Redetection and description of the European dagger nematode Xiphinema diversicaudatum on peach (Prunus persica L.) in Canada.

The study reports the detection of Xiphinema diversicaudatum in a peach field in Ontario, Canada. Comprehensive population characterization involved morphological and molecular analyses using 18S and 28S rDNA sequences. Morphological and molecular analysis demonstrated a close relationship between the Ontario population and those from Central Europe. This is the first report of X. diversicaudatum from peaches (Prunus persica) in Canada and in North America.

First Report of Mesocriconema xenoplax from Apricots (Prunus armeniaca) in Ontario, Canada.

Plant-parasitic nematodes have been causing more economic losses and related damages to fruit crops in the Province of Ontario than previous estimates. We have been conducting surveys since 2019 for the presence of plant-parasitic nematodes in different orchards in the Niagara region. Collected soil samples were extracted for nematodes following Jenkins (1964). Among the plant-parasitic nematodes identified, we detected ring nematodes (Mesocriconema species) in 87% of the soil samples collected from different apricots fields, ranging from 20 to 200 ring nematode per kg of soil. The apricot cultivar was Haroblush and did not show any symptoms of nematode infestation. The morphometric measurements (mean in µm ± standard deviation, range) of the ring nematodes (adult females), n = 27), body length = (605 +/- 23, 550 - 650), stylet = (75 +/- 8, 65 - 79), a (total body length divided by maximum body diameter = 12 +/- 1.5, 10.2 - 13.9), b (total body length divided by pharyngeal length = 3.6 +/- 0.5, 3.2 - 3.9), c = 20, V (percentage of length from anterior end to vulva position in total body length) = 92 +/- 1.3,91 - 93), R (total body annules = 105 +/- 5, 100 - 110), Rvan (total annules between vulva and anus = 2), and Ran (total annules between anus and tail terminus = 5). Lips sclerotized, with conspicuous and elevated labial disc surrounding the oral aperture, sub-lateral lobes also conspicuous. The stylet was long and robust. Excretory pore was located between 30-31 annuli from the anterior end. The vulva located either between 6 and 7, but mostly between 7 and 8 annuli from the posterior end. The juveniles were similar to the females in body shape with: sub-lateral lobes present, stylet 55-60 µm long, total annuls 80-90, and fine longitudinal lines were observed. Based on the morphology (Figure 1) and morphometric characteristics, the nematode species was identified as Mesocriconema xenoplax, which was consistent with the description by Raski (1952). To further confirm, DNA was extracted from a total of 20 nematodes (females and juveniles), the D2-D3 expansion segment of 28S rDNA amplified using the primer pair: D2A: 5´-ACAAGTACCGTGAGGGAAAGT TG-3', D3B: 5'-TCCTCGGAAGGAACCAGCTACTA-3' (Nunn, 1992). The PCR product was purified and sequenced. The resulting sequence was deposited into the GenBank database and assigned with accession number ON130754. A BLAST search in the NCBI database revealed 99% similarity to sequences of M. xenoplax with accession numbers MH819082.1 and FN433854.1 from South Africa and California, respectively. Eight Haroblush plants were each inoculated with 100 ring nematodes/kg in 5-gallon pots. Haroblush without nematode were maintained as control. The nematode reproduction and symptoms were assessed after 6 and 24 months. The plants did not show below or aboveground symptoms after 6 months. However, root growth reduction, necrosis and death of feeder roots were observed after 24 months when compared to the control. Nematodes extracted from the soil had a mean of 512±48 and 1593±162 ring nematode/kg after 6 and 24 months, respectively, an indication of susceptibility. Mesocriconema xenoplax can cause 85% reduction in root biomass, and yield losses can occur at higher infestations (Pinkerton et al., 2004). Regardless, the potential impact and distribution of this nematode on apricots growth and yield in Ontario are not documented yet. To the best of our knowledge, this is the first report of the ring nematode M. xenoplax on apricots from Ontario, Canada.

Genome Characterization and Development of Real-Time PCR Assays for Ditylenchus Dipsaci and D. Weischeri.

