PPNID : a reference database and molecular identification pipeline for plant-parasitic nematodes

Motivation: The phylum Nematoda comprises the most cosmopolitan and abundant metazoans on Earth and plant-parasitic nematodes represent one of the most significant nematode groups, causing severe losses in agriculture. Practically, the demands for accurate nematode identification are high for ecological, agricultural, taxonomic and phylogenetic researches. Despite their importance, the morphological diagnosis is often a difficult task due to phenotypic plasticity and the absence of clear diagnostic characters while molecular identification is very difficult due to the problematic database and complex genetic background. Results: The present study attempts to make up for currently available databases by creating a manually-curated database including all up-to-date authentic barcoding sequences. To facilitate the laborious process associated with the interpretation and identification of a given query sequence, we developed an automatic software pipeline for rapid species identification. The incorporated alignment function facilitates the examination of mutation distribution and therefore also reveals nucleotide autapomorphies, which are important in species delimitation. The implementation of genetic distance, plot and maximum likelihood phylogeny analysis provides more powerful optimality criteria than similarity searching and facilitates species delimitation using evolutionary or phylogeny species concepts. The pipeline streamlines several functions to facilitate more precise data analyses, and the subsequent interpretation is easy and straightforward

Aptitud de la solarización para incrementar la temperatura del suelo en campo e invernadero en el Alto Valle de Río Negro y Neuquén: un ejemplo de aplicación agronómica

Durante 1998 y 1999, se estudió la evolución de las temperaturas del suelo durante el proceso de solarización en invernadero y en el campo en la región del Alto Valle de Río Negro y Neuquén. En todos los ensayos se incluyó un control sin tratar. En invernadero se incluyó un tratamiento con bromuro de metilo. Se registraron diariamente las temperaturas a los 10 cm y 30 cm de profundidad. En invernadero, a las 15 hs y 18 hs se presentaron diferencias significativas entre los tratamientos a los 10 cm y 30 cm de profundidad en ambos años. En el campo, a los 10 cm de profundidad la solarización incrementó la temperatura del suelo solarizado en 10 ºC a las 15 hs y en 11ºC a las 18 hs. A los 30 cm de profundidad el incremento fue de 9ºC a las 15 hs y 18 horas. En otros trabajos de los integrantes o colaboradores del grupo de investigación, estos incrementos de la temperatura del suelo posibilitaron reducir las poblaciones de hongos patógenos, Meloidogyne spp., malezas y microorganismos benéficos entre 25% y 60 %.Asociación Argentina de Energías Renovables y Medio Ambiente (ASADES

Aptitud de la solarización para incrementar la temperatura del suelo en campo e invernadero en el Alto Valle de Río Negro y Neuquén: un ejemplo de aplicación agronómica

Durante 1998 y 1999, se estudió la evolución de las temperaturas del suelo durante el proceso de solarización en invernadero y en el campo en la región del Alto Valle de Río Negro y Neuquén. En todos los ensayos se incluyó un control sin tratar. En invernadero se incluyó un tratamiento con bromuro de metilo. Se registraron diariamente las temperaturas a los 10 cm y 30 cm de profundidad. En invernadero, a las 15 hs y 18 hs se presentaron diferencias significativas entre los tratamientos a los 10 cm y 30 cm de profundidad en ambos años. En el campo, a los 10 cm de profundidad la solarización incrementó la temperatura del suelo solarizado en 10 ºC a las 15 hs y en 11ºC a las 18 hs. A los 30 cm de profundidad el incremento fue de 9ºC a las 15 hs y 18 horas. En otros trabajos de los integrantes o colaboradores del grupo de investigación, estos incrementos de la temperatura del suelo posibilitaron reducir las poblaciones de hongos patógenos, Meloidogyne spp., malezas y microorganismos benéficos entre 25% y 60 %.Asociación Argentina de Energías Renovables y Medio Ambiente (ASADES

The Genus Pratylenchus (Nematoda: Pratylenchidae) in Israel: From Taxonomy to Control Practices

