Pine wilt disease: what do we know from proteomics?

Pine wilt disease (PWD) is a devastating forest disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, a migratory endoparasite that infects several coniferous species. During the last 20 years, advances have been made for understanding the molecular bases of PWN-host trees interactions. Major advances emerged from transcriptomic and genomic studies, which revealed some unique features related to PWN pathogenicity and constituted fundamental data that allowed the development of postgenomic studies. Here we review the proteomic approaches that were applied to study PWD and integrated the current knowledge on the molecular basis of the PWN pathogenicity. Proteomics has been useful for understanding cellular activities and protein functions involved in PWN-host trees interactions, shedding light into the mechanisms associated with PWN pathogenicity and being promising tools to better clarify host trees PWN resistance/susceptibility.

First report of root knot nematodes Meloidogyne incognita and M. javanica parasitizing sweet potato, Ipomoea batatas L., in Portugal.

Sweet potato, Ipomoea batatas L., is a tuberous root vegetable rich in low glycemic sugars, vitamins and fibers (Galvão et al., 2021). Although it is widely cropped and consumed in tropical regions, in Europe consumer demand is growing exponentially (CBI, 2021). In Portugal, the production area of sweet potato increased from 588 ha in 2011 to 954 ha in 2017, and exports increased from 2404 tons in 2011 to 13412 tons in 2019 (FAOSTAT, 2021). During a survey carried out in August 2019, sweet potato plants were collected in Almada (38°39'40"N 9°10'54"W) and Belmonte (38°39'40"N 9°10'54"W), South and Centre regions of Portugal, respectively. No symptoms were observed on leaves, however, roots presented numerous galls and/or small spots (females and respective egg masses) were observed in the tuberous root flesh, suggestive of root knot nematodes (RKN, Meloidogyne spp.) infection. At least 8 individual females and respective egg masses were handpicked from roots of each sample and characterized biochemically by electrophoretic analysis of esterases (Pais & Abrantes, 1989). Phenotypes I2 and J3, attributed to M. incognita and M. javanica, respectively, were present in samples from Almada, whereas only phenotype I2 was found from Belmonte sample (Santos et al., 2019). Pure RKN cultures were established on tomato cv. Coração-de-Boi to obtain inoculum for molecular characterization and host suitability assays. Molecular characterization was performed by DNA amplification with M. incognita (Mi-F/Mi-R) and M. javanica (Fjav/Rjav) species-specific primers (Zijlstra et al., 2000; Meng et al., 2004). DNA amplification resulted in unique bands of ≈900 bp and ≈650 bp, respectively, confirming the RKN species identification. The host suitability of sweet potato cvs. Lira (local variety, purple skin, yellow flesh) and Murasaki (purple skin, white/pale to yellow flesh) to M. javanica (Almada) and M. incognita (Belmonte) isolates was assessed. Sweet potato slips with ≈10 cm roots were transplanted to 500 cm3 pots (one slip/pot) and after 2 weeks, each plant was inoculated with 5000 eggs + second-stage juveniles (Pi, initial population density) and maintained in a growth chamber (25±2°C; 12:12 h photoperiod). Tomato cv. Coração-de-Boi was included as a positive control. Each RKN species-plant germplasm combination was repeated 6 times. At 60 days after inoculation, host suitability was evaluated on the basis of root gall index (GI) and reproduction factor (Rf=final population density/Pi) (Sasser et al., 1984). Sweet potato cv. Lira was susceptible (GI=5; Rf=111.8) to M. incognita and resistant (GI=2; Rf=0.11) to M. javanica; while cv. Murasaki was hypersusceptible (GI=5; Rf=0.9) to M. incognita and susceptible (GI=5; Rf=5.5) to M. javanica. Although cultivars varied in their response to M. incognita and M. javanica isolates and variation in the final population density was high, both RKN isolates reproduced in these sweet potato cultivars. In previous studies, cv. Murasaki was considered resistant to M. enterolobii and to M. incognita (La Bonte et al. 2008; Schwarz et al., 2021). Depending on the RKN species, cultivation of cvs. Murasaki and Lira may thus benefit succeeding crops, but they should be combined with other management strategies to further reduce RKN populations in the field. In Portugal, M. incognita and M. javanica have been found associated with economically important horticultural crops, such as tomato and potato, trees and weeds (Santos et al., 2019; Maleita et al., 2021). To our knowledge, these species are reported for the first time parasitizing sweet potato in Portugal and this is the first report on the occurrence of M. incognita and M. javanica infecting sweet potato in Europe. Although findings were not totally unexpected due to the wide distribution and host range of these RKN species, they are of crucial importance since the sweet potato production in Europe has almost doubled from 50 (2011) to 97 thousand tons (2017), with Spain, Portugal, Italy and Greece being the largest producers (FAO, 2021). Our findings also reveal that sweet potato cropped in Portugal have different susceptibility levels to these common RKN species, reinforcing the importance of cultivar selection in RKN management.

