Carla Maleita

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.

Host status of cultivated plants to Meloidogyne hispanica

The reproduction of a Meloidogyne hispanica isolate from Portugal was evaluated in 63 plant species/cultivars, in pot assays at 25 ± 2.0°C, on the basis of root gall index (GI) and reproduction factor (Rf = final/initial egg density) at 60 days after inoculation. Cultivars of aubergine, bean, beetroot, broccoli, carnation, corn, cucumber, French garlic, lettuce, melon, onion, parsley, pea, potato, spinach, and tobacco and two of cabbage were susceptible (3 ≤ GI ≤ 5; 1.15 ≤ Rf ≤ 262.86). Cabbage cv. Bacalan, cauliflower cv. Temporão and pepper cv. Zafiro R2 were hypersusceptible or poor hosts (Rf < 1; GI > 2) and pepper cvs. Aurelio and Solero were resistant (0.0 ≤ GI ≤ 0.4; 0.00 ≤ Rf ≤ 0.03). The response of the pepper cultivars and the Mi-1 resistant tomato cv. Rossol was also conducted in pots using two inoculum levels and four temperatures, three growth chamber (25 ± 2.7°C, 29.3 ± 1.8°C and 33.6 ± 1.2°C) and one outdoors (24.4 ± 8.2°C). At 24.4 ± 8.2°C and 25 ± 2.7°C, the reproduction on the resistant tomato was significantly lower compared to the susceptible cv. Easypeel. At all temperatures, resistance was evident for the pepper cultivars, despite the fact they were not found to contain any of the Me1, Me3, Me7 and N genes. The eggs obtained on cv. Aurelio at 33.6 ± 1.2°C were used to get a selected resistance breaking isolate of M. hispanica that was able to reproduce on the three pepper cultivars. Our results suggest that the initial M. hispanica isolate is a mixture of virulent and avirulent individuals. The pepper cultivars tested, have potential to reduce M. hispanica populations in agro-ecosystems under certain conditions, but they should be used as a part of an integrated management strategy in order to prevent the development of virulent populations.

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.

Characterization of the venom allergen—like protein (vap-1) and the fatty acid and retinol binding protein (far-1) genes in Meloidogyne hispanica

The root-knot nematode (RKN) Meloidogyne hispanica has been found in all continents associated with a wide host range, including economically, important plants and can be considered a species of emerging importance. Considerable progress has been made to identify nematode effector genes as they are important targets for the development of novel control strategies. The effector genes, venom allergen-like protein (vap-1) and fatty acid and retinol binding protein (far-1), were identified, isolated and sequenced in M. hispanica (Mhi-vap-1 and Mhi-far-1) using the genome information available for the RKNs M. incognita and M. hapla. These genes are differentially expressed during M. hispanica development and their amplification products were observed from cDNA of the eggs, second-stage juveniles (J2) and adult females. However, Mhi-vap-1 showed the highest level of expression in J2. In situ hybridization analysis revealed that the Mhi-vap-1 and Mhi-far-1 transcripts are accumulated within the J2 subventral oesophageal glands. The specific expression in the subventral oesophagel glands and presence of the secretion signal peptide for both genes suggests that these proteins are secreted by the J2 and may play a role in the early parasitic stage of the infection process. These genes were also isolated and sequenced in M. arenaria, M. incognita and M. javanica; and phylogenetic analysis revealed that the predicted protein sequences belonging to M. hispanica and several other species of plant-parasitic nematodes have a high degree of conservation.