The stem and bulb nematode Ditylenchus dipsaci is a destructive nematode pest on many crops and is internationally quarantined in many countries, whereas Ditylenchus weischeri, only known to infect a weed plant (Cirsium arvense), is an unregulated nematode species with no known economic importance. In this study, we used comparative genomics to identify multiple gene regions and developed novel real-time PCR assays for the detection of D. dipsaci and D. weischeri. We sequenced the genomes of two mixed-stage nematode populations of D. dipsaci and two mixed-stage nematode populations of D. weischeri. The assembled genomes of D. dipsaci were 228.2 Mb and 239.5 Mb, and the genomes of D. weischeri were 177.0 Mb and 196.3 Mb. Depending on the species, 21,403-27,365 gene models were predicted. Using orthologous group analysis, single-copy and species-specific genes were identified. Primers and probes were designed targeting two species-specific genes in each species. The assays detected as low as 12 pg of DNA from the target species, or as few as five nematodes, with a Cq of 31 cycles or less. Our study provides genome data for two additional D. dipsaci isolates and two D. weischeri isolates, and four new and validated molecular assays to be used for rapid detection and identification of the two species.

Genome sequencing and comparison of five Tilletia species to identify candidate genes for the detection of regulated species infecting wheat.

Tilletia species cause diseases on grass hosts with some causing bunt diseases on wheat (Triticum). Two of the four species infecting wheat have restricted distributions globally and are subject to quarantine regulations to prevent their spread to new areas. Tilletia indica causes Karnal bunt and is regulated by many countries while the non-regulated T. walkeri is morphologically similar and very closely related phylogenetically, but infects ryegrass (Lolium) and not wheat. Tilletia controversa causes dwarf bunt of wheat (DB) and is also regulated by some countries, while the closely related but non-regulated species, T. caries and T. laevis, both cause common bunt of wheat (CB). Historically, diagnostic methods have relied on cryptic morphology to differentiate these species in subsamples from grain shipments. Of the DNA-based methods published so far, most have focused on sequence variation among tested strains at a single gene locus. To facilitate the development of additional molecular assays for diagnostics, we generated whole genome data for multiple strains of the two regulated wheat pathogens and their closest relatives. Depending on the species, the genomes were assembled into 907 to 4633 scaffolds ranging from 24 Mb to 30 Mb with 7842 to 9952 gene models predicted. Phylogenomic analyses confirmed the placement of Tilletia in the Exobasidiomycetes and showed that T. indica and T. walkeri were in one clade whereas T. controversa, T. caries and T. laevis grouped in a separate clade. Single copy and species-specific genes were identified by orthologous group analysis. Unique species-specific genes were identified and evaluated as suitable markers to differentiate the quarantine and non-quarantine species. After further analyses and manual inspection, primers and probes for the optimum candidate genes were designed and tested in silico, for validation in future wet-lab studies.

Nitrogen-fixing bacteria with multiple plant growth-promoting activities enhance growth of tomato and red pepper.

As a suitable alternative to chemical fertilizers, the application of plant growth-promoting rhizobacteria has been increasing in recent years due to their potential to be used as biofertilizers. In the present work, 13 nitrogen-fixing bacterial strains belonging to 11 different genera were tested for their PGP attributes. All of the strains were positive for 1-aminocyclopropane-1-carboxylate deaminase (ACCD), indole-3-acetic acid (IAA), salicylic acid, and ammonia production while negative for cellulase, pectinase, and hydrocyanic acid production. The strains Pseudomonas sp. RFNB3 and Serratia sp. RFNB14 were the most effective in solubilizing both tri-calcium phosphate and zinc oxide. In addition, all strains except Pseudomonas sp. RFNB3 were able to oxidize sulfur, and six strains were positive for siderophore synthesis. Each strain tested in this study possesses at least four PGP properties in addition to nitrogen fixation. Nine strains were selected based on their multiple PGP potential, particularly ACCD and IAA production, and evaluated for their effects on early growth of tomato and red pepper under gnotobiotic conditions. Bacterial inoculation considerably influenced root and shoot length, seedling vigor, and dry biomass of the two crop plants. Three strains that demonstrated substantial effects on plant performance were further selected for greenhouse trials with red pepper, and among them Pseudomonas sp. RFNB3 resulted in significantly higher plant height (26%) and dry biomass (28%) compared to control. The highest rate of nitrogen fixation, as determined by acetylene reduction assay, occurred in Novosphingobium sp. RFNB21 inoculated red pepper root (49.6 nM of ethylene/h/g of dry root) and rhizosphere soil (41.3 nM of ethylene/h/g of dry soil). Inoculation with nitrogen-fixing bacteria significantly increased chlorophyll content, and the uptake of different macro- and micro-nutrient contents enhancing also in red pepper shoots, in comparison with uninoculated controls. The population estimation studies showed that nitrogen-fixing as well as total heterotrophic bacteria were also noticeably increased in soil and plant samples. The findings of this study suggest that certain nitrogen-fixing strains possessing multiple PGP traits could be applied in the development of biofertilizers.

Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification.

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes.

Comparative analysis of complete mitochondrial genome sequences confirms independent origins of plant-parasitic nematodes.