Protein markers of Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Nickle, 1970) populations using quantitative proteomics and character compatibility.

The Pine Wood Nematode (PWN) Bursaphelenchus xylophilus is a severe forest pathogen in countries where it has been introduced and is considered a worldwide quarantine organism. In this study, protein markers for differentiating populations of this nematode were identified by studying differences among four selected Iberian and one American population. These populations were compared by quantitative proteomics (iTRAQ). From a total of 2860 proteins identified using the public database from the B. xylophilus genome project, 216 were unambiguous and significantly differentially regulated in the studied populations. Comparisons of their pairwise ratio were statistically treated and supported in order to convert them into discrete character states, suggesting that 141 proteins were not informative as population specific markers. Application of the Character Compatibility methodology on the remaining 75 proteins (belonging to families with different biological functions) excludes 27 which are incompatible among them. Considering only the compatible proteins, the method selects a subset of 30 specific unique protein markers which allowed the compared classification of the Iberian isolates. This approach makes it easier search for diagnostic tools and phylogenetic inference within species and populations of a pathogen exhibiting a high level of genetic diversity.

Morphological, Biometrical, Biochemical, and Molecular Characterization of the Coffee Root-Knot Nematode Meloidogyne megadora.

Meloidogyne megadora infects coffee trees, an economically important crop worldwide. The accurate identification of M. megadora is essential for the development of preventive measures to avoid the dispersion of this pathogen and establishment of efficient and sustainable integrated pest management programs. One M. megadora isolate was studied by biometrical, biochemical, and molecular characteristics (random amplified polymorphic DNA [RAPD] and PCR of internal transcribed spacer [ITS] region). Biometrical characteristics of M. megadora females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern for M. megadora esterases (Me3) that allowed for species differentiation. Three RAPD primers (OPG-4, OPG-5, and OPG-6) produced specific bands to all Meloidogyne spp. studied: M. megadora, M. arenaria, M. incognita, and M. javanica. Molecular analysis of the ITS region resulted in an amplification product of 700 bp. The phylogenetic relationship between M. megadora and several Meloidogyne spp. sequences was analyzed, revealing that M. megadora clearly differs from the most common root-knot nematode species. Based on the studies conducted, isozyme analysis remains a useful and efficient methodology for M. megadora identification when females are available. Further studies will be needed to convert the M. megadora differential DNA fragment obtained by RAPD and develop a species-specific sequence-characterized amplified region PCR assay for its diagnosis based on second-stage juveniles.

Relation between magnetic parameters and nematode abundance in agricultural soils of Portugal--a multidisciplinary study in the scope of environmental magnetism.