{null=Effect of the Mi gene on reproduction of Meloidogyne hispanica on tomato genotypes, en=Effect of the Mi gene on reproduction of Meloidogyne hispanica on tomato genotypes}

{null=The root-knot nematode resistance (Mi) gene was screened in 25 tomato genotypes of Solanum lycopersicum, by amplification of REX-1 and Mi23 markers. Ten heterozygous tomato genotypes (Mimi), nine homozygous (MiMi) at the Mi locus and six lacking the Mi gene for resistance to root-knot nematode were identified using the marker REX-1. The results obtained with Mi23 marker confirmed the Mi gene status of the tomato genotypes, except for genotype Valouro RZ F1 that was homozygous (MiMi) and heterozygous (Mimi) at the Mi locus when using the REX-1 and Mi23 markers, respectively. The pathogenicity of Meloidogyne hispanica on the 25 tomato genotypes was assessed 60 days after inoculation with 5000 eggs on the basis of root gall index (GI) and reproduction factor (Rf). All the tomato genotypes were susceptible (excellent or good hosts), with GI > 4 and Rf > 2, except the genotype Rapit (Mimi), considered as resistant/hypersensitive (poor host). In this genotype, the nematode induced galls (GI = 4) on its roots and a small number of eggs were produced (Pf = 3085 ± 485). Significant differences in reproduction were detected between the Mi allelic conditions and genotypes within Mi allelic conditions. The increasing number of Mi alleles (0, 1 or 2) is associated with decreasing Rf, which suggests a possible dosage effect of the Mi gene. The variability observed in the Rf values for MiMi tomato genotypes may reflect an influence of the genetic background of the plants containing the Mi gene. Ten of the 25 tomato genotypes with Mi gene are commercially available. However, only Rapit can be used to control the three most common Meloidogyne spp. and inhibit the increasing of M. hispanica populations, and may have potential to be included in an integrated pest management programme. However, it is advisable to evaluate the pathogenicity of local populations of this nematode species associated with different environmental factors., en= The root-knot nematode resistance ( Mi ) gene was screened in 25 tomato genotypes of Solanum lycopersicum , by amplification of REX-1 and Mi23 markers. Ten heterozygous tomato genotypes (Mimi), nine homozygous (MiMi) at the Mi locus and six lacking the Mi gene for resistance to root-knot nematode were identified using the marker REX-1. The results obtained with Mi23 marker confirmed the Mi gene status of the tomato genotypes, except for genotype Valouro RZ F1 that was homozygous (MiMi) and heterozygous (Mimi) at the Mi locus when using the REX-1 and Mi23 markers, respectively. The pathogenicity of Meloidogyne hispanica on the 25 tomato genotypes was assessed 60 days after inoculation with 5000 eggs on the basis of root gall index (GI) and reproduction factor (Rf). All the tomato genotypes were susceptible (excellent or good hosts), with GI>4 and Rf>2, except the genotype Rapit (Mimi), considered as resistant/hypersensitive (poor host). In this genotype, the nematode induced galls (GI=4) on its roots and a small number of eggs were produced (Pf=3085±485). Significant differences in reproduction were detected between the Mi allelic conditions and genotypes within Mi allelic conditions. The increasing number of Mi alleles (0, 1 or 2) is associated with decreasing Rf, which suggests a possible dosage effect of the Mi gene. The variability observed in the Rf values for MiMi tomato genotypes may reflect an influence of the genetic background of the plants containing the Mi gene. Ten of the 25 tomato genotypes with Mi gene are commercially available. However, only Rapit can be used to control the three most common Meloidogyne spp. and inhibit the increasing of M. hispanica populations, and may have potential to be included in an integrated pest management programme. However, it is advisable to evaluate the pathogenicity of local populations of this nematode species associated with different environmental factors. }

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.