BACKGROUND: The nematode infraorder Tylenchomorpha (Class Chromadorea) includes plant parasites that are of agricultural and economic importance, as well as insect-associates and fungal feeding species. Among tylenchomorph plant parasites, members of the superfamily Tylenchoidea, such as root-knot nematodes, have great impact on agriculture. Of the five superfamilies within Tylenchomorpha, one (Aphelenchoidea) includes mainly fungal-feeding species, but also some damaging plant pathogens, including certain Bursaphelenchus spp. The evolutionary relationships of tylenchoid and aphelenchoid nematodes have been disputed based on classical morphological features and molecular data. For example, similarities in the structure of the stomatostylet suggested a common evolutionary origin. In contrast, phylogenetic hypotheses based on nuclear SSU ribosomal DNA sequences have revealed paraphyly of Aphelenchoidea, with, for example, fungal-feeding Aphelenchus spp. within Tylenchomorpha, but Bursaphelenchus and Aphelenchoides spp. more closely related to infraorder Panagrolaimomorpha. We investigated phylogenetic relationships of plant-parasitic tylenchoid and aphelenchoid species in the context of other chromadorean nematodes based on comparative analysis of complete mitochondrial genome data, including two newly sequenced genomes from Bursaphelenchus xylophilus (Aphelenchoidea) and Pratylenchus vulnus (Tylenchoidea). RESULTS: The complete mitochondrial genomes of B. xylophilus and P. vulnus are 14,778 bp and 21,656 bp, respectively, and identical to all other chromadorean nematode mtDNAs in that they contain 36 genes (lacking atp8) encoded in the same direction. Their mitochondrial protein-coding genes are biased toward use of amino acids encoded by T-rich codons, resulting in high A+T richness. Phylogenetic analyses of both nucleotide and amino acid sequence datasets using maximum likelihood and Bayesian methods did not support B. xylophilus as most closely related to Tylenchomorpha (Tylenchoidea). Instead, B. xylophilus, was nested within a strongly supported clade consisting of species from infraorders Rhabditomorpha, Panagrolaimomorpha, Diplogasteromorpha, and Ascaridomorpha. The clade containing sampled Tylenchoidea (P. vulnus, H. glycines, and R. similis) was sister to all analyzed chromadoreans. Comparison of gene arrangement data was also consistent with the phylogenetic relationships as inferred from sequence data. Alternative tree topologies depicting a monophyletic grouping of B. xylophilus (Aphelenchoidea) plus Tylenchoidea, Tylenchoidea plus Diplogasteromorpha (Pristionchus pacificus), or B. xylophilus plus Diplogasteromorpha were significantly worse interpretations of the mtDNA data. CONCLUSIONS: Phylogenetic trees inferred from nucleotide and amino acid sequences of mtDNA coding genes are in agreement that B. xylophilus (the single representative of Aphelenchoidea) is not closely related to Tylenchoidea, indicating that these two groups of plant parasites do not share an exclusive most recent common ancestor, and that certain morphological similarities between these stylet-bearing nematodes must result from convergent evolution. In addition, the exceptionally large mtDNA genome size of P. vulnus, which is the largest among chromadorean nematode mtDNAs sequenced to date, results from lengthy repeated segments in non-coding regions.

Comparisons of direct extraction methods of microbial DNA from different paddy soils.

Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18-20.17 µg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical-enzymatic-mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A260/A230 and A260/A280 ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical-enzymatic and chemical-mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals.

The complete mitochondrial genome sequence of Oncicola luehei (Acanthocephala: Archiacanthocephala) and its phylogenetic position within Syndermata.

In the present study, we determined the complete mitochondrial genome sequence of Oncicola luehei (14,281bp), the first archiacanthocephalan representative and the second complete sequence from the phylum Acanthocephala. The complete genome contains 36 genes including 12 protein coding genes, 22 transfer RNA (tRNA) genes and 2 ribosomal RNA genes (rrnL and rrnS) as reported for other syndermatan species. All genes are encoded on the same strand. The overall nucleotide composition of O. luehei mtDNA is 37.7% T, 29.6% G, 22.5% A, and 10.2% C. The overall A+T content (60.2%) is much lower, compared to other syndermatan species reported so far, due to the high frequency (18.3%) of valine encoded by GTN in its protein-coding genes. Results from phylogenetic analyses of amino acid sequences for 10 protein-coding genes from 41 representatives of major metazoan groups including O. luehei supported monophyly of the phylum Acanthocephala and of the clade Syndermata (Acanthocephala+Rotifera), and the paraphyly of the clade Eurotatoria (classes Bdelloidea+Monogononta from phylum Rotifera). Considering the position of the acanthocephalan species within Syndermata, it is inferred that obligatory parasitism characteristic of acanthocephalans was acquired after the common ancestor of acanthocephalans diverged from its sister group, Bdelloidea. Additional comparison of complete mtDNA sequences from unsampled acanthocephalan lineages, especially classes Polyacanthocephala and Eoacanthocephala, is required to test if mtDNA provides reliable information for the evolutionary relationships and pattern of life history diversification found in the syndermatan groups.

Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences.

BACKGROUND: The orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution. RESULTS: For this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomia siamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result. CONCLUSION: The phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda.

The mitochondrial genome sequence of Enterobius vermicularis (Nematoda: Oxyurida)--an idiosyncratic gene order and phylogenetic information for chromadorean nematodes.

The complete mitochondrial genome sequence was determined for the human pinworm Enterobius vermicularis (Oxyurida: Nematoda) and used to infer its phylogenetic relationship to other major groups of chromadorean nematodes. The E. vermicularis genome is a 14,010-bp circular DNA molecule that encodes 36 genes (12 proteins, 22 tRNAs, and 2 rRNAs). This mtDNA genome lacks atp8, as reported for almost all other nematode species investigated. Phylogenetic analyses (maximum parsimony, maximum likelihood, neighbor joining, and Bayesian inference) of nucleotide sequences for the 12 protein-coding genes of 25 nematode species placed E. vermicularis, a representative of the order Oxyurida, as sister to the main Ascaridida+Rhabditida group. Tree topology comparisons using statistical tests rejected an alternative hypothesis favoring a closer relationship among Ascaridida, Spirurida, and Oxyurida, which has been supported from most studies based on nuclear ribosomal DNA sequences. Unlike the relatively conserved gene arrangement found for most chromadorean taxa, E. vermicularis mtDNA gene order is very unique, not sharing similarity to any other nematode species reported to date. This lack of gene order similarity may represent idiosyncratic gene rearrangements unique to this specific lineage of the oxyurids. To more fully understand the extent of gene rearrangement and its evolutionary significance within the nematode phylogenetic framework, additional mitochondrial genomes representing a greater evolutionary diversity of species must be characterized.

Molecular identification and phylogenetic analysis of nuclear rDNA sequences among three opisthorchid liver fluke species (Opisthorchiidae: Trematoda).

In this study, we describe the development of a fast and accurate molecular identification system for human-associated liver fluke species (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) using the PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region. Based on sequence variation in the target rDNA region, we selected three species-specific restriction enzymes within the ITS1 regions, generating different restriction profiles among the species: MunI for O. viverrini, NheI for O. felineus, and XhoI for C. sinensis, respectively. Each restriction enzyme generated different-sized fragments specific to the species examined, but no intraspecific polymorphism or cross-reaction between the species was detected in their restriction pattern. These results indicate that PCR-linked restriction analysis of the ITS1 region allows for the rapid and reliable molecular identification among these opisthorchid taxa. In addition, phylogenetic analysis of rDNA sequences using different methods (MP, ML, NJ, and Bayesian inference) displayed O. viverrini and O. felineus as a sister group, but this relationship was not strongly supported. The failure of recovering a robust phylogeny may be due to the relatively small number of synapomorphic characters shared among the species, yielding weak phylogenetic signal. Alternatively, rapid speciation within a very short period time could be another explanation for the relatively poorly resolved relationships among these species. Our data are insufficient for discriminating between sudden cladogenesis and other potential causes of poor resolution. Further information from independent loci might help resolve this phylogeny.

Characterization of the complete mitochondrial genome of Diphyllobothrium nihonkaiense (Diphyllobothriidae: Cestoda), and development of molecular markers for differentiating fish tapeworms.

We sequenced and characterized the complete mitochondrial genome of the Japanese fish tapeworm D. nihonkaiense. The genome is a circular-DNA molecule of 13607 bp (one nucleotide shorter than that of D. latum mtDNA) containing 12 protein-coding genes (lacking atp8), 22 tRNA genes and two rRNA genes. Gene order and genome content are identical to those of the other cestodes reported thus far, including its congener D. latum. The only exception is Hymenolepis diminuta in which the positions of trnS2 and trnL1 are switched. We tested a PCR-based molecular assay designed to rapidly and accurately differentiate between D. nihonkaiense and D. latum using species-specific primers based on a comparison of their mtDNA sequences. We found the PCR-based system to be very reliable and specific, and suggest that PCR-based identification methods using mtDNA sequences could contribute to the study of the epidemiology and larval ecology of Diphyllobothrium species.