Soil is composed of different types of particles which are either natural or of anthropogenic origin. Anthropogenic particles are often related to the presence of heavy metals and thus provide information on soil quality. Magnetic parameters can detect the presence of such particles and may be used as a proxy for environmental pollution. This study explores the relationships between magnetic particles and the nematofauna of agricultural soils. Magnetic, pedological, microscopy and nematological analyses were conducted in soils collected from major regions of potato production in Portugal. The magnetic characterisation of soils identified regions with magnetic particles with possible anthropogenic origin. Microscopy analysis revealed the presence of spherical particles dominantly composed of Fe, O and C. A positive and significant relationship was found between saturation isothermal remanent magnetisation (SIRM) and mass-specific susceptibility (χ), confirming the importance the ferrimagnetic fraction to magnetic properties. The nematode communities were composed of nematodes belonging to four trophic groups (bacterial feeding, plant feeders, fungal feeders and omnivores/predators). The relationships between magnetic parameters and the nematodes showed that (1) S-25 has a linear correlation with number of nematodes per kilogram of soil and with plant feeders' trophic group and (2) SIRM correlates with the bacterial feeders trophic group. This study reveals that magnetic proxies may provide means for detecting regions with higher levels of pollution, possibly related to heavy metals. Due to the large background variability found in magnetic parameters, the sampling spacial mesh should to be further refined and the input of magnetic minerals needs to be locally calibrated.

Transcriptome analysis reveals the high ribosomal inhibitory action of 1,4-naphthoquinone on Meloidogyne luci infective second-stage juveniles.

The root-knot nematode (RKN) Meloidogyne luci presents a threat to the production of several important crops. This nematode species was added to the European Plant Protection Organization Alert list in 2017. The scarce availability of efficient nematicides to control RKN and the phasing out of nematicides from the market have intensified the search for alternatives, such as phytochemicals with bionematicidal properties. The nematicidal activity of 1,4-naphthoquinone (1,4-NTQ) against M. luci has been demonstrated; however, knowledge of the potential mode(s) of action of this compound is still scarce. In this study, the transcriptome profile of M. luci second-stage juveniles (J2), the infective stage, in response to 1,4-NTQ exposure was determined by RNA-seq to identify genes and pathways that might be involved in 1,4-NTQ's mode(s) of action. Control treatments, consisting of nematodes exposed to Tween® 80 (1,4-NTQ solvent) and to water, were included in the analysis. A large set of differentially expressed genes (DEGs) was found among the three tested conditions, and a high number of downregulated genes were found between 1,4-NTQ treatment and water control, reflecting the inhibitory effect of this compound on M. luci, with a great impact on processes related to translation (ribosome pathway). Several other nematode gene networks and metabolic pathways affected by 1,4-NTQ were also identified, clarifying the possible mode of action of this promising bionematicide.

Biometrical, Biochemical, and Molecular Diagnosis of Portuguese Meloidogyne hispanica Isolates.

Meloidogyne hispanica infects many economically important crops worldwide. The accurate identification of this pathogen is essential for the establishment of efficient and sustainable integrated pest management programs. Portuguese M. hispanica isolates were studied by biometrical, biochemical, and molecular characteristics. Biometrical characteristics of M. hispanica females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern (Hi4) for M. hispanica esterases that allowed for species differentiation. Molecular analysis of the mtDNA region from COII and 16S rRNA genes resulted in amplification products (1,800 bp) similar to M. hispanica, M. ethiopica, and M. javanica, and the described HinfI was unable to discriminate M. hispanica from the other two species. Analysis of the mtDNA sequences revealed altered nucleotides among the isolates that created new restriction sites for AluI and DraIII. The resulting restriction patterns successfully discriminated between the three species, providing a new tool for Meloidogyne identification. Finally, the phylogenetic relationship between M. hispanica and several Meloidogyne spp. sequences was analyzed using mtDNA, confirming the divergence between meiotic and mitotic species and revealing the proximity of M. hispanica to closely related species. Based on the studies conducted, the application of isozyme or polymerase chain reaction restriction fragment length polymorphism analysis would be a useful and efficient methodology for M. hispanica identification.

Juglone and 1,4-Naphthoquinone-Promising Nematicides for Sustainable Control of the Root Knot Nematode Meloidogyne luci.

