The Bursaphelenchus xylophilus candidate effector BxLip-3 targets the class I chitinases to suppress immunity in pine.

Lipase is involved in lipid hydrolysis, which is related to nematodes' energy reserves and stress resistance. However, the role of lipases in Bursaphelenchus xylophilus, a notorious plant-parasitic nematode responsible for severe damage to pine forest ecosystems, remains largely obscure. Here, we characterized a class III lipase as a candidate effector and named it BxLip-3. It was transcriptionally up-regulated in the parasitic stages of B. xylophilus and specifically expressed in the oesophageal gland cells and the intestine. In addition, BxLip-3 suppressed cell death triggered by the pathogen-associated molecular patterns PsXEG1 and BxCDP1 in Nicotiana benthamiana, and its Lipase-3 domain is essential for immunosuppression. Silencing of the BxLip-3 gene resulted in a delay in disease onset and increased the activity of antioxidant enzymes and the expression of pathogenesis-related (PR) genes. Plant chitinases are thought to be PR proteins involved in the defence system against pathogen attack. Using yeast two-hybrid and co-immunoprecipitation assays, we identified two class I chitinases in Pinus thunbergii, PtChia1-3 and PtChia1-4, as targets of BxLip-3. The expression of these two chitinases was up-regulated during B. xylophilus inoculation and inhibited by BxLip-3. Overall, this study illustrated that BxLip-3 is a crucial virulence factor that plays a critical role in the interaction between B. xylophilus and host pine.

Molecular characterization and functional analysis of glutathione S-transferase genes of pine wood nematode (Bursaphelenchus xylophilus) for avermectin.

Pine wilt disease (PWD), caused by Bursaphelenchus xylophilus (pine wood nematodes, PWNs), is a forest disease that seriously threatens the health of Pinus forestry. Glutathione S-transferases (GSTs) play important roles in xenobiotic metabolism, lipophilic compound transport, antioxidative stress reactions, anti-mutagenesis, and antitumor activity. The analysis and investigation of the specific functions of GSTs in the metabolism of toxic substances in nematodes are important for identifying potential target genes to control the spread and transmission of B. xylophilus. In this study, 51 Bx-GSTs were found in the genome of B. xylophilus. Two key Bx-gsts (Bx-gst12 and Bx-gst40) were analyzed when B. xylophilus was exposed to avermectin. The expression of Bx-gst12 and Bx-gst40 was significantly increased when B. xylophilus was exposed to 1.6 and 3.0 mg/mL avermectin solutions. Notably, combined silencing of both Bx-gst12 and Bx-gst40 did not further increase the mortality rates under avermectin exposure. Mortality rates were significantly increased in nematodes treated with dsRNA compared to control nematodes (p < 0.05) after RNAi. The feeding ability of nematodes was also significantly reduced after treatment with dsRNA. These results suggested that Bx-gsts are associated with the detoxification process and feeding behavior of B. xylophilus. Silencing Bx-gsts leads to increased susceptibility to nematicides and reduces the feeding ability of B. xylophilus. Therefore, Bx-gsts will be a new control target of PWNs in the future.

Development of a Droplet Digital PCR Assay for Detection and Quantification of Stubby Root Nematode, Paratrichodorus allius, in Soil.

