative Ca v β subunit gene of D. destructor , namely, DdCa v β , was subjected to molecular characterization. In situ hybridization assays showed that DdCa v β was expressed in all nematode tissues. Transcriptional analyses showed that DdCa v β was expressed during each developmental stage of D. destructor , and the highest expression level was recorded in the third-stage juveniles. The crucial role of DdCa v β was verified by dsRNA soaking-mediated RNA interference (RNAi). Silencing of DdCa v β or HVA Ca v α 1 alone and co-silencing of the DdCa v β and HVA Ca v α 1 genes resulted in defective locomotion, stylet thrusting, chemotaxis, protein secretion and reproduction in D. destructor . Co-silencing of the HVA Ca v α 1 and Ca v β subunits showed stronger interference effects than single-gene silencing. This study provides insights for further study of VGCCs in plant-parasitic nematodes.

RNA interference (RNAi) is a powerful tool for gene functional analysis of plant-parasitic nematodes (PPNs). RNAi involving soaking in a dsRNA solution and in planta methods is commonly applied in the study of gene function in PPNs. However, certain problems restrict the application of these methods. Therefore, more convenient and effective RNAi methods need to be established for different PPNs according to their biological characteristics. In this study, the fatty acid and retinoid binding protein genes ( Ab-far-1, Ab-far-4 , and combinatorial Ab-far-1 and Ab-far-4 ) of the rice white tip nematode (RWTN), Aphelenchoides besseyi , were used as target genes to construct a fungal RNAi vector, and the Ab-far-n dsRNA transgenic Botrytis cinerea (ARTB n ) were generated using Agrobacterium -mediated transformation technology. After RWTN feeding on ARTB n , the expression of Ab-far-1 and Ab-far-4 in the nematodes was efficiently silenced, and the reproduction and pathogenicity of the nematodes were clearly inhibited. The Ab-far-1 and Ab-far-4 co-RNAi effects were better than the effects when each gene was individually targeted with RNAi. Additionally, the RNAi induced when RWTNs fed on ARTB 1 were persistent and heritable. Thus, a new method of fungus-mediated RNAi was established for fungivorous PPNs and was verified as effective and applicable to the study of nematode gene function. This technique will remove the technological bottlenecks and provide a new method to studying the multiple genes with polygene co-RNAi in fungivorous PPNs. This study also provides a theoretical basis and new thought for further study of the gene function in PPNs. Abbreviations: FAR(Fatty acid and retinol-binding proteins); RWTN (The rice white tip nematode, Aphelenchoides besseyi); Ab-far-n (Fatty acid and retinol binding protein gene of A. besseyi) ; ARTB 1 ( Ab-far-1 hpRNA transgenic Botrytis cinerea ); ARTB 4 ( Ab-far-4 hpRNA transgenic Botrytis cinerea ); ARTB 1 / 4 (combinatorial Ab-far-1 and Ab-far-4 hpRNA transgenic B. cinerea ); EVTB (Empty vector transgenic B. cinerea ); GRTB (eGFP hpRNA transgenic B. cinerea ); WTB (Wild-type B. cinerea ).

Background: Nacobbus aberrans (Tylenchida: Pratylenchidae) is one of the main plant-parasitic nematodes species that affects crops in Mexico, generating substantial economic losses. Traditionally, the control of the nematodes is carried out using chemical products; however, research efforts are presently focused on the search for new methods for the control of this pest. Natural products derived from plants are an alternative for the control of populations of plant-parasitic nematodes. The genus Heterotheca (Asteraceae) is characterized by containing sesquiterpenes with cadinane skeleton, and some species of this genus exert nematicidal activity., Results: We determined the effects of selected Heterotheca inuloides plant metabolites and some semisynthetic derivatives on the hatching of eggs isolated from the gelatinous matrix and infective second-stage juveniles (J2) of the false root-knot nematode N. aberrans using an in vitro experimental model. Among the evaluated compounds, nematodes were more susceptible to hydroxylated and quinone compounds, whereas the remaining compounds showed moderate or no activity. The presence of the hydroxyl group is essential for nematicidal potential, with changes at the hydroxyl group modifying the nematicidal activity., Conclusion: Flowers of Heterotheca inuloides contain bioactive compounds that showed nematicidal activity against N. aberrans. Here we report the nematicidal activities of cadinenes isolated from the flowers of H. inuloides and their semisynthetic derivatives against the false root-knot nematode N. aberrans. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Heterodera glycines is the most pervasive soybean pests worldwide. Biocontrol provides a strategy to sustainably control nematodes. In this study, 22 fungal isolates were obtained and identified from cysts of Heterodera spp. Among them, Aspergillus niger NBC001 showed high nematicidal activity against H. glycines. The 2-fold dilution of NBC001 culture filtrate caused 89% mortality of second-stage juveniles and inhibited more than 98% of egg hatching in vitro. In both pot and field experiments, the numbers of H. glycines cysts in soybean seedlings dressed with the the 5-fold concentrated culture filtrate of NBC001 were significantly reduced by 43% and 28%, respectively. In addition, application of NBC001 remarkably reduced the penetration of nematodes into the roots. Histochemical and fluorometric staining analyses indicate that application of NBC001 stimulated hydrogen peroxide activity in the roots and triggered callose deposition in the leaves and roots. Transcription of the PR1a and EREBP genes in the salicylic acid and ethylene signaling pathways was upregulated in soybean plants treated with NBC001. However, the application of concentrated culture filtrate of NBC001 had no significant impacts on the soil microbial community based on next generation DNA sequencing technology. In summary, NBC001 may be a good biocontrol agent against H. glycines via stimulation of the immunity/defense of the plant host.