The scarce availability of efficient and eco-friendly nematicides to control root-knot nematodes (RKN), Meloidogyne spp., has encouraged research toward the development of bionematicides. Naphthoquinones, juglone (JUG) and 1,4-naphthoquinone (1,4-NTQ), are being explored as alternatives to synthetic nematicides to control RKN. This study expands the knowledge on the effects of these natural compounds toward M. luci life cycle (mortality, hatching, penetration, reproduction). M. luci second-stage juveniles (J2)/eggs were exposed to each compound (250, 150, 100, 50, and 20 ppm) to monitor nematode mortality and hatching during 72 h and 15 days, respectively. Tomato seedlings were then inoculated with 200 J2, which had been exposed to JUG/1,4-NTQ for 3 days. The number of nematodes inside the roots was determined at 3 days after inoculation, and the final population density was assessed at 45 days after inoculation. Moreover, the potential mode of action of JUG/1,4-NTQ was investigated for the first time on RKN, through the assessment of reactive oxygen species (ROS) generation, acetylcholinesterase (AChE) in vitro inhibitory activity and expression analysis of ache and glutathione-S-transferase (gst) genes. 1,4-NTQ was the most active compound, causing ≥50% J2 mortality at 250 ppm, within 24 h. At 20 and 50 ppm, hatching was reduced by ≈50% for both compounds. JUG showed a greater effect on M. luci penetration and reproduction, decreasing infection by ≈80% (50 ppm) on tomato plants. However, 1,4-NTQ-induced generation of ROS and nematode vacuolization was observed. Our study confirms that JUG/1,4-NTQ are promising nematicidal compounds, and new knowledge on their physiological impacts on Meloidogyne was provided to open new avenues for the development of innovative sustainable nematicides.

Differential Impact of the Pinewood Nematode on Pinus Species Under Drought Conditions.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, responsible for the pine wilt disease (PWD), is a major threat to pine forests worldwide. Since forest mortality due to PWN might be exacerbated by climate, the concerns regarding PWD in the Mediterranean region are further emphasized by the projected scenarios of more drought events and higher temperatures. In this context, it is essential to better understand the pine species vulnerability to PWN under these conditions. To achieve that, physiological responses and wilting symptoms were monitored in artificially inoculated Pinus pinaster (P. pinaster), Pinus pinea (P. pinea), and Pinus radiata (P. radiata) saplings under controlled temperature (25/30°C) and water availability (watered/water stressed). The results obtained showed that the impact of PWN is species-dependent, being infected P. pinaster and P. radiata more prone to physiological and morphological damage than P. pinea. For the more susceptible species (P. pinaster and P. radiata), the presence of the nematode was the main driver of photosynthetic responses, regardless of their temperature or water regime conditions. Nevertheless, water potential was revealed to be highly affected by the synergy of PWN and the studied abiotic conditions, with higher temperatures (P. pinaster) or water limitation (P. radiata) increasing the impact of nematodes on trees' water status. Furthermore, water limitation had an influence on nematodes density and its allocation on trees' structures, with P. pinaster revealing the highest nematode abundance and inner dispersion. In inoculated P. pinea individuals, nematodes' population decreased significantly, emphasizing this species resistance to PWN. Our findings revealed a synergistic impact of PWN infection and stressful environmental conditions, particularly on the water status of P. pinaster and P. radiata, triggering disease symptoms and mortality of these species. Our results suggest that predicted drought conditions might facilitate proliferation and exacerbate the impact of PWN on these two species, through xylem cavitation, leading to strong changes in pine forests of the Mediterranean regions.

Carbofuran effects in soil nematode communities: using trait and taxonomic based approaches.

