Bacterial Communities and Virulence Associated with Pine Wood Nematode Bursaphelenchus xylophilus from Different Pinus spp.

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is a destructive threat to pine forests. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. This study investigated variation in bacterial communities and the virulence of surface-sterilized B. xylophilus from different Pinus spp. The predominant culturable bacteria of nematodes from different pines were Stenotrophomonas and Pseudomonas. Biolog EcoPlate analysis showed that metabolic diversity of bacteria in B. xylophilus from P. massoniana was the highest, followed by P. thunbergii and P. densiflora. High-throughput sequencing analysis indicated that bacterial diversity and community structure in nematodes from the different pine species varied, and the dominant bacteria were Stenotrophomonas and Elizabethkingia. The virulence determination of B. xylophilus showed that the nematodes from P. massoniana had the greatest virulence, followed by the nematodes from P. thunbergii and P. densiflora. After the nematodes were inoculated onto P. thunbergii, the relative abundance of the predominant bacteria changed greatly, and some new bacterial species emerged. Meanwhile, the virulence of all the nematode isolates increased after passage through P. thunbergii. These inferred that some bacteria associated with B. xylophilus isolated from different pine species might be helpful to adjust the PWN's parasitic adaptability.

A Rapid Diagnostic for Detection of Aphelenchoides besseyi and A. fujianensis Based on Real-Time PCR.

Aphelenchoides besseyi and A. fujianensis have been frequently found in mixed populations associated with forage grass seed in Brazil. The morphological similarity between both species has previously led A. fujianensis to be erroneously identified as A. besseyi. A. besseyi is a quarantine pest in many countries that import Brazilian forage seed; however, there is no current evidence suggesting that A. fujianensis is a plant-parasitic species. Two real-time polymerase chain reaction (qPCR) diagnostics were developed to detect each species and an operational envelope was established. A set of primers and hydrolysis probes for each species was designed targeting the large subunit (LSU) region. To assess their specificity, primers and probes sets were tested with samples of nontarget Aphelenchoides and Paraphelenchus sp. also frequently associated with forage seed. Experiments using dilutions of purified plasmid standards underpinned the sensitivity of the qPCR assays, which detected as few as 10 copies of target nematode ribosomal DNA. Thus, the developed diagnostics were sufficiently sensitive to detect DNA extracted from a fragment of a single target nematode. There was a positive correlation between copy number of the target species and nematode abundance, suggesting the potential of this method for quantification. Evidence of intra-individual variability among cloned sequences of the LSU region in a single A. besseyi population is also reported.

Molecular and morphological characterization of Paurodontoides siddiqii n. sp. (Nematoda: Hexatylina, Sphaerulariidae) associated with bark samples of Pinus eldarica from western Iran.

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).

Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus, and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine endophyte with antagonistic effects.

The aim of this study is to understand the biological role of Serratia quinivorans BXF1, a bacterium commonly found associated with Bursaphelenchus xylophilus, the plant parasitic nematode responsible for pine wilt disease. Therefore, we studied strain BXF1 effect in pine wilt disease. We found that strain BXF1 promoted in vitro nematode reproduction. Moreover, the presence of bacteria led to the absence of nematode chitinase gene (Bxcht-1) expression, suggesting an effect for bacterial chitinase in nematode reproduction. Nevertheless, strain BXF1 was unable to colonize the nematode interior, bind to its cuticle with high affinity or protect the nematode from xenobiotic stress. Interestingly, strain BXF1 was able to promote tomato and pine plant-growth, as well as to colonize its interior, thus, acting like a plant-growth promoting endophyte. Consequently, strain BXF1 failed to induce wilting symptoms when inoculated in pine shoot artificial incisions. This bacterium also presented strong antagonistic activities against fungi and bacteria isolated from Pinus pinaster. Our results suggest that B. xylophilus does not possess a strict symbiotic community capable of inducing pine wilt disease symptoms as previously hypothesized. We show that bacteria like BXF1, which possess plant-growth promoting and antagonistic effects, may be opportunistically associated with B. xylophilus, possibly acquired from the bacterial endophytic community of the host pine.