The pine wood nematode (PWN), Bursaphelenchus xylophilus , is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN ( Bx-cpls ) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN ( Bx-cpl ) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpl s in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1 , Bx-cpl-2 , and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpl s could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpl s in the egg stage was highest. After infection, the expression levels of Bx-cpl s increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpl s play multiple roles in the development and pathogenic processes of PWN.

A new species of Paurodontoides, P. siddiqii n. sp., is described and illustrated based on its morphological, morphometric, and molecular characters. The new species is characterized by a female 550-729 μm long, lip region continuous with body contour, stylet length 7.0-8.0 μm long or c. 1.0-1.2 times the lip region diameter, lateral fields with four smooth incisures, excretory pore at 85-125 μm from anterior end located at the base of the pharyngeal bulb or posterior to it, basal pharyngeal bulb with a short posterior extension projecting into the intestine, monodelphic-prodelphic reproductive system with prominent 19-22 μm long post-uterine sac, and elongate conoid tail with a filiform terminus. The new species is compared with two known species of the genus. It differs from the type species of the genus, P. linfordi, by having slightly shorter stylet, lateral field with smooth incisures, different position of the excretory pore, and absence of male. Compared to P. latus, the new species has a shorter body, shorter stylet, different position of the excretory pore, female tail shape and absence of male. The new species was also compared with close species of the genus Paurodontus because of lateral field marked with four lines, asymmetrical stylet knobs and absence of male. Molecular phylogenetic studies of the new species using partial sequences of 18S rDNA revealed that it forms a clade with a species of the genus Ficotylus. In phylogenetic analyses using partial sequences of the 28S rDNA D2-D3 domain, the new species formed a monophyletic group with a species of the genus Veleshkinema and Sphaerularia spp. (Sphaerulariinae).

The harlequin ladybird Harmonia axyridis is native to Asia but has been introduced into many countries as a biological control agent. It is now considered an invasive pest, threatening the biodiversity of native ladybirds globally, in part because of its superior immune system. H. axyridis is infected and killed by the parasitic nematode Parasitylenchus bifurcatus, which could therefore be developed as a biological strategy to counter the spread of this insect pest. However, effective control requires an understanding of the tripartite relationship between H. axyridis, P. bifurcatus and their potential bacterial mutualists. Here we describe the isolation of two species of nematode-associated bacteria (Serratia marcescens and Providencia rettgeri) which were highly virulent against H. axyridis in survival experiments. In addition, contact between the nematodes and beetles led to the sex-specific modulation of multiple host immunity-related genes after 24 and 48 h, with many genes encoding antimicrobial peptides rapidly and stably repressed in females whereas the same genes were initially induced in males before suppression at the later time point. These data provide evidence that the female immune system responds much more strongly to the nematodes and provokes, in turn, a more robust invasion strategy involving the bacterial mutualists.

Background: Bursaphelenchus xylophilus is a serious quarantined pest that causes severe damage and major economic losses to pine forests. Because of the adverse effects of some traditional nematicides on humans and the environment, the search for new plant toxicants against these nematodes has intensified. Nematicidal activity of trans-2-hexenal, which is a six-carbon aldehyde present in many plants, was tested against the nematode., Results: trans-2-Hexenal showed significant efficacy against B. xylophilus in a dose range of 349.5-699 g m -3 by fumigation of pinewood logs. Additionally, it had significant nematicidal activity against different life stages of B. xylophilus in an in vitro test, with second-stage larvae (L2s) being the most sensitive, with an LC 50 value of 9.87 µg mL -1 at 48 h. Egg hatch was also significantly inhibited. Further studies revealed that trans-2-hexenal inhibited the reproductive activity of B. xylophilus, with negative effects on reproduction rate and egg numbers. Moreover, trans-2-hexenal reduced the body length of B. xylophilus. Respiratory rate and thrashing behaviour of B. xylophilus also decreased following treatment with this compound., Conclusion: trans-2-Hexenal had significant nematicidal activity against B. xylophilus, providing a basis for elucidation of the mode of action of trans-2-hexenal against plant-parasitic nematodes in future studies. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Two new species of Seinura are described and illustrated using both morphological and molecular characters. Seinura persica n. sp. is characterized by having females with a distinct cephalic region, stylet 19.5 (18-20) μm long, four incisures in the lateral fields, position of excretory pore anterior to the base of metacorpus, post-uterine sac 54.8 (50-60) μm long, and an elongate-conoid tail with a shallow depression on the dorsal side ending in a sharply pointed tip. Males have body length of 467 and 592 μm, spicules 15 and 16 μm long with a prominent triangular-shaped rostrum, developed condylus and no cucullus. Seinura hyrcania n. sp. is characterized by having an offset lip region, stylet 21.5 (18-24) μm long, three incisures in the lateral fields, excretory pore anterior to the base of the metacorpus, post-uterine sac 19 (12-25) μm long and conical tail ending in a filiform terminus. Morphological and morphometric diagnostic characters together with molecular data based on partial sequences of SSU and LSU of the ribosomal DNA gene are provided for these new species.

