Jonathan D. Eisenback

First Insights into the Genetic Diversity of the Pinewood Nematode in Its Native Area Using New Polymorphic Microsatellite Loci

The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed.

The Pratylenchus penetrans Transcriptome as a Source for the Development of Alternative Control Strategies: Mining for Putative Genes Involved in Parasitism and Evaluation of in planta RNAi

The root lesion nematode Pratylenchus penetrans is considered one of the most economically important species within the genus. Host range studies have shown that nearly 400 plant species can be parasitized by this species. To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population, as well as nematode reads detected in infected soybean roots 3 and 7 days after nematode infection. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,715 transcripts. Homology searches showed significant hit matches to 58% of the total number of transcripts using different protein and EST databases. In general, the transcriptome of P. penetrans follows common features reported for other root lesion nematode species. We also explored the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected nematode genes. Different comparisons were performed to identify putative nematode genes with a role in parasitism, resulting in the identification of transcripts with similarities to other nematode parasitism genes. Focusing on the predicted nematode secreted proteins found in this transcriptome, we observed specific members to be up-regulated at the early time points of infection. In the present study, we observed an enrichment of predicted secreted proteins along the early time points of parasitism by this species, with a significant number being pioneer candidate genes. A representative set of genes examined using RT-PCR confirms their expression during the host infection. The expression patterns of the different candidate genes raise the possibility that they might be involved in critical steps of P. penetrans parasitism. This analysis sheds light on the transcriptional changes that accompany plant infection by P. penetrans, and will aid in identifying potential gene targets for selection and use to design effective control strategies against root lesion nematodes.

Worldwide invasion routes of the pinewood nematode: What can we infer from population genetics analyses?

Identifying the invasion routes and determining the origin of new outbreaks of invasive species are of crucial importance if we are to understand the invasion process, improve or establish regulatory measures and, potentially, limit the damage. We focused here on the invasion of Europe by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer, 1934; Nickle 1970; Nematoda: Aphelenchoididae), a major pest of forest ecosystems, native to North America and already invasive in Asia since the beginning of the twentieth century. We evaluated the genetic diversity and structure of worldwide field PWN samples by classical and Bayesian population genetics methods to determine the source of the European invasive populations and the number of introduction events in Europe. We found (1) a very strong spatial genetic structure in native PWN populations, (2) a very low level of polymorphism in each of the invaded areas and (3) contrasted results concerning the origin of European invasive populations. Our findings provide evidence for: (1) a large effect of genetic drift on the biological cycle of the PWN, due to intense demographic bottlenecks during tree infections, not compensated for by effective dispersal of its vector; (2) a single introduction event for each of the invaded areas in Japan and Europe and a small effective size for the introduced populations and (3) a mainland Portuguese origin for PWN populations from Madeira. However, more sophisticated methods of invasion route inference and broader sampling are required to conclusively determine the origin of the European outbreak.

Meloidogyne brasilensis n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitising tomato cv. Rossol in Brazil

Meloidogyne brasilensis n. sp., Londrina and Brasilia populations, is described and illustrated from specimens from tomato cv. Rossol and pea cv. Mikado, respectively, in Brazil. Characteristically, the perineal pattern is elongated to ovoid with a flattened to very high, squarish dorsal arch with widely spaced, coarse striae. The lateral fields may have wing-like striae on one or both sides. The female stylet is 14 μm long with narrow and elongated knobs that are distinctly set-off from the shaft. The excretory pore is variable in location, but generally opens near the anterior portion of the median bulb about 50 μm from the head end. The male is 1.89 mm long and has a high head cap that slopes posteriorly. The labial disc is separated from the medial lips by a deep, rounded groove. The delicate stylet of the male is 23 μm long and has small, rounded knobs that are distinctly set-off from the shaft which has numerous small, rounded projections. Mean second-stage juvenile length is 434 μm. The juvenile head cap is highly elevated, the medial lips are crescent-shaped and unequal in size, and the head region is not annulated. The stylet is 11 μm long and has small, rounded, posteriorly sloping knobs. The tail is 53 μm long; it is marked with large, irregular annules and ends in a bluntly rounded tip. The hyaline tail terminus is short (13 μm). Reproduction occurred on NC95 tobacco, tomato, pea, and bean, whereas pepper, watermelon, peanut, cotton, corn, and soybean were not hosts.

Meloidogyne phaseoli n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitising bean in Brazil

Meloidogyne phaseoli n. sp. is described and illustrated from specimens parasitising bean cv. Carioca in Brasilia, Brazil. The perineal pattern of the female is rounded to oval-shaped with a dorsal arch that is flattened to moderately high and squarish, sometimes with rounded shoulders. The striae are moderately spaced and often distinctly forked in the lateral field. The female stylet is 14-19 μm long and has broad, distinctly set-off knobs and several small, rounded projections on the shaft. The excretory pore opens 34-82 μm from the head near the level of metacorpus. Males are 999-2105 μm in length and have a high, wide head cap that slopes posteriorly. The labial disc and medial lips are partially fused to form an elongated lip structure. The robust stylet is 20-26 μm long and has wide knobs that are distinctly set-off from the shaft which is marked by several small, round projections. Mean second-stage juvenile length is 464 μm. The head region is not annulated and the large labial disc and crescent-shaped medial lips are fused to form a dumbbell-shaped head cap. The stylet is 10.5-12 μm long and has rounded, posteriorly sloping knobs. The slender tail, 46-64 μm long, has large irregular-sized annules in the posterior region and a slightly rounded tip. The hyaline tail terminus is long, 10.5-19.3 μm. The esterase isozyme is a unique E3 phenotype having one weak and two strong bands. Tomato, tobacco, bean and pea are good hosts, corn is a very poor host, whilst pepper, watermelon, peanut, cotton and soybean are non-hosts.