This work intends to implement the use of native soil nematode communities in ecotoxicological tests using a model pesticide and two geographically nematode communities (Mediterranean and sub-tropical) in order to obtain new perspectives on the evaluation of the toxic potential of chemical substances. The environmental condition of the nematode communities was described using a trait-based approach (grouping the organisms according to their feeding traits) and a traditional taxonomic method (identification to family level). Effects on total nematode abundance, number of families and abundance of nematode feeding groups as well as potential shifts in both trophic and family structure were assessed. Agricultural soils from Curitiba (Brazil) and Coimbra (Portugal) were sampled and the corresponding nematode communities were extracted. Part of the collected soil was defaunated and spiked with four doses of a carbofuran commercial formulation. Afterwards each of the replicates was inoculated with a nematode suspension containing ≈200 or 300 nematodes. After 14 and 28 d of exposure the nematodes were extracted, counted and identified at family level and separately classified according to their feeding traits. The patterns of nematode responses revealed a decrease in the total abundance and a reduction in the number of families. Despite the similar effects observed for both communities, statistically significant toxic effects were only found within the Portuguese community. The total nematode abundance was significantly reduced at the highest carbofuran concentrations and significant shifts in the family structure were detected. However, the trophic structure, i.e., the contribution of each feeding group for the overall community structure, did not significantly change along the contamination gradient. Results showed that using such a trait-based approach may increase the ecological relevance of toxicity data, by establishing communalities in the response to a chemical from two different taxonomic communities, although with potential loss of information on biodiversity of the communities.

Pratylenchus penetrans Parasitizing Potato Crops: Morphometric and Genetic Variability of Portuguese Isolates.

The root lesion Pratylenchus penetrans is an economically important pest affecting a wide range of plants. The morphometry of five P. penetrans isolates, parasitizing potato roots in Portugal, was compared and variability within and between isolates was observed. Of the 15 characters assessed, vulva position (V%) in females and the stylet length in both females/males showed the lowest coefficient of intra and inter-isolate variability. Moreover, DNA sequencing of the internal transcribed spacers (ITS) genomic region and cytochrome c oxidase subunit 1 (COI) gene was performed, in order to evaluate the intraspecific genetic variability of this species. ITS revealed higher isolate genetic diversity than the COI gene, with 15 and 7 different haplotypes from the 15 ITS and 14 COI sequences, respectively. Intra- and inter-isolate genetic diversity was found considering both genomic regions. The differentiation of these isolates was not related with their geographical origin. In spite of the high intraspecific variability, phylogenetic analyses revealed that both ITS region and COI gene separate P. penetrans from other related species. Our findings contribute to increasing the understanding of P. penetrans variability.

Meloidogyne graminicola-A Threat to Rice Production: Review Update on Distribution, Biology, Identification, and Management.

Rice (Oryza sativa L.) is one of the main cultivated crops worldwide and represents a staple food for more than half of the world population. Root-knot nematodes (RKNs), Meloidogyne spp., and particularly M. graminicola, are serious pests of rice, being, probably, the most economically important plant-parasitic nematode in this crop. M. graminicola is an obligate sedentary endoparasite adapted to flooded conditions. Until recently, M. graminicola was present mainly in irrigated rice fields in Asia, parts of the Americas, and South Africa. However, in July 2016, it was found in northern Italy in the Piedmont region and in May 2018 in the Lombardy region in the province of Pavia. Following the first detection in the EPPO region, this pest was included in the EPPO Alert List as its wide host range and ability to survive during long periods in environments with low oxygen content, represent a threat for rice production in the European Union. Considering the impact of this nematode on agriculture, a literature review focusing on M. graminicola distribution, biology, identification, and management was conducted.

Species-Specific Molecular Detection of the Root Knot Nematode Meloidogyne luci.

Meloidogyne luci has been identified in various countries around the world parasitizing economically important crops and, due to its potential to cause serious damage to agriculture, was included in the European and Mediterranean Plant Protection Organization Alert List in 2017. This species shares morphological and molecular similarities with M. ethiopica and M. inornata, and a M. ethiopica group was therefore established. Although specific primers for the DNA amplification of species belonging to the M. ethiopica group have been developed previously, the primers were not species-specific, so molecular markers for the specific detection of M. luci are still needed. The objective of this study was to develop a SCAR marker for the detection of M. luci and the discrimination from other Meloidogyne spp. based on the intraspecific variability found in RAPD markers. RAPD screening of M. luci and M. ethiopica genome was used for the identification of a specific amplification product on M. luci, which was cloned, sequenced and converted into a SCAR marker. The specificity of the designed primers (Mlf/r) was tested and produced a fragment (771 bp) for all nine M. luci isolates with no amplification for the other nine Meloidogyne spp., including M. ethiopica and M. inornata. Additionally, the proper amplification of the M. luci SCAR-marker was also successful with DNA from galls of M. luci infected tomato roots. The results obtained in this study reveal that the specific molecular detection of M. luci was achieved and that the developed methodology can be used for routine diagnosis purposes, which are essential to monitoring the distribution and spread of M. luci in order to implement future effective and integrated nematode pest management programs.