This article belongs to the Special Issue Plant Parasitic Nematodes.Due to Israel’s successful agricultural production and diverse climatic conditions, plant-parasitic nematodes are flourishing. The occurrence of new, previously unidentified species in Israel or of suggested new species worldwide is a consequence of the continuous withdrawal of efficient nematicides. Among plant-parasitic nematodes, migratory endoparasitic species of the genus Pratylenchus are widely distributed in vegetable and crop fields in Israel and are associated with major reductions in quality and yield. This review focuses on the occurrence, distribution, diagnosis, pathogenicity, and phylogeny of all Pratylenchus species recorded over the last few decades on different crops grown throughout Israel—covering early information from nematologists to recent reports involving the use of molecular phylogenetic methodologies. We explore the accepted distinction between Pratylenchus thornei and Pratylenchus mediterraneus isolated from Israel’s northern Negev region, and address the confusion concerning the findings related to these Pratylenchus species. Our recent sampling from the northern Negev revealed the occurrence of both P. thornei and P. mediterraneus on the basis of molecular identification, indicating P. mediterraneus as a sister species of P. thornei and their potential occurrence in a mixed infection. Finally, the efficiencies of common control measures taken to reduce Pratylenchus’ devastating damage in protected crops and field crops is discussed.This research was funded by The Chief Scientist Ministry of Agriculture and Rural Development grant number 131-4544.Peer reviewe

Bioactive Volatiles from an Endophytic Daldinia cf. concentrica Isolate Affect the Viability of the Plant Parasitic Nematode Meloidogyne javanica.

Plant-parasitic nematodes form one of the largest sources of biotic stress imposed on plants, and are very difficult to control; among them are the obligate parasites, the sedentary root-knot nematodes (RKNs)-Meloidogyne spp.-which are extremely polyphagous and exploit a very wide range of hosts. Endophytic fungi are organisms that spend most of their life cycle within plant tissue without causing visible damage to the host plant. Many endophytes secrete specialized metabolites and/or emit volatile organic compounds (VOCs) that exhibit biological activity. Recently, we demonstrated that the endophytic fungus Daldinia cf. concentrica secrets biologically active VOCs. Here we examined the ability of the fungus and its VOCs to control the RKN M. javanica both in vitro and greenhouse experiments. The D. cf. concentrica VOCs showed bionematicidal activity against the second-stage juveniles (J2s) of M. javanica. We found that exposure of J2s to fungal volatiles caused 67% reduction in viability, and that application of a synthetic volatile mixture (SVM), comprising 3-methyl-1-butanol, (±)-2-methyl-1-butanol, 4-heptanone, and isoamyl acetate, in volumetric ratio of 1:1:2:1 further reduced J2s viability by 99%. We demonstrated that, although each of the four VOCs significantly reduced the viability of J2s relative to the control, only 4-heptanone elicited the same effect as the whole mixture, with nematicidal activity of 90% reduction in viability of the J2s. Study of the effect of the SVM on egg hatching demonstrated that it decreased eggs hatching by 87%. Finally, application of the SVM to soil inoculated with M. javanica eggs or J2s prior to planting susceptible tomato plants resulted in a significantly reduced galling index and fewer eggs produced on each root system, with no effect on root weight. Thus, D. cf. concentrica and/or SVM based on fungal VOCs may be considered as a novel alternative approach to controlling the RKN M. javanica

SlWRKY16 and SlWRKY31 of tomato, negative regulators of plant defense, involved in susceptibility activation following root-knot nematode Meloidogyne javanica infection

Abstract The involvement of WRKY transcription factors in plant-nematode interactions, and in particular, how these WRKYs participate in regulating the complex morphological and physiological changes occurring after nematode infection, are the topic of active research. We characterized the functional role of the unstudied tomato WRKY genes SlWRKY16 and SlWRKY31 in regulating tomato roots’ response to infection by the root-knot nematode Meloidogyne javanica. Using promoter–GUS reporter gene fusions and qRT-PCR, we show that both SlWRKYs are predominantly expressed during the first half of the parasitic life stages, when feeding-site induction and construction occur. Expression of SlWRKY16 increased sharply 15 days after inoculation, whereas SlWRKY31 was already induced earlier, but reached its maximum expression at this time. Both genes were downregulated at the mature female stage. To determine biological function, we produced transgenic lines overexpressing SlWRKY16 and SlWRKY31 in tomato hairy roots. Overexpression of both genes resulted in enhanced M. javanica infection, reflected by increased galling occurrence and reproduction. Expression profiling of marker genes responsive to defense-associated phytohormones indicated reductions in salicylic acid defense-related PR-1 and jasmonic acid defense-related PI in inoculated roots overexpressing SlWRK16 and SlWRKY31, respectively. Our results suggest that SlWRKY16 and SlWRKY31 function as negative regulators of plant immunity induced upon nematode infection