Identification of nematode species by using conventional methods requires fixation of the isolated material and a suitable preparation for further analyses. Tentative identification using microscopic methods should also be performed prior to initiating molecular studies. In the literature, various methods are described for the preparation of nematodes from the genus Aphelenchoides for identification and microscopic studies. The most commonly used fixatives are formalin (Timm 1969; Szczygieł & Cid del Prado Vera 1981, Crozzoli et al. 2008, Khan et al. 2008), FAA (Wasilewska 1969; Vovlas et al. 2005, Khan et al. 2007) and TAF (Hooper 1958, Chizhov et al. 2006, Jagdale & Grewal 2006).

The Kamona strain of the nematode Deladenus siricidicola has been extensively used as a biological control agent against invasive Sirex noctilio woodwasps in the Southern Hemisphere, where it sterilizes female hosts. In North America, a non-sterilizing (NS) strain of D. siricidicola, thought to have been introduced with S. noctilio, is commonly found parasitizing this invasive woodwasp. Species of Deladenus that parasitize Sirex have a parasitic form, as well as a mycophagous form. The mycophagous form feeds on Sirex fungal symbionts in the genus Amylostereum. The goal of this study was to compare reproduction of NS and Kamona D. siricidicola when feeding on four isolates of Amylostereum areolatum (three introduced and one native in North America) and one native strain of Amylostereum chailletii isolated from Sirex nigricornis. Mycophagous forms of the two D. siricidicola strains displayed relatively similar production of offspring when feeding on most of the A. areolatum found associated with S. noctilio in this continent, except for strain BD on which NS produced more offspring than the biological control strain Kamona. Growth of both nematodes was greater on the introduced versus the native A. areolatum isolates., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Few Aphelenchoides spp. are facultative plant-parasites (foliar and bulb nematodes); three of them are well known in agricultural systems, namely Aphelenchoides besseyi, A. fragariae and A. ritzemabosi. Ten other plant-parasitic species, A. arachidis, A. bicaudatus, A. blastophthorus, A. dalianensis, A. ensete, A. nechaleos, A. paranechaleos, A. saprophilus, A. sphaerocephalus and A. subtenuis, have been reported from a limited number of plant species. We compiled a new database of the associated plants for these thirteen species, a comprehensive list that includes 1104 reports from 126 botanical families. A. besseyi, A. fragariae and A. ritzemabosi represent 94% of the reports, circa 83% and 16% of the total reports correspond to flowering plants and ferns, respectively, with three records on conifers and two from other botanical groups also listed. Most plant-parasitic Aphelenchoides show a remarkably broad diversity of associated plants. Most species appear to have no specific plant hosts (i.e. are generalists). The broad host ranges of these species and absence of more intimate interactions with the associated plants highlights the primitive mode of parasitism in Aphelenchoides species, making them potentially interesting in the study of the evolution of plant parasitism. Even though the compiled list of associated plants is long, it probably only represents a fraction of the potential range. The complete compilation has been uploaded to http://nematodes.myspecies.info/.

Laimaphalenchus hyrcanus n. sp., from bark samples of cypress (Cupressus sp.) and jujube (Ziziphus jujube Miller) from Gorgan, Golestan province in northern Iran, is described and illustrated based on morphological and molecular characters. The new species is characterized by females with 670-870 μm body length, slender stylet of 11-12 µm, six-lobed cephalic region not divided by ribs, three incisures in the lateral field with slightly areolated outer lines in the posterior region, cuticular vulval flap, males with two pairs of caudal papillae, and subcylindrical tail in both sexes ending in a single stalk-like terminus with four pedunculate tubercles bearing 9-10 finger-like projections. The new species most closely resembles L. persicus and L. penardi but it can be differentiated by its having three incisures in the lateral field and two pairs of caudal papillae in males. Molecular analysis of the D2/D3 region of the 28S ribosomal RNA gene indicates a sister-taxon relationship with L. penardi (Steiner, 1914) Filipjev & Schuurmans Stekhoven, 1941 and L. deconincki Elmiligy & Geraert, 1972.