Comparative Analysis of Bursaphelenchus xylophilus Secretome Under Pinus pinaster and P. pinea Stimuli.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the pine wilt disease's (PWD) causal agent, is a migratory endoparasitic nematode skilled to feed on pine tissues and on fungi that colonize the trees. In order to study B. xylophilus secretomes under the stimulus of pine species with different susceptibilities to disease, nematodes were exposed to aqueous pine extracts from Pinus pinaster (high-susceptible host) and P. pinea (low-susceptible host). Sequential windowed acquisition of all theoretical mass spectra (SWATH-MS) was used to determine relative changes in protein amounts between B. xylophilus secretions, and a total of 776 secreted proteins were quantified in both secretomes. From these, 22 proteins were found increased in the B. xylophilus secretome under the P. pinaster stimulus and 501 proteins increased under the P. pinea stimulus. Functional analyses of the 22 proteins found increased in the P. pinaster stimulus showed that proteins with peptidase, hydrolase, and antioxidant activities were the most represented. On the other hand, gene ontology (GO) enrichment analysis of the 501 proteins increased under the P. pinea stimulus revealed an enrichment of proteins with binding activity. The differences detected in the secretomes highlighted the diverse responses from the nematode to overcome host defenses with different susceptibilities and provide new clues on the mechanism behind the pathogenicity of this plant-parasitic nematode. Proteomic data are available via ProteomeXchange with identifier PXD024011.

Virulence Biomarkers of Bursaphelenchus xylophilus: A Proteomic Approach.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, one of the most serious forest pests worldwide, is considered the causal agent of the pine wilt disease (PWD). The main host species belong to the genus Pinus, and a variation in the susceptibility of several pine species to PWN infection is well-known. It is also recognized that there is variation in the virulence among B. xylophilus isolates. In the present study, we applied a quantitative mass spectrometry-based proteomics approach to perform a deep characterization of proteomic changes across two B. xylophilus isolates with different virulence from different hosts and geographical origins. A total of 1,456 proteins were quantified and compared in the two isolates secretomes, and a total of 2,741 proteins were quantified and compared in the nematode proteomes in pine tree extract and fungus stimuli conditions. From the proteomic analyses, a group of proteins was selected and identified as potential virulence biomarkers and shed light on putative most pathogenic proteins of this plant-parasitic nematode. Proteomic data are available via ProteomeXchange with identifier PXD029377.

Ficus microcarpa Bonsai "Tiger bark" Parasitized by the Root-Knot Nematode Meloidogyne javanica and the Spiral Nematode Helicotylenchus dihystera, a New Plant Host Record for Both Species.

In December 2017, a Ficus microcarpa "Tiger bark" bonsai tree was acquired in a shopping center in Coimbra, Portugal, without symptoms in the leaves, but showing small atypical galls of infection caused by root-knot nematodes (RKN), Meloidogyne spp. The soil nematode community was assessed and four Tylenchida genera were detected: Helicotylenchus (94.02%), Tylenchus s.l. (4.35%), Tylenchorynchus s.l. (1.09%) and Meloidogyne (0.54%). The RKN M. javanica was identified through analysis of esterase isoenzyme phenotype (J3), PCR-RFLP of mitochondrial DNA region between COII and 16S rRNA genes and SCAR-PCR. The Helicotylenchus species was identified on the basis of female morphology that showed the body being spirally curved, with up to two turns after relation with gentle heat, a key feature of H. dihystera, and molecular characterization, using the D2D3 expansion region of the 28S rDNA, which revealed a similarity of 99.99% with available sequences of the common spiral nematode H. dihystera. To our knowledge, M. javanica and H. dihystera are reported for the first time as parasitizing F. microcarpa. Our findings reveal that more inspections are required to detect these and other plant-parasitic nematodes, mainly with quarantine status, to prevent their spread if found.