The stubby root nematode Paratrichodorus allius is an important plant-parasitic nematode species within the Trichodoridae family. It can directly harm the plants by feeding on the roots or indirectly by transmitting Tobacco rattle virus. These nematodes are mostly diagnosed either by traditional microscopic methods or a polymerase chain reaction (PCR)-based method. Droplet digital PCR (ddPCR) is a novel PCR technique which is sensitive and precise in quantifying DNA templates of the test samples. In this study, we developed a ddPCR assay to detect and quantify P. allius in soil. The specificity and sensitivity of the assay was first determined using P. allius nematode DNA or DNA from sterilized soil artificially inoculated with P. allius, and the assay was used to quantify P. allius populations in field soils. The assay did not detect nematodes other than P. allius, thus showing high specificity. It was able to detect P. allius equivalent to a 0.01 and 0.02 portion of a single nematode in soil DNA and nematode DNA extracts, respectively. Highly linear relationships between DNA copy numbers from ddPCR and serial dilutions of known concentrations were observed with DNA from P. allius nematodes (R2 = 0.9842) and from artificially infested soil (R2 = 0.9464). The P. allius populations from field soils determined by ddPCR were highly correlated with traditional microscopic counts (R2 = 0.7963). To our knowledge, this is the first report of applying ddPCR to detect and quantify stubby root nematode in soil. The results of this study support the potentiality of a ddPCR assay as a new research tool in diagnostics of plant-parasitic nematodes.

Roles of Species-Specific Legumains in Pathogenicity of the Pinewood Nematode Bursaphelenchus xylophilus.

Peptidases are very important to parasites, which have central roles in parasite biology and pathogenesis. In this study, by comparative genome analysis, genome-wide peptidase diversities among plant-parasitic nematodes are estimated. We find that genes encoding cysteine peptidases in family C13 (legumain) are significantly abundant in pine wood nematodes Bursaphelenchus genomes, compared to those in other plant-parasitic nematodes. By phylogenetic analysis, a clade of B. xylophilus-specific legumain is identified. RT-qPCR detection shows that these genes are highly expressed at early stage during the nematode infection process. Utilizing transgene technology, cDNAs of three species-specific legumain were introduced into the Arabidopsis γvpe mutant. Functional complementation assay shows that these B. xylophilus legumains can fully complement the activity of Arabidopsis γVPE to mediate plant cell death triggered by the fungal toxin FB1. Secretory activities of these legumains are experimentally validated. By comparative transcriptome analysis, genes involved in plant cell death mediated by legumains are identified, which enrich in GO terms related to ubiquitin protein transferase activity in category molecular function, and response to stimuli in category biological process. Our results suggest that B. xylophilu-specific legumains have potential as effectors to be involved in nematode-plant interaction and can be related to host cell death.

A Bursaphelenchus xylophilus Effector, BxSCD3, Suppresses Plant Defense and Contributes to Virulence.

Bursaphelenchus xylophilus is the most economically important species of migratory plant-parasitic nematodes (PPNs) and causes severe damage to forestry in China. The successful infection of B. xylophilus relies on the secretion of a repertoire of effector proteins. The effectors, which suppress the host pine immune response, are key to the facilitation of B. xylophilus parasitism. An exhaustive list of candidate effectors of B. xylophilus was predicted, but not all have been identified and characterized. Here, an effector, named BxSCD3, has been implicated in the suppression of host immunity. BxSCD3 could suppress pathogen-associated molecular patterns (PAMPs) PsXEG1- and INF1-triggered cell death when it was secreted into the intracellular space in Nicotiana benthamiana. BxSCD3 was highly up-regulated in the early infection stages of B. xylophilus. BxSCD3 does not affect B. xylophilus reproduction, either at the mycophagous stage or the phytophagous stage, but it contributes to the virulence of B. xylophilus. Moreover, BxSCD3 significantly influenced the relative expression levels of defense-related (PR) genes PtPR-3 and PtPR-6 in Pinus thunbergii in the early infection stage. These results suggest that BxSCD3 is an important toxic factor and plays a key role in the interaction between B. xylophilus and host pine.