Three new species of Fergusobia, respectively collected from shoot bud galls on narrow-leaved Melaleuca spp. in Australia, are described. Fergusobia armillarisae n. sp. Davies is characterised by the combination of an arcuate to open C-shaped parthenogenetic female with an extensile uterus and a short, conoid tail, an arcuate infective female with a broadly rounded tail tip, and an arcuate male with an angular spicule and bursa arising at 50-80% of body length. Fergusobia decorae n. sp. Davies has an arcuate parthenogenetic female with a non-extensile uterus and a broadly conoid tail, an arcuate infective female with most curvature behind the vulva and a short tail with a broadly rounded tip, and an arcuate male with an arcuate spicule and bursa arising at 40-50% of body length. Fergusobia linariifoliae n. sp. Davies is characterised by the combination of an arcuate parthenogenetic female with an extensile uterus and a short, conoid tail with a bluntly rounded tip, a barely arcuate infective female with a broadly rounded tail tip, and an arcuate male with an angular spicule and bursa arising at 40-50% of body length. Earlier molecular analyses inferred from DNA sequencing of 28S rDNA D2/D3 domains and a portion of mitochondrial DNA cytochrome oxidase subunit I (mtCOI) are further discussed.

Two new species of Fergusobia, collected from 'rosette' shoot bud galls on Melaleuca quinquenervia, and from leaf, stem, leaf and flower bud galls on Syzygium luehmannii, both from the Cairns region of Queensland, Australia, are described. Fergusobia rosettae Davies n. sp. is characterised by the combination of a small, arcuate parthenogenetic female having a short conoid tail with a bluntly rounded tip, an arcuate, relatively slender, infective female with an almost hemispherical tail tip, and arcuate males with arcuate to angular (not heavily sclerotised) spicules and leptoderan bursa arising at 40-50% of body length from tail tip. Fergusobia tolgaensis Davies n. sp. is characterised by the combination of a small open C-shaped parthenogenetic female with a broadly conoid tail, an arcuate infective female with a broadly rounded tail tip, and arcuate males with angular (not heavily sclerotised) spicules and short to mid-length leptoderan bursa. These two species of nematodes are associated with fly larvae that have dorsal shields comprising bars of raised cuticular ridges and spicules, similar to that of fly larvae from the M. leucadendra species group. The shield morphologies of these fly larvae and their possible genetic relationships are discussed. Possible evolutionary relationships of the Fergusobia nematodes from these galls are discussed, considering their morphology, DNA sequences, and the relationships of the associated Fergusonina flies and host plants.

Fergusobia tumifaciens (Currie 1937) Wachek 1955, the type species for the genus Fergusobia, is re-described from specimens collected from 'leafy' leaf bud galls on Eucalyptus bridgesiana near Albury in New South Wales, Australia. It is morphologically characterized by the combination of an open C-shaped parthenogenetic female with a small broadly conoid tail, a C-shaped infective female with a bluntly rounded tail tip, and an arcuate to J-shaped male with angular spicules, not heavily sclerotised, and short to mid-length peloderan bursa. Two new species of Fergusobia, collected from 'leafy' leaf bud galls on, respectively, Eucalyptus planchoniana in Queensland, and E. viminalis in South Australia, Australia, are described. Fergusobia planchonianae Davies n. sp. is characterised by the combination of a C-shaped parthenogenetic female with a conoid tail, an arcuate infective female with an hemispherical tail tip, and an almost straight to arcuate to C-shaped male with an angular spicule, a long peloderan bursa and a narrow tail. Fergusobia viminalisae Davies n. sp. is characterised by the combination of an open C-shaped parthenogenetic female with a broadly conoid tail, a C-shaped infective female with a bluntly rounded tail tip, and an arcuate to J-shaped male with an angular (not heavily sclerotised) spicule and short to mid-length peloderan bursa. The shield morphologies of the fly larvae associated with the 'leafy' leaf bud galls and their possible relationships are outlined. Possible evolutionary relationships of the Fergusobia nematodes from these galls are discussed, considering their morphology, DNA sequences, and the relationships of the associated Fergusonina flies and host plants.