Molecular Characterization of Meloidogyne hispanica (Nematoda, Meloidogynidae) by Phylogenetic Analysis of Genes Within the rDNA in Meloidogyne spp.

In the past, the distribution of Meloidogyne hispanica, the Seville root-knot nematode, appeared to be restricted to the southern part of Spain and Prunus spp.; however, its distribution has been confirmed to be worldwide because it occurs in all continents (Europe, Africa, Asia, Australia, and North, Central, and South America). Differentiation of M. hispanica from other Meloidogyne spp., mainly M. arenaria, can be very difficult using morphological and biological traits data. These species are quite similar and can be regularly confused in inaccurate taxonomic comparisons. In this study, species-specific polymerase chain reaction (PCR) and phylogenetic analysis of sequences from three ribosomal (r)DNA regions (18S, internal transcribed spacer [ITS]1-5.8S-ITS2, and D2-D3 of 28S) were used to characterize three M. hispanica isolates from different geographical origins (Brazil, Portugal, and Spain). Molecular analyses showed identical sequences for all three isolates for the three rDNA regions. Maximum parsimony analysis of the three rDNA regions and the species-specific PCR demonstrated and supported the differentiation of M. hispanica from M. incognita, M. javanica, and M. arenaria and from all described root-knot nematode species.

Cysteine proteases secreted by the pinewood nematode, Bursaphelenchus xylophilus: In silico analysis.

The pinewood nematode, Bursaphelenchus xylophilus, is an important plant-parasitic nematode responsible for the development of the pine wilt disease and recognised as a major forest pest. Previous studies on the comparison of B. xylophilus and B. mucronatus secretomes obtained under maritime pine, Pinus pinaster, wood extract stimulus revealed that several cysteine proteases were increased in B. xylophilus secretome. In nematodes, proteases are known to play critical roles in parasitic processes like tissue penetration, digestion of host tissues for nutrition and evasion of host immune response. To gain further insight into the possible role of cysteine proteases on B. xylophilus pathogenicity, the molecular characterisation of four secreted cysteine peptidases was performed. BxCP3 and BxCP11 were identified as cathepsin L-like proteins and BxCP7 and BxCP8 as cathepsin B proteins. Only BxCP8 revealed high homology with another B. xylophilus cathepsin B referred on GenBank, all the others differ from the closer proteins deposited in this database. In silico three-dimensional structures of the four BxCP suggest that these proteins are pro-enzymes that become active when the pro-peptide is cleaved. BxCP7 and BxCP8 predicted structures revealed the presence of an occluding loop that occludes the active site cleft, typical of cathepsin B proteases.

First report of Meloidogyne graminis on golf courses turfgrass in Brazil.

Plant-parasitic nematodes of the genus Meloidogyne, known as root-knot nematodes (RKN), have an important economic impact on golf course turfgrasses. The most prevalent RKN species associated with grasses are M. chitwoodi, M. graminicola, M. graminis, M. incognita, M. marylandi, M. microtyla, M. minor, M. naasi and M. sasseri. In 2010, slight thickening of the roots and RKN females with unusual features were observed in turfgrass roots on golf courses in Araras, São Paulo state, Brazil. This population (MgARA) was maintained in the lab and studied including morphological, morphometrical, biochemical and molecular markers. Morphology and morphometry were variable and not useful for identification, although perineal pattern morphology showed highly similarity with M. graminis description. Concerning to biochemical characterisation, the esterase phenotype Mg1, characterised by a very slow and fainter band, was detected in some protein homogenates. Regarding to molecular analysis, D2-D3 region of 28S rDNA gene and cytochrome oxidase subunit II region from mitochondrial DNA were amplified by PCR and sequenced. Phylogenetic analysis revealed that the Brazilian isolate, found associated with turfgrass, grouped with M. graminis isolates (98-99% bootstrap; variation of 8-11 and 0-24 bp, respectively), close to M. marylandi, supporting its identification as M. graminis. This is the first report of M. graminis on golf courses in Brazil.