<i>SlWRKY45</i>, nematode-responsive tomato <i>WRKY</i> gene, enhances susceptibility to the root knot nematode; <i>M. javanica</i> infection

<p>The fluctuation of tomato's WRKY defense regulators during infection by the root knot nematode <i>Meloidogyne javanica</i> was analyzed: and the spatial and temporal expression of <i>SlWRKY45</i> was studied in depth with regard to its response to nematode infection, phytohormones, and wounding. Expression of <i>WRKY45</i> increased substantially within 5 d upon infection and continued through feeding-site development and gall maturation. Histological analysis of nematode feeding sites indicated that <i>WRKY45</i> was highly expressed within the feeding cells and associated vascular parenchyma cells. Responses of SlWRKY45 promoters to several phytohormones showed that <i>WRKY45</i> was highly induced by specific phytohormones, including cytokinin, auxin, and the defense-signaling molecule salicylic acid (SA), but not by the jasmonates. Overexpressing tomato lines were generated, and infection tests showed that, significantly, roots over-expressing SlWRKY45 contained substantially increased number of females, indicating that <i>WRKY45</i> overexpression supported faster nematode development. qRT-PCR tests have shown roots overexpressing <i>WRKY45</i> suppressed the jasmonic acid and salicylic acid marker genes, proteinase inhibitor (PI), and pathogenesis related protein (PR1), respectively, and also the cytokinin response factors <i>CRF1</i> and <i>CRF6</i>. Overall, this study indicated SlWRKY45 to be a potential transcription factor whose manipulation by the invading nematode might be critical for coordination of hormone signals supporting favorable condition for nematode development in root tissue<i>.</i></p

Effect of <i>D</i>. <i>cf</i>. <i>concentrica</i> culture plate on <i>M</i>. <i>javanica</i> J2 viability.

<p>J2s were exposed to 0–3 culture plates of <i>D</i>. <i>cf</i>. <i>concentrica</i> for 48 h. The numbers on the x-axis represent the numbers of Petri plates (50 mm in diameter, containing 5 mL of growth medium, and the fungal culture) in each 1-L sealed box. The number 0 indicates control treatment in which the nematodes were not exposed to the fungal culture plate. The viable J2s were separated using 30 μm sieves, and the numbers on the y-axis represent the numbers (mean ± SE) of viable J2s counted following the incubation. There were 10 repetitions, each using 300 J2s. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at <i>P ≤</i> 0.05. The experiment was independently repeated three times, each time with similar results.</p

Effect on <i>M</i>. <i>javanica</i> J2 viability of a SVM compared with that of <i>D</i>. <i>cf</i>. <i>concentrica</i> culture.

<p>J2s were exposed for 48 h either to the SVM at 1 mL/L (V/V) [3-methyl-1-butanol (0.2 mL, 1.84 mmole), (±)-2-methyl-1-butanol (0.2 mL, 1.86 mmole), 4-heptanone (0.4 mL, 2.86 mmole), and isoamyl acetate (0.2 mL, 1.35 mmole)] or to three 50-mm-diameter fungal culture plates, each with 5 mL of growth medium, and then the viable J2s were separated using 30 μm sieves. The numbers on the y-axis represent the numbers (mean ± SE) of viable J2s counted following the incubation. Each treatment was composed of 10 technical repetitions using 300 J2s in each repetition. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at <i>P ≤</i> 0.05. The experiment was independently repeated three times, each time with similar results.</p

Activity of the SVM in loamy soil.

<p>A 0.4 mL stock of SVM [3-methyl-1-butanol (0.08 mL, 0.73 mmole), (±)-2-methyl-1-butanol (0.08 mL, 0.74 mmole), 4-heptanone (0.16 mL, 1.14 mmole), and isoamyl acetate (0.08 mL, 0.54 mmole)] was aliquoted, loaded on perlite particles, and mixed with 60 g of soil in a sealed 50-mL cup, before the addition of 500 J2s. The cups (five repetitions) were sealed and incubated for 48 h, after which viable J2s were determined according to the Baermann funnel method. The numbers on the y-axis represent the means (± SE) of viable J2s extracted from the soil. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at <i>P</i> ≤ 0.05. The experiment was independently repeated twice, each time with similar results.</p