Additional notes on the morphology of Meloidogyne spartinae (Nematoda: Meloidogynidae)

Females, males and second-stage juveniles of Meloidogyne spartinae were examined by light and scanning electron microscopy. The morphology of M. spartinae is very typical of the genus for most characters in general. However, as expected, some minor differences occur, particularly in the shape of the head, stylet and tail, and morphometrics of several characters. It is not clear at present how the karyotype of seven chromosomes in M. spartinae is related to the 13-18 chromosomes of other apparently related Meloidogyne species. The M. spartinae karyotype may represent an ancestral form from which the other species have evolved, possibly as polyploid-aneuploids. More studies are needed to clarify this point. However, M. spartinae is remarkably similar in morphology to other species and is justly placed within the genus Meloidogyne.

Characterization and silencing of the fatty acid- and retinol-binding Pp-far-1 gene in Pratylenchus penetrans

&NA; Fatty acid‐ and retinol‐binding proteins (FARs) are unique to nematodes, and are implicated in a wide range of metabolic and parasitic related functions. Along with the in silico analyses performed in this study, three different FAR members of this family were identified in Pratylenchus penetrans. The cDNA corresponding to the fatty acid‐ and retinol‐binding Pp‐far‐1 gene was cloned and herein characterized at the molecular level for the first time for this genus. The translated 185 amino acid sequence of P. penetrans FAR‐1 sequence, with a predicted molecular weight of 20.82 kDa and a pI of 5.49, shares highest sequence identity to FARs of other migratory nematodes of the Pratylenchidae family (90% to P. vulnus and 80% to Radopholus similis). In situ hybridization localizes Pp‐far‐1 transcripts in the hypodermis of the nematode. RT‐qPCR detected Pp‐far‐1 transcripts for all nematode developmental stages, with highest expression levels found in juveniles, adult females and adult males, respectively. Pp‐far‐1 is also highly expressed during infection and establishment of P. penetrans in roots of different host plants, such as corn, lily and soybean. The importance of Pp‐far‐1 was studied by in planta RNA interference (RNAi) assays using stable soybean hairy root lines. Targeting Pp‐far‐1 decreased expression of the nematode Pp‐far‐1 and significantly reduced the reproduction of nematodes, with a total of between 44% and 70% fewer nematodes in comparison to the average number of nematodes counted for control lines. The results indicate that suppressing the expression levels of Pp‐far‐1 can act as an effective target gene to control P. penetrans.

Bursaphelenchus hofmanni Braasch, 1998 associated with peat growth substrate in hops nurseries in the Czech Republic

To date, seven Bursaphelenchus species have been reported in surveys of the Czech Republic (Čermák et al., 2013). However, the occurrence of some species, such as Bursaphelenchus hofmanni Braasch, 1998, is limited to single detections in imported coniferous wood (unpubl. data in Braasch, 2001). During a survey to determine pathogenic agents on hops (Humulus lupulus L.) conducted by State Phytosanitary Administration (CZ) in hops fields and nurseries in 2012, B. hofmanni was found in a mixture of peat and soil in a hops seedling nursery in Žatec (Bohemia, Czech Republic). The occurrence of Bursaphelenchus species in non-woody plants or substrates has been previously reported for species of the fungivorus group, such as B. gonzalezi Loof, 1964, B. hunti Giblin & Kaya, 1983 and B. fungivorus Franklin & Hooper, 1962. Species belonging to the hofmanni group are often associated with wood products such as packaging material (Gu et al., 2006) and, to our knowledge, this is the first time that this species has been found associated with peat substrate and soil. Nematodes were isolated from 60 g of peat substrate (3 parts peat to 1 part soil) associated with hops seedlings,

Improved preparation of perineal patterns of root-knot nematodes for Scanning Electron Microscopy

The perinealpatternisperhapsthemost importantcharacter for the identie cation of species of root-knot nematodes (Meloidogyne spp.). The morphology is usually examined with the light microscope (LM) which has the advantage of revealing internal structures, but resolution and depth of focus are extremely limited. The scanning electron microscope (SEM) has a greater depth of focus and more resolving power,but it is limited to imaging only the surface morphology. Several techniques for preparing perineal patterns for SEM have been described: Mulvey et al. (1975), Spaull

Electronic Taxonomic Databases for Bursaphelenchus and Other Aphelenchid Nematodes

Undoubtedly, the most important resources for nematode taxonomy are the original species descriptions. Unfortunately, nematode descriptions have been published since the middle of the eighth century in a variety of forms including many relatively obscure journals and proceedings and sometimes in lengthy special publications. As a result, the task of collecting all of the descriptions of a single genus is daunting and has been repeated by numerous nematode taxonomists around the world. Furthermore, many of the papers were printed on acid containing paper and are rapidly deteriorating by yellowing and becoming brittle. The purpose of the present project is to collect all of the original species descriptions of the Aphelenchs including the genus Bursaphelenchus. The original papers are digitized with a flatbed scanner and converted to the PDF documents. The image is converted into text with optical character recognition software that is built into Adobe® Acrobat; thus each document becomes a fully searchable text that includes all of the photographs and drawings that are the same quality as the original. Theses collections are put together into monographs that are published as individual CD ROMs.