The use of real-time polymerase chain reaction with high resolution melting (real-time PCR-HRM) analysis for the detection and discrimination of nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus.

The real-time PCR-HRM analysis was developed for the detection and discrimination of the quarantine nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. A set of primers was designed to target the ITS region of rDNA. The results have demonstrated that this analysis is a valuable tool for differentiation of these both species.

Musca autumnalis (Diptera: Muscidae) and the host-specific nematode Paraiotonchium autumnale (Tylenchida: Iotonchiidae) in southern California.

Musca autumnalis DeGeer were collected in the summer and fall of 2011 and 2012 from a beef cattle herd in southern California Visual counts of Musca spp. on cattle faces were documented, and sweep net samples of face flies and other Diptera were also collected from cattle faces. Face flies dominated in the net collections, and 5-30 flies per face were common between early July and October 2011. Adult female M. autumnalis were dissected and examined for the presence of the host-specific nematode Paraiotonchium autumnale (Nickle). Overall, 67 of 887 (7.6%) adult face fly females were parasitized. M. autumnalis' ability to survive in such a southerly latitude (34 degree N) could reflect the rather temperate weather (coastal effects) and frequently irrigated pastures at the experimental site in southern California Preliminary observations suggest that face flies disappear from cattle during winter, despite generally favorable temperatures for fly activity. This is a possible indication of diapause and should be examined further.

Phylogenetic analysis of Deladenus nematodes parasitizing northeastern North American Sirex species.

The parasitic nematode Deladenus siricidicola is a biological control agent of the invasive woodwasp, Sirex noctilio. Since the discovery of S. noctilio in pine forests of northeastern North America in 2005, a biological control program involving the Kamona strain of D. siricidicola has been under consideration. However, North American pine forests have indigenous Sirex spp. and likely harbor a unique assemblage of associated nematodes. We assessed phylogenetic relationships among native Deladenus spp. in the northeastern United States and the Kamona strain of D. siricidicola. We sequenced three genes (mtCO1, LSU, and ITS) from nematodes extracted from parasitized Sirex spp. collected inside and outside of the range of S. noctilio. Our analyses suggest cospeciation between four North American Sirex spp. and their associated nematode parasites. Within two S. noctilio individuals we found nematodes that we hypothesize are normally associated with Sirex nigricornis. One individual of the native S. nigricornis contained Deladenus normally associated with S. noctilio. We discuss nematode-host fidelity in this system and the potential for non-target impacts of a biological control program using D. siricidicola against S. noctilio.

Molecular characterization of Portuguese populations of the pinewood nematode Bursaphelenchus xylophilus using cytochrome b and cellulase genes.

Bursaphelenchus xylophilus is the causal agent of pine wilt disease and a worldwide pest with high economic impact. Since its first diagnosis in Portugal in 1999, it has been subjected to quarantine measures with impact on forest health and ecosystem stability, significantly affecting international trade of wood products. The disease was detected in the north and centre of continental Portugal and, since 2008, the whole country has been considered an affected area. Recently, it was detected in Madeira Island. In order to avoid new outbreaks, it has become of major importance to understand the patterns of spread, introduction points and to characterize the new populations from continental Portugal and Madeira Island. Mitochondrial cytochrome b (cytb) and parasitic cellulase gene sequences were used to evaluate the genetic relationships among isolates that could indicate possible origins of the new outbreaks. Portuguese isolates were compared with isolates from USA, China, Japan and South Korea, in order to investigate possible infection pathways and disease spread patterns in Portugal. Phylogenetic trees based on both genes show that Portuguese isolates group with Asian isolates. Isolates from USA are in a separate position in both gene trees. However, the phylogenetic tree based on the cellulase gene sequences shows higher differentiation among Portuguese isolates than that of cytb. These results agree with those previously obtained using inter-simple sequence repeats (ISSR). This was the first study to use cytb and cellulase genes to characterize pinewood nematode (PWN) populations. This study suggests that cellulase is a better marker than cytb to study genetic diversity in B. xylophilus.