Transcriptomics and coexpression network profiling of the effects of levamisole hydrochloride on Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is one of the most dangerous forest pathogens in the world, causing devastating pine forest deaths with considerable economic losses. In this study, we investigated the B. xylophilus RNA sequence responses of two different concentrations of levamisole hydrochloride (LH). We observed that body-wall muscle twitching, paralysis and, ultimately, death. 2.5 mg/ml and 3.5 mg/ml LH have toxicological effects on B. xylophilus, with mortality increasing significantly with concentration (p < 0.05). RNA sequencing, differential gene expression analysis, and cluster analysis were performed, and 336, 384, 6 genes with significant variance in expression were identified. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the 12 intersecting genes revealed that these genes are mostly involved in metabolism of xenobiotics and have essential roles in drug sensitivity. Through the trend analysis of DEGs, it was divided into 8 modules, and the significant modules were selected to construct the co-expression network as the central genes of the drug metabolism-cytochrome P450 pathway (ko00982) and metabolism of xenobiotics by cytochrome P450 (ko00980). Eight highly related genes were identified, including cuticle collagen, cystathionine beta-synthase, endochitinase, pyruvate dehydrogenase E1 component subunit beta, aldehyde dehydrogenase, lipase, and zinc metalloproteinase. The expression levels of these genes were upregulated significantly at low concentrations and were significantly related to the resistance of B. xylophilus to LH. This study shows that B. xylophilus gene family expansions occurred in xenobiotic detoxification pathways through gene expression and potential horizontal correlated gene transfer with LH and helps to elucidate LH lethality and the evolutionary mechanisms underlying the adaptations of B. xylophilus to the environment. These results contributing to our understanding of B. xylophilus under LH and provide a data platform to providing a basis for its control.

Potential Molecular Mimicry Proteins Responsive to α-pinene in Bursaphelenchus xylophilus.

: Bursaphelenchus xylophilus is a nematode species that has damaged pine trees worldwide, but its pathogenesis has not been fully characterized. α-pinene helps protect host species during the early B. xylophilus infection and colonization stages. In this study, we identified potential molecular mimicry proteins based on a comparative transcriptomic analysis of B. xylophilus. The expression levels of three genes encoding secreted B. xylophilus proteins were influenced by α-pinene. We cloned one gene encoding a thaumatin-like protein, Bx-tlp-2 (accession number MK000287), and another gene encoding a cysteine proteinase inhibitor, Bx-cpi (accession number MK000288). Additionally, α-pinene appeared to induce Bx-tlp-1 expression, but had the opposite effect on Bx-cpi expression. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees revealed that the α-pinene content was consistent with the expression levels of Bx-tlp-1 (Bx-cpi) and Pm-tlp (Pm-cpi) over time. Thus, these genes likely have important roles contributing to the infection of pine species by B. xylophilus. The results of this study may be relevant for future investigations of the functions of Bx-tlp-1, Bx-tlp-2 and Bx-cpi, which may provide a point to explore the relationship between B. xylophilus and host pines.

The genome of the migratory nematode, Radopholus similis, reveals signatures of close association to the sedentary cyst nematodes.

Radopholus similis, commonly known as the burrowing nematode, is an important pest of myriad crops and ornamentals including banana (Musa spp.) and Citrus spp. In order to characterize the potential role of putative effectors encoded by R. similis genes we compared predicted proteins from a draft R. similis genome with other plant-parasitic nematodes in order to define the suite of excreted/secreted proteins that enable it to function as a parasite and to ascertain the phylogenetic position of R. similis in the Tylenchida order. Identification and analysis of candidate genes encoding for key plant cell-wall degrading enzymes including GH5 cellulases, PL3 pectate lyases and GH28 polygalactouranase revealed a pattern of occurrence similar to other PPNs, although with closest phylogenetic associations to the sedentary cyst nematodes. We also observed the absence of a suite of effectors essential for feeding site formation in the cyst nematodes. Clustering of various orthologous genes shared by R. similis with other nematodes showed higher overlap with the cyst nematodes than with the root-knot or other migratory endoparasitic nematodes. The data presented here support the hypothesis that R. similis is evolutionarily closer to the cyst nematodes, however, differences in the effector repertoire delineate ancient divergence of parasitism, probably as a consequence of niche specialization. These similarities and differences further underscore distinct evolutionary relationships during the evolution of parasitism in this group of nematodes.

