Regina M. D. G. Carneiro

Phylogeny of Meloidogyne spp. based on 18S rDNA and the intergenic region of mitochondrial DNA sequences

The 18S rDNA of 19 populations of Meloidogyne spp. was amplified and directly sequenced. The region of mitochondrial DNA, located in the 3′ portion of the gene that codes for cytochrome oxidase subunit II (COII) through a portion of the 16S rRNA (lRNA) gene, from 16 of these populations was cloned and sequenced. Heteroplasmic sequences were identified from both rDNA and mtDNA regions for several taxa. Several sequences sampled from nominal taxa differed from previously published accounts. Phylogenetic trees based on alignments of these sequences were constructed using distance, parsimony and likelihood optimality criteria. For 18S rDNA data, three main clades were identified. One well supported clade (86–91% bootstrap) included the most common and widely disseminated species, e.g.,M. arenaria, M. javanica and M