Rapid microscopic and molecular method Aphelenchoides species identification.

A. ritzemabosi and A. fragariae collected from Poland were compared with A. besseyi to check the difference between features for quick identification. Although 10 features were typed for comparison, it needs to be stressed that body length, tail length, PUS and the value 'a' are the most helpful to distinguish these species. Conditions of execution for quantitative molecular analyses were also qualified.

First case of fatal equine meningoencephalitis caused by Halicephalobus gingivalis in Mexico.

Aberrant nematode larval migration in the CNS of horses is rare but frequently fatal; one of the main etiological agents involved in this illness is Halicephalobus gingivalis. This soil nematode has been associated with several fatal equine meningoencephalitis reports worldwide; however, it had never been diagnosed in horses of Mexico. A 10 year-old Andalusian horse presented dysphagia, fever, weakness, prostration and ataxia; the patient expired during the medical attention. Post mortem examination was performed and no gross alterations were found. Histopathology revealed meningoencephalitis, vasculitis and intralesional adult nematodes, larvae and eggs compatible with Halicephalobus spp. A polymerase chain reaction (PCR) for the nuclear large subunit ribosomal RNA gene (LSU rDNA) of nematodes was performed from formalin-fixed and paraffin wax-embedded sections of brain. Posterior nucleotide sequence analysis of the amplified fragment identified the agent as H. gingivalis. To our knowledge, this is the first confirmed report of Halicephalobiasis in Mexico.

Point-of-care diagnostic (POCD) method for detecting Bursaphelenchus xylophilus in pinewood using recombinase polymerase amplification (RPA) with the portable optical isothermal device (POID).

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a causative agent of pine wilt disease (PWD). To date, although several molecular diagnostic methods have been developed, rapid on-site diagnostic tools for detecting PWN in pinewood are limited. In this study, a point of care diagnostic (POCD) method for detecting PWN in pinewood using recombinase polymerase amplification (RPA) assay was developed. This method comprises quick gDNA extraction buffer (DAP buffer) for the direct extraction of gDNA of PWN from pinewood and a battery-mounted portable optical isothermal device (POID) for the detection of PWD in the field. The RPA assay can distinguish between the PWN and its conspecies which exist in pinewood and can complete diagnostic procedures within 25 min in the field. Moreover, the RPA assay can detect PWN in old wood samples in both natural and stored conditions. The POCD-RPA assay to detect PWN will be useful for epidemiological investigations in the field as well as for quarantine processes in the wood trade.

A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification.

ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions.

[Dispersion of the nematodes belonging to the Bursaphelenchus xylophilus species group with saw timber in Russian Federation].

Four new records of Bursaphelenchus mucronatus in saw-timbers from the Asian part of Russia (Irkutsk Oblast and Krasnoyarsk Krai, larch, pine and spruce wood) intercepted in St. Petersburg, are described, measured and illustrated. The diagnosis of B. mucronatus is amended and its relationships within species group xylophilus, and especially its differences from a quarantine pests B. xylophilus, are given. The record of the B. mucronatus transition from Asiatic into European part of Russia suggests possible transcontinental way of the xylophilus group penetration with saw-timber. Pest risk analysis of B. xylophilus for the European part of Russia is discussed in scope of the global warming.

Comparative Transcriptome Analysis of Pinus densiflora Following Inoculation with Pathogenic (Bursaphelenchus xylophilus) or Non-pathogenic Nematodes (B. thailandae).