Identification of a novel effector BxSapB3 that enhances the virulence of pine wood nematode Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pine wood nematode Bursaphelenchus xylophilus, leads to severe damage to pine forests in China. In our previous study, effectors secreted by this pathogen were shown to play roles in the different infection stages of pine wilt disease, and a series of candidate effectors were predicted by transcriptome sequencing. This study identified and characterized a novel effector, BxSapB3, which was among these candidate effectors. Agrobacterium-mediated transient expression was used to identify BxSapB3. BxSapB3 was secreted by B. xylophilus and found to be capable of inducing cell death in Nicotiana benthamiana. Quantitative real-time PCR (qRT-PCR) analysis revealed that BxSapB3 was upregulated in a highly virulent strain of B. xylophilus and expressed at lower levels in a weakly virulent strain at the early stages of infection. When BxSapB3 was silenced in B. xylophilus, the process of infection was delayed. These results indicate that BxSapB3 acts as an effector and contributes to virulence at the early stages of B. xylophilus infection.

Cathepsin L-like Cysteine Proteinase Genes Are Associated with the Development and Pathogenicity of Pine Wood Nematode, Bursaphelenchus xylophilus.

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-cpls 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-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls 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-cpls play multiple roles in the development and pathogenic processes of PWN.

An Effector, BxSapB1, Induces Cell Death and Contributes to Virulence in the Pine Wood Nematode Bursaphelenchus xylophilus.

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused serious damage to pine forests in China. Effectors secreted by phytonematodes play a role in host infection. We identified and characterized an effector, BxSapB1, based on the B. xylophilus transcriptome at the early stages of infection and the transient expression of proteins in Nicotiana benthamiana. BxSapB1 triggered cell death in N. benthamiana when secreted into the apoplast, and this effect was independent of N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1) and suppressor of BIR1-1 (NbSOBIR1). The signal peptide of BxSapB1 was proven to be functional in yeast using the yeast signal sequence trap system and BxSapB1 was strongly expressed in the subventral gland cells of B. xylophilus, as revealed by in-situ hybridization. In addition, based on local BLAST analysis, the BxSapB1 showed 100% identity to BUX.s00139.62, which was identified from the B. xylophilus secretome during Pinus thunbergii infection. BxSapB1 was upregulated in a highly virulent strain and downregulated in a weakly virulent strain of PWN at the early stages of infection. RNA interference assays showed that silencing BxSapB1 resulted in decreased expression of pathogenesis-related genes (PtPR-1b, PtPR-3, and PtPR-5) as well as delayed onset of symptoms in P. thunbergii infected by B. xylophilus. The combined data suggest that BxSapB1 can trigger cell death in N. benthamiana and that it contributes to the virulence in B. xylophilus during parasitic interaction.

STATAWAARS: a promoter motif associated with spatial expression in the major effector-producing tissues of the plant-parasitic nematode Bursaphelenchus xylophilus.

BACKGROUND: Plant-parasitic nematodes cause severe damage to a wide range of crop and forest species worldwide. The migratory endoparasitic nematode, Bursaphelenchus xylophilus, (pinewood nematode) is a quarantine pathogen that infects pine trees and has a hugely detrimental economic impact on the forestry industry. Under certain environmental conditions large areas of infected trees can be destroyed, leading to damage on an ecological scale. The interactions of B. xylophilus with plants are mediated by secreted effector proteins produced in the pharyngeal gland cells. Identification of effectors is important to understand mechanisms of parasitism and to develop new control measures for the pathogens. RESULTS: Using an approach pioneered in cyst nematodes, we have analysed the promoter regions of a small panel of previously validated pharyngeal gland cell effectors from B. xylophilus to identify an associated putative regulatory promoter motif: STATAWAARS. The presence of STATAWAARS in the promoter region of an uncharacterized gene is a predictor that the corresponding gene encodes a putatively secreted protein, consistent with effector function. Furthermore, we are able to experimentally validate that a subset of STATAWAARS-containing genes are specifically expressed in the pharyngeal glands. Finally, we independently validate the association of STATAWAARS with tissue-specific expression by directly sequencing the mRNA of pharyngeal gland cells. We combine a series of criteria, including STATAWAARS predictions and abundance in the gland cell transcriptome, to generate a comprehensive effector repertoire for B. xylophilus. The genes highlighted by this approach include many previously described effectors and a series of novel "pioneer" effectors. CONCLUSIONS: We provide a major scientific advance in the area of effector regulation. We identify a novel promoter motif (STATAWAARS) associated with expression in the pharyngeal gland cells. Our data, coupled with those from previous studies, suggest that lineage-specific promoter motifs are a theme of effector regulation in the phylum Nematoda.