Collection data and biological information is presented on the Fergusobia (Nematoda: Neotylenchidae)/ Fergusonina (Diptera: Fergusoninidae) mutualism inducing galls on Angophora in Australia. Three species and two morphospecies have been recognised. Fergusobia colbrani Davies n. sp. is described from soft spheroid leaf galls on Angophora floribunda. It is characterised by a combination of morphological characters including a small C-shaped parthenogenetic female with a short broadly conoid tail, an arcuate infective female with an almost hemispherical tail tip, and an arcuate to barely J-shaped male with an angular spicule having a notched tip and mid-length leptoderan bursa. A key to the species and morphospecies of nematodes collected from Angophora is presented. Possible relationships of these organisms are discussed based on evidence from the nematode morphology, gall forms, and the morphology of the dorsal shield of the associated Fergusonina fly larvae.

Morphological and morphometric analyses were conducted on the genus Paratylenchus, based on documented data in the literature and characters of Iranian populations of twelve species, including P. arculatus, P. colinus, P. conicephalus, P. coronatus, P. neoamblycephalus, P. neoprojectus, P. obtusicaudatus, P. paraperaticus, P. similis, P. straeleni, P. variabilis and P. veruculatus. Moreover, males of P. obtusicaudatus are described for the first time and additional data provided for males of P. paraperaticus. The variability of the most important taxonomic characters for species identification in the genus was estimated by calculation of coefficient of variation (CV) for 49 species and the ratio of extremes in the range of measured females (max/min ratios) for 82 species of the genus, based on 66 and 170 populations, respectively. A list of 117 valid species of Paratylenchus is provided, along with a dichotomous identification key and a diagnostic compendium.

Esteya vermicola, an endoparasitic fungus of Bursaphelenchus xylophilus, the pinewood nematode (PWN), exhibits great potential as a biological control agent against this nematode. E. vermicola produces blastospores in liquid media and aerial conidia on solid media. The agent was mass-produced using two kinds of culture media: S (50 % wheat bran and 50 % pine wood powder), L (0.5 g wheat bran and 0.5 g pinewood powder in 200 ml of potato dextrose broth), and two controls: SC (potato dextrose agar), LC (potato dextrose broth). Yields, multiple stress tolerance, storage life, new generation conidial number, and PWN mortality rates of the spores were measured in each of these four media and compared. The spore yields, new generation conidial number, and nematode mortality rates of blastospores were higher than those of conidia. Nevertheless, the conidia had a higher germination rate than the blastospores during the storage process and multiple stress treatments. Considering the number of spores surviving from the process of the storage and multiple stress treatments per unit of mass media, the blastospores from L survived most. Comprehensive analysis indicates that the L culture medium is the most optimal medium for mass production relatively.

The pinewood nematode Bursaphelenchus xylophilus has caused severe damage to pine forests in large parts of the world [1-4]. Dispersal of this plant-parasitic nematode occurs when the nematode develops into the dispersal fourth larval stage (LIV) upon encountering its insect vector, the Monochamus pine sawyer beetle, inside an infected pine tree [5-9]. Here, we show that LIV formation in B. xylophilus is induced by C16 and C18 fatty acid ethyl esters (FAEEs), which are produced abundantly on the body surface of the vector beetle specifically during the late development pupal, emerging adult, and newly eclosed adult stages. The LIV can then enter the tracheal system of the adult beetle for dispersal to a new pine tree. Treatment of B. xylophilus with long-chain FAEEs, or the PI3 kinase inhibitor LY294002, promotes LIV formation, while Δ7-dafachronic acid blocks the effects of these chemicals, suggesting a conserved role for the insulin/IGF-1 and DAF-12 pathways in LIV formation. Our work provides a mechanism by which LIV formation in B. xylophilus is specifically coordinated with the life cycle of its vector beetle. Knowledge of the chemical signals that control the LIV developmental decision could be used to interfere with the dispersal of this plant-parasitic nematode., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The nematode family Aphelenchoididae (Rhabditida: Tylenchomorpha) includes species with various feeding habitats. Bursaphelenchus, a member of the family, has for a long time been considered as a home for plant parasitic or mycophagous species (or both). However, recent intensive biological studies on the family revealed that the genus contains several insect parasitic species. Dauer juveniles of Bursaphelenchus doui were isolated from Acalolepta fraudatrix during a field study of longhorn beetle-Bursaphelenchus nematode associations. Two different insect-associated forms, an "entomoparasitic adult form" and a regular dauer juvenile, were isolated from a single individual beetle in a subsequent laboratory investigation of the B. doui-A. fraudatrix relationship. Thus these 2 distinct, insect-associated forms were confirmed to occur simultaneously. The entomoparasitic form is morphologically similar to that of Bursaphelenchus luxuriosae, with a dome-shaped head and vacuole-like spots assumed to be an internal structure of sensory organ, a stylet, a metacorpus (median bulb), and a moderately-developed and seemingly fully functional reproductive system. It is distinguishable from B. luxuriosae based on male spicule morphology and female tail morphology. A degenerate ingestive-digestive system distinguishes the entomoparasitic form from the propagative form and, unlike dauer juveniles, it has a moderately-developed reproductive system. The presence of this characteristic parasitic adult form is known only in these 2 Bursaphelenchus species. However, these 2 species did not form a clear monophyletic clade within the Bursaphelenchus xylophilus group and, thus, this characteristic parasitic form may occur independently in each species.