Meloidogyne luci, a new root-knot nematode parasitizing potato in Portugal

In 2013, during a field survey conducted in Portugal on potato, Solanum tuberosum L., an unusual esterase (EST) phenotype was detected in a root-knot nematode (RKN) from potato roots collected in Coimbra. This Portuguese isolate was purified and maintained on tomato, S. lycopersicum L., and studied by morphological, biochemical and molecular characteristics. Perineal pattern morphology was highly variable, similar to M. ethiopica and not useful for the identification. The EST phenotype, from young egg-laying females, displayed three bands similar to the Brazilian M. luci (L3) and distinct from M. ethiopica (E3). Phylogenetic analyses of mitochondrial cytochrome oxidase subunit I and mitochondrial DNA region between COII and 16S rRNA genes revealed that the Portuguese isolate grouped with M. luci isolates close to M. ethiopica isolates. However, considering the ITS1-5.8S-ITS2 region the Portuguese isolate grouped with isolates of M. luci, M. ethiopica and M. hispanica, which limits the confidence of this region for M. luci diagnosis, and its differentiation from other species with morphological similarities. The M. luci pathogenicity to potato were also assessed in 16 commercial cultivars and compared with M. chitwoodi, considered a quarantine RKN species by EPPO. All potato cultivars were susceptible to both Meloidogyne species with gall indices of 5 and higher reproduction factor values ranging from 12.5 to 122.3, which suggest that M. luci may constitute a potential threat to potato production. In the present study, M. luci is reported for the first time attacking potato in Portugal. This article is protected by copyright. All rights reserved.

Laimaphelenchus suberensis sp. nov. associated with Quercus suber in Portugal

Laimaphelenchus suberensis sp. nov. obtained from declining Quercus suber trees of Herdade da Gouveia de Baixo, Alentejo, Portugal, is described and illustrated based on morphological, biometrical and molecular characters. The diagnosis of Laimaphelenchus species has been commonly based on the presence or absence of a vulval flap and on the shape structure of the tail tip. The species described here has been included in the Laimaphelenchus group without vulval flap, and can be distinguished from morphologically similar species by its tail tip shape structure that has a stalk-like terminus and three diffuse tubercles with 4–6 finger-like protrusions. For the molecular analyses, the mitochondrial DNA region from the cytochrome oxidase subunit I (mtCOI), the D2-D3 expansion segments of the large subunit (LSU) and small subunit (SSU) of rRNA gene were amplified and sequenced. Sequences of L. suberensis sp. nov. clustered separately from all Laimaphelenchus spp. with available sequences in Genbank, confirming its identification as a new species. This is the second report of the genus Laimaphelenchus in Portugal, associated with Q. suber: L. heidelbergi and L. suberensis sp. nov.

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.

Molecular characterization of putative parasitism genes in the plant-parasitic nematode Meloidogyne hispanica

Meloidogyne hispanica (Mhi) is a difficult-to-control polyphagous root-knot nematode (RKN) species of emerging importance for economically valuable crops. Nematode secretions are likely to be the first signals perceived by the plant and are thought to be involved in various aspects of the plant-nematode interaction. The aims of this work were to identify and characterize M. hispanica parasitism genes: cathepsin L cysteine protease (cpl-1), calreticulin (crt-1), β-1,4-endoglucanase-1 (eng-1) and manganese superoxide dismutase (mnsod). As there are no genomic data available for M. hispanica, primers were designed from the conserved regions of the putative parasitism genes in M. incognita and M. hapla and used to amplify the genes in M. hispanica, which led to the successful amplification of these genes in M. hispanica. Partial gene sequences were also obtained for M. arenaria, M. hapla, M. hispanica, M. incognita and M. javanica cpl-1, crt-1, eng-1 and mnsod genes, and their phylogenetic relationship analysed. In order to determine whether these genes are differentially expressed during M. hispanica development, cDNA was amplified from mRNA isolated from eggs, second-stage juveniles (J2) and females. Amplification products were observed from cDNA of all developmental stages for the Mhi-cpl-1 and Mhi-crt-1 genes. However, the gene Mhi-crt-1 exhibited intense amplification bands in females, while the Mhi-eng-1 gene was equally amplified in eggs and J2 and the Mhi-mnsod gene was only expressed in eggs. In comparison to the other RKN species, the genes Mhi-eng-1 and Mhi-mnsod showed transcription in different nematode developmental stages.