Integrative taxonomy of root-knot nematodes reveals multiple independent origins of mitotic parthenogenesis.

During sampling of several Coffea arabica plantations in Tanzania severe root galling, caused by a root-knot nematode was observed. From pure cultures, morphology and morphometrics of juveniles and females matched perfectly with Meloidogyne africana, whereas morphology of the males matched identically with those of Meloidogyne decalineata. Based on their Cox1 sequence, however, the recovered juveniles, females and males were confirmed to belong to the same species, creating a taxonomic conundrum. Adding further to this puzzle, re-examination of M. oteifae type material showed insufficient morphological evidence to maintain its status as a separate species. Consequently, M. decalineata and M. oteifae are synonymized with M. africana, which is herewith redescribed based on results of light and scanning electron microscopy, ribosomal and mitochondrial DNA sequences, isozyme electrophoresis, along with bionomic and cytogenetic features. Multi-gene phylogenetic analysis placed M. africana outside of the three major clades, together with M. coffeicola, M. ichinohei and M. camelliae. This phylogenetic position was confirmed by several morphological features, including cellular structure of the spermatheca, egg mass position, perineal pattern and head shape. Moreover, M. africana was found to be a polyphagous species, demonstrating that "early-branching" Meloidogyne spp. are not as oligophagous as had previously been assumed. Cytogenetic information indicates M. africana (2n = 21) and M. ardenensis (2n = 51-54) to be a triploid mitotic parthenogenetic species, revealing at least four independent origins of mitotic parthenogenesis within the genus Meloidogyne. Furthermore, M. mali (n = 12) was found to reproduce by amphimixis, indicating that amphimictic species with a limited number of chromosomes are widespread in the genus, potentially reflecting the ancestral state of the genus. The wide variation in chromosome numbers and associated changes in reproduction modes indicate that cytogenetic evolution played a crucial role in the speciation of root-knot nematodes and plant-parasitic nematodes in general.

Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism.

Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the H. glycines genome and may have different roles during infection.

A new stem nematode, Ditylenchus oncogenus n. sp. (Nematoda: Tylenchida), parasitizing sowthistle from Adriatic coast dunes in southern Italy.

Morphological and molecular analyses of a stem nematode causing a severe disease on infected sowthistle (Sonchus bulbosus) plants, involving the formation of gall-like structures on infected leaves and stems, have led to the description of a new species named Ditylenchus oncogenus n. sp. Morphologically, the new species is characterized by a medium to large body size (all adults more than 1 mm in length); a delicate stylet (9.0-11.0 µm long) with minute, rounded knobs; a long post-vulval uterine sac (c. 65% of the vulva-anus distance); six incisures at the lateral fields and characteristic D. destructor-pattern of spicules (with pronounced ventral tumulus and anteriorly pointed, less sclerotized, cuticle parts present within the lamina). The results of molecular analysis of rRNA gene sequences, including the D2-D3 expansion regions of 28S rRNA, internal transcribed spacer (ITS) rRNA, partial 18S rRNA gene, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI), and the heat-shock protein 90 (hsp90) gene, support the new species status. The results of a host-suitability test indicated that the new species does not parasitize potato (Solanum tuberosum) tubers and broad bean (Vicia faba) seedlings. Histopathological observations on naturally infected sowthistle tissues revealed that D. oncogenus n. sp. causes floral stem neoplasia and midrib leaf gall formation on the type, and to date only known, host. The galls were characterized by extensive hyperplasia, where several necrotic cells in the neoplasic area were directly damaged by feeding of the nematode, whereas a number of adjacent cells showed typical cytological changes, such as granulated cytoplasm with hypertrophied nuclei and nucleoli.