Pinus densiflora (Korean red pine) is a species of evergreen conifer that is distributed in Korea, Japan, and China, and of economic, scientific, and ecological importance. Korean red pines suffer from pine wilt disease (PWD) caused by Bursaphelenchus xylophilus, the pinewood nematode (PWN). To facilitate diagnosis and prevention of PWD, studies have been conducted on the PWN and its beetle vectors. However, transcriptional responses of P. densiflora to PWN have received less attention. Here, we inoculated Korean red pines with pathogenic B. xylophilus, or non-pathogenic B. thailandae, and collected cambium layers 4 weeks after inoculation for RNA sequencing analysis. We obtained 72,864 unigenes with an average length of 869 bp (N50 = 1,403) from a Trinity assembly, and identified 991 differentially expressed genes (DEGs). Biological processes related to phenylpropanoid biosynthesis, flavonoid biosynthesis, oxidation-reduction, and plant-type hypersensitive response were significantly enriched in DEGs found in trees inoculated with B. xylophilus. Several transcription factor families were found to be involved in the response to B. xylophilus inoculation. Our study provides the first evidence of transcriptomic differences in Korean red pines inoculated with B. xylophilus and B. thailandae, and might facilitate early diagnosis of PWD and selection of PWD-tolerant Korean red pines.

Nematode parasites of Duttaphrynus stomaticus (Lutken, 1864) (Amphibia: Anura) with description of a new species of Rhabdias Stiles and Hassall, 1905 (Nematoda: Rhabdiasidae) from Dehradun (Uttarakhand), India.

Rhabdias stomatica sp. nov. from the lungs of Duttaphrynus stomaticus (Lutken, 1864) from Dehradun, Uttarakhand, India is described and illustrated. Rhabdias stomatica sp. nov. is the 16th species described from the Oriental biogeographical region and the 8th species from India. The new species is differentiated from the closely related Oriental species in having 4 weakly developed lips, a trapezoidal shaped buccal cavity, different position of nerve ring and in the esophagus/body length ratio. In addition, to the new species found in the lungs, mature specimens of Aplectana macintoshii (Stewart, 1914) Travassos, 1931 and larvae representing two unidentified species of nematode were found in the large intestine of the D. stomaticus.

Molecular and morphological characterisation of Rhabdias picardiae Junker, Lhermitte-Vallarino et Bain, 2010 (Nematoda: Rhabdiasidae) from Delaland's River Frog, Amietia delalandii (Duméril et Bibron, 1841) (Amphibia: Pyxicephalidae) in South Africa.

Rhabdias picardiae previously known from the Guttural Toad, Sclerophrys gutturalis (Bufonidae) is described from a new host, Delaland's River Frog, Amietia delalandii (Pyxicephalidae). The species identification is confirmed by analysis of the COI gene sequence and morphological comparison with the original description. Morphological details of apical structures in adult parasitic R. picardiae, namely, the shape and position of lips, the number and position of external labial papillae, and the buccal capsule morphology are described using light and scanning electron microscopy. The buccal capsule width and length, the distance from anterior end to nerve ring and excretory pore, and ratios of distance to vulva to body length and body length to width were identified as less variable metrical characters in the studied sample of 30 specimens. Adult specimens demonstrated differences in the position of lips in relation to the oral opening and position of the buccal capsule in relation to the oesophagus depending on the size (age) of worms. The infective larvae of R. picardiae are described for the first time and characterised by two lateral alae consisting of two ridges, triangular pseudolabia with rounded tops, and presence of ornamentation on the rounded tail tip.

The infection of Harmonia axyridis by a parasitic nematode is mediated by entomopathogenic bacteria and triggers sex-specific host immune responses.

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.

Species discovery and diversity in Lobocriconema (Criconematidae: Nematoda) and related plant-parasitic nematodes from North American ecoregions.