Pratylenchus ekrami from maize (Zea mays) roots in Shenyang and luffa (Luffa cylindrica) roots in Dalian, China, are described in this paper. Nematodes from the two areas were identified consistently, and were characterized by a heavy cephalic sclerotization, extending posteriorly up to two body annuli, stylet 11-13 μm long, elongating conoid tail, and becoming thinner from vulva. Males were not found. Pratylenchus ekrami is close to Pratylenchus vulnus, but the most critical characteristics between the two species were the number of lip annuli, stylet size, and shape of stylet knobs. This species is the first reported in China., Pratylenchus ekrami from maize ( Zea mays ) roots in Shenyang and luffa ( Luffa cylindrica ) roots in Dalian, China, are described in this paper. Nematodes from the two areas were identified consistently, and were characterized by a heavy cephalic sclerotization, extending posteriorly up to two body annuli, stylet 11-13 μm long, elongating conoid tail, and becoming thinner from vulva. Males were not found. Pratylenchus ekrami is close to Pratylenchus vulnus , but the most critical characteristics between the two species were the number of lip annuli, stylet size, and shape of stylet knobs. This species is the first reported in China.

Background: Species of Cephalotaxus (the plum yews) produce nematotoxic compounds of unknown identity. Consequently, bioassay-guided fractionation was employed to identify the compound(s) in Cephalotaxus fortunei twigs and leaves with activity against plant-parasitic nematodes., Results: A crude alkaloid extract, particularly drupacine, was responsible for much of the nematotoxicity. The ED50 of drupacine for Bursaphelenchus xylophilus was 27.1 µg mL⁻¹, and for Meloidogyne incognita it was 76.3 µg mL⁻¹. Immersion of M. incognita eggs in 1.0 mg mL⁻¹ crude alkaloid extract (the highest tested concentration) reduced hatch by 36%; immersion of second-stage juveniles (J2) resulted in 72-98% immobility. Crude alkaloid extract and drupacine suppressed protease activity in extracts of the microbivorous nematode Panagrellus redivivus by 50% and 80%, respectively. Application of 0.02-0.5 mg mL⁻¹ crude alkaloid extract to soil with M. incognita inoculum did not significantly reduce pepper plant shoot length or weight, compared with nematode-inoculated, water-treated controls, but the number of eggs and J2 per root system respectively decreased by 69% and 73% at 0.5 mg mL⁻¹., Conclusion: Drupacine and a crude alkaloid extract suppress nematode hatch, activity of mixed life stages, and population numbers on plant roots. This is the first demonstration of nematotoxicity of crude Cephalotaxus alkaloids and drupacine., (© 2013 Society of Chemical Industry.)

After devastating vast areas of pine forests in Asian countries, the pine wilt disease spread into European forests in 1999 and is causing worldwide concern. This disease involves very complicated interactions between a pathogenic nematode, its vector beetle, host pine species, and fungi in dead hosts. Pathogenicity of the pine wood nematode is determined not only by its physical and chemical traits but also by its behavioral traits. Most life history traits of the pine wood nematode, such as its phoretic relationship with vector beetles, seem to be more effective in virulent than in avirulent isolates or species. As the pathogenicity determinants, secreted enzymes, and surface coat proteins are very important, they have therefore been studied intensively. The mechanism of quick death of a large pine tree as a result of infection by a tiny nematode could be ascribed to the dysfunction of the water-conducting system caused by the death of parenchyma cells, which must have originally evolved as an inherent resistant system.

Fifty-two fungal isolates obtained from seawater were screened for anti-nematode activity. One isolate, an Acremonium sp., exhibited the highest activity against the pinewood nematode, Bursaphelenchus xylophilus. When it was grown at pH 7.0 and 24-26 °C, nematicidal metabolites were produced. These were heat-stable, water-soluble molecules with MWs <6 kDa. Nematicidal activity was highest at neutral pH.

A new record species Heterodera ripae was found in the rhizosphere soil of nettle (Urtica sp.) on the bank of the Mangniu River in Shenyang City, Liaoning Province, China. The new record species can be characterized by vulval cone bifenestrate with circular or subcircular semifenestrae, vulval slit length averaging 34.2 μm, vulval bridge mostly broad, underbridge weak, bullae absent; the second-stage larvae stylet knobs rather wide and slightly projecting anteriorly, tail length averaging 43.7 μm and hyaline region length averaging 22.3 μm.