Genome wide comprehensive analysis and web resource development on cell wall degrading enzymes from phyto-parasitic nematodes.

BACKGROUND: The plant cell wall serves as a primary barrier against pathogen invasion. The success of a plant pathogen largely depends on its ability to overcome this barrier. During the infection process, plant parasitic nematodes secrete cell wall degrading enzymes (CWDEs) apart from piercing with their stylet, a sharp and hard mouthpart used for successful infection. CWDEs typically consist of cellulases, hemicellulases, and pectinases, which help the nematode to infect and establish the feeding structure or form a cyst. The study of nematode cell wall degrading enzymes not only enhance our understanding of the interaction between nematodes and their host, but also provides information on a novel source of enzymes for their potential use in biomass based biofuel/bioproduct industries. Although there is comprehensive information available on genome wide analysis of CWDEs for bacteria, fungi, termites and plants, but no comprehensive information available for plant pathogenic nematodes. Herein we have performed a genome wide analysis of CWDEs from the genome sequenced phyto pathogenic nematode species and developed a comprehensive publicly available database. RESULTS: In the present study, we have performed a genome wide analysis for the presence of CWDEs from five plant parasitic nematode species with fully sequenced genomes covering three genera viz. Bursaphelenchus, Glorodera and Meloidogyne. Using the Hidden Markov Models (HMM) conserved domain profiles of the respective gene families, we have identified 530 genes encoding CWDEs that are distributed among 24 gene families of glycoside hydrolases (412) and polysaccharide lyases (118). Furthermore, expression profiles of these genes were analyzed across the life cycle of a potato cyst nematode. Most genes were found to have moderate to high expression from early to late infectious stages, while some clusters were invasion stage specific, indicating the role of these enzymes in the nematode's infection and establishment process. Additionally, we have also developed a Nematode's Plant Cell Wall Degrading Enzyme (NCWDE) database as a platform to provide a comprehensive outcome of the present study. CONCLUSIONS: Our study provides collective information about different families of CWDEs from five different sequenced plant pathogenic nematode species. The outcomes of this study will help in developing better strategies to curtail the nematode infection, as well as help in identification of novel cell wall degrading enzymes for biofuel/bioproduct industries.

Nematodes from galls on Myrtaceae. IX. Fergusobia rosettae n. sp. on Melaleuca quinquenervia and F. tolgaensis n. sp. on Syzygium luehmannii, from Queensland.

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.

Nematodes from galls on Myrtaceae. X. Fergusobia from galls on narrow-leaved Melaleuca spp. in Australia, with descriptions of three new species .

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. 

Nematodes from galls on Myrtaceae. VII. Fergusobia from 'leafy' leaf bud galls in Australia, with re-description of Fergusobia tumifaciens (Currie 1937) Wachek 1955 and descriptions of Fergusobia planchonianae n. sp. and Fergusobia viminalisae n. sp.

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. 

Computational discovery of soybean promoter cis-regulatory elements for the construction of soybean cyst nematode-inducible synthetic promoters.

Computational methods offer great hope but limited accuracy in the prediction of functional cis-regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)-inducible motif discovery among promoters of 18 co-expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean-SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co-localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN-inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN-inducible, leading to the discovery of 23 core motifs of 5- to 7-bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W-AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high-throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.