There are many nematode species that, following formal description, are seldom mentioned again in the scientific literature. Lobocriconema thornei and L. incrassatum are two such species, described from North American forests, respectively 37 and 49 years ago. In the course of a 3-year nematode biodiversity survey of North American ecoregions, specimens resembling Lobocriconema species appeared in soil samples from both grassland and forested sites. Using a combination of molecular and morphological analyses, together with a set of species delimitation approaches, we have expanded the known range of these species, added to the species descriptions, and discovered a related group of species that form a monophyletic group with the two described species. In this study, 148 specimens potentially belonging to the genus Lobocriconema were isolated from soil, individually measured, digitally imaged, and DNA barcoded using a 721 bp region of cytochrome oxidase subunit 1 (COI). One-third of the specimens were also analyzed using amplified DNA from the 3' region of the small subunit ribosomal RNA gene (18SrDNA) and the adjacent first internal transcribed spacer (ITS1). Eighteen mitochondrial haplotype groups, falling into four major clades, were identified by well-supported nodes in Bayesian and maximum likelihood trees and recognized as distinct lineages by species delimitation metrics. Discriminant function analysis of a set of morphological characters indicated that the major clades in the dataset possessed a strong morphological signal that decreased in comparisons of haplotype groups within clades. Evidence of biogeographic and phylogeographic patterns was apparent in the dataset. COI haplotype diversity was high in the southern Appalachian Mountains and Gulf Coast states and lessened in northern temperate forests. Lobocriconema distribution suggests the existence of phylogeographic patterns associated with recolonization of formerly glaciated regions by eastern deciduous forest, but definitive glacial refugia for this group of plant parasitic nematodes have yet to be identified. Unlike agricultural pest species of plant-parasitic nematodes, there is little evidence of long-distance dispersal in Lobocriconema as revealed by haplotype distribution. Most haplotype groups were characterized by low levels of intragroup genetic variation and large genetic distances between haplotype groups. The localization of nematode haplotypes together with their characteristic plant communities could provide insight into the historical formation of these belowground biotic communities.

Transcriptional and morphological changes in the transition from mycetophagous to phytophagous phase in the plant-parasitic nematode Bursaphelenchus xylophilus.

Drastic physiological and morphological changes in parasites are crucial for the establishment of a successful infection. The nematode Bursaphelenchus xylophilus is the pathogenic agent of pine wilt disease, and little is known about the physiology and morphology in this nematode at the initial stage of infection. In this study, we devised an infection system using pine stem cuttings that allowed us to observe transcriptional and morphological changes in the host-infecting phytophagous phase. We found that 60 genes enriched in xenobiotic detoxification were up-regulated in two independent post-inoculation events, whereas down-regulation was observed in multiple members of collagen gene families. After 48 h of inoculation, the tails in some of the adult females exposed to the host changed in morphology. These results suggest that B. xylophilus may change its physiology and morphology to protect itself and to adapt to the host pine wood environment.

Description of Cryptaphelenchus iranicus n. sp. (Nematoda: Ektaphelenchinae) recovered from bark samples of Pinus nigra from Iran.

Cryptaphelenchus iranicus n. sp., recovered from bark and wood samples of a weakened Pinus nigra in Kermanshah Province, western Iran, is described. The new species has females with body length of 250-330 µm and males 230-275 µm long, lip region set-off from body contour, 7-8 µm long stylet with small basal swellings, excretory pore located at 1.5-2.0 body diam. posterior to median bulb, post-vulval uterine sac short, 7-10 µm long and conical female posterior body end (tail) ending to a pointed tip. Males of the new species have seven (1+2+2+2) caudal papillae and a short mucro at tail tip. Based on morphological characters, the new species is close to C. cirrus, C. latus and C. leptocaudus. Phylogenetic analyses using the D2/D3 fragment of 28S rDNA show the new species forming a clade with other Cryptaphelenchus species with maximal (1.00) Bayesian posterior probability (BPP) in Bayesian inference and 100% bootstrap value (BS) in the maximum likelihood method. The Cryptaphelenchus clade forms a monophyletic group with members of subfamily Ektaphelenchinae.