Attraction of the Bursaphelenchus xylophilus nematode toward 18 volatiles of Pinus species was evaluated by a Petri-dish bioassay under laboratory conditions to develop a rapid diagnostic kit. Among these compounds, α-pinene, β-pinene, and camphor showed significantly higher attractiveness to B. xylophilus in both the reproductive and dispersal stages, whereas these compounds were not active against Bursaphelenchus mucronatus . A trap tube was developed as a diagnostic kit, which consisted of a tube filled with 0.8% agar and a matrix impregnated with an attractant: α-pinene, β-pinene, or camphor. All tested compounds attracted a significantly higher number of B. xylophilus than that in the control treatment. No significant difference was observed among attractants. The cotton-ball matrix was significantly more effective than the filter-paper matrix for attracting B. xylophilus in the artificial pupal chamber bioassay. In a bioassay with pine wood nematode (PWN)-infected pine tree logs, B. xylophilus was initially attracted after an 8 h trap period and the number of B. xylophilus increased with time. The trap tube using camphor and the cotton-ball matrix were most effective for attracting B. xylophilus . The semiochemical-based tube-trapping method is simple to use, requires minimal labor, and is economical and effective for detecting B. xylophilus living in host pine trees during field sampling.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Postembryonic development of Paraphelenchus myceliophthorus has been studied. The first molt takes place inside the egg-shell; outside the latter 3 molts were revealed, dividing 2nd, 3rd, 4th stage juveniles, and the adults. Descriptions of morphology of all juvenile stages were given, as well as morphology of all molting juveniles and adults. Stages of development differ in the genital primordium structure and in body length. In juveniles of 3rd and 4th stage the sex of juvenile can be distinguished--male juveniles have cloacal primordium, whereas female juveniles have vulval primordium. In male 3rd stage juveniles the large germinal cell is situated in the posterior part of the genital primordium, whereas female 3rd stage juvenile has the large germinal cell in the anterior part of the genital primordium. Adults differ from juveniles in the presence of copulative structures--vulva in adult female and spicules in male. The adult male has caudal alae (bursa) in the form of lateral cutucular folds and 4 pairs of caudal papillae, one preanal pair at mid-spicule level and 3 postanal pairs--p1 situated just posterior to cloacal opening, p2 at mid tail, p3 and p4 on the tail terminus. An unpaired papilla is also present just anterior to the cloacal opening. Gubernaculum is present. The tracery inner tail framework supporting the spicules has been described. In females 2 pairs of tail tip tuberculi are present, and 2 ventral ones are distinctly longer than 2 dorsal ones. These tuberculi correspond to p3 and p4 pairs of papillae in male and are homologous to tail tip papillae of the Aphelenchus females, which earlier were considered to be phasmids. These 4 tuberculi are joined with 4 one-celled glands of tail terminus.

Postembryonic development of Bursaphelenchus mucronatus has been studied. First molt takes place within the egg-shell, and three subsequent molts after hatching. Structure of all juvenile stages are described, as well as molting juveniles and adults. Juvenile stages differ in genital primordium structure, copulatory structures primordial and the body length. In juveniles of 3rd and 4th stage the sex of juvenile was distinguished: male juveniles have cloacal primordium, whereas female juveniles have vulval primordium. Adults differ from juveniles in presence of vulva in females and spicules in males. The key to identify juvenile stages and their sex is given. Male has the terminal bursal flap and 3 pairs of iail papillae: p1 anadanal; p2 and p3 at the bursal flap base: p2 close to mid-line of the ventral body surface, p3 located laterally to p2 at the same level. Unpaired papilla is present on the upper cloacal lip, on the ventral mid-line.

A putative "entomoparasitic adult form" of Bursaphelenchus luxuriosae was found in the tracheal system and body cavity of its cerambycid vector beetle, Acalolepta luxuriosa. Morphologically, this form is intermediate between the usual mycophagous adult and the phoretic dauer juvenile, i.e., it shares the primary and secondary reproductive features with the mycophagous phase but shares a degenerate digestive tract with JIV dauer juveniles. In addition, the "parasitic form" has specialized characters relative to the mycophagous phase analogous to dicyclic Hexatylina, e.g., vacuole-like dots (assumed to be sensory organs) at the anterior end, a very long vulval flap, conical female tail, and elongated male spicules. The presence of insect-parasitic juveniles has been reported in several Bursaphelenchus species, but this is the first confirmed report of a putative "entomoparasitic adult form" in the genus. Thus, the "parasitic adult form" of B. luxuriosae is hypothesized to be an autapomorphic character of this species in the genus. The physiological impact of the parasitism on the host beetle is assumed to be weak because no clear symptoms were observed in the infested beetles. Also, no nematode eggs or propagative juveniles were observed in the beetle host, suggesting that more research is needed to confirm the nature of the association (parasitic vs. endophoretic).

RNA interference (RNAi) was performed on several essential genes in the pinewood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. Double-stranded RNA (dsRNA) was delivered to larvae or adult worms by soaking, electroporation, or microinjection. Soaking and electroporation of L2-L3 stage worms in solutions containing dsRNA for essential genes induced over 25% lethality after 5 days, and gene-specific phenotypes were observed. This lethality agreed with significant reductions of the targeted transcripts, as assayed by reverse-transcription coupled with real time PCR. Microinjection was the most efficient route as measured by the hatching rate of F1 embryos, which was reduced by 46%. When adult worms were soaked in dsRNA, lethality was induced in the F1 larvae, revealing the persistence of knockdown phenotypes. The penetrance of the RNAi phenotypes for essential genes was relatively low but consistent, indicating that RNAi should be useful for studying the in vivo functions of B. xylophilus gene products.

For obligate plant-parasitic nematodes, cryopreservation has advantages over the usual preservation methods on whole plants or axenic culture systems, because the latter two are labourious and time and space consuming. In addition, cross contamination among different isolates can occur easily. Moreover, specific genetic studies require maintenance of the original population. The nematode under investigation, Radopholus similis, is a plant-parasitic nematode from the humid tropics. Therefore, any treatment at low temperatures is likely to add extra stress to the nematode, making the development of a cryopreservation protocol extremely difficult. In this paper, we describe experiments to achieve a successful cryopreservation protocol for the tropical nematode R. similis using vitrification solution-based methods based on a well defined mixture of cryoprotectants in combination with ultra-rapid cooling and thawing rates. A two-step treatment was used consisting of an incubation in glycerol followed by the application of a vitrifying mixture of methanol, glycerol and glucose. After cryopreservation, the pathogenicity of the nematodes was not altered, since they could infect and reproduce on carrot discs after recovery in the Ringer solution. The cryopreservation method described can be used for routine cryopreservation of R. similis lines from different origins.

Knowledge of rRNA structure is increasingly important to assist phylogenetic analysis through reconstructing optimal alignment, utilizing molecule features as an additional source of data and refining appropriate models of evolution of the molecule. We describe a procedure of optimization for alignment and a new coding method for nucleotide sequence data using secondary structure models of the D2 and D3 expansion fragments of the LSU-rRNA gene reconstructed for fifteen nematode species of the agriculturally important and diverse family Hoplolaimidae, order Tylenchida. Using secondary structure information we converted the original sequence data into twenty-eight symbol codes and submitted the transformed data to maximum parsimony analysis. We also applied the original sequence data set for Bayesian inference. This used the doublet model with sixteen states of nucleotide doublets for the stem region and the standard model of DNA substitution with four nucleotide states for loops and bulges. By this approach, we demonstrate that using structural information for phylogenetic analyses led to trees with lower resolved relationships between clades and likely eliminated some artefactual support for misinterpreted relationships, such as paraphyly of Helicotylenchus or Rotylenchus. This study as well as future phylogenetic analyses is herein supported by the development of an on-line database, NEMrRNA, for rRNA molecules in a structural format for nematodes. We also have developed a new computer program, RNAstat, for calculation of nucleotide statistics designed and proposed for phylogenetic studies.

Background: Heterodera glycines (soybean cyst nematode [SCN]), the major pathogen of Glycine max (soybean), undergoes muscle degradation (sarcopenia) as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc) family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87 (calponin) during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia., Results: We developed a bioinformatics database that compares expressed sequence tag (est) and genomic data of C. elegans and H. glycines (CeHg database). We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin), unc-89, unc-15 (paromyosin), unc-27 (troponin I), unc-54 (myosin), and the potassium channel unc-110 (twk-18). Less reduction is observed for the focal adhesion complex gene Hg-unc-97., Conclusion: The CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.

Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.

A study was done on the taxonomy and morphology of plant parasitic nematodes (Tylenchida) found in Sennar State (Sudan). Sixty samples of different crops were collected in the sugarcane area. Thirty samples originated from soil around the roots of Saccharum officinarum (sugarcane) from different ratoons and thirty samples were collected from other crops (Mangifera indica; Citrus limon; Citrus aurantifolia; Citrus paradisi; Citrus sinensis, Phoenix dactylifera, Musa sapentium; Cassia italica, Capsicum annuum, Sorghum bicolor, Sorghum sudanensis, Gossypium barbadense, Ficus nitida, Khaya senegalensis, Eucalyptus microtheca, Acacia nilotica, Acacia seyal, Azardichta indica, Cajanus cajana, Caltropsis spp. and Liguster ovalifolium). Seven species belonging to seven different genera of Tylenchida were identified: Paratrophurus lobatus, Scutellonema clathricaudatum, Hoplolaimus aegypti and Filenchus cylindricus. Helicotylenchus plumariae, Pratylenchus thornei and Malenchus andrassyi are new records for Sudan. These seven species were compared with the descriptions given in the literature and differences and variations were discussed. Additional morphological data were described by means of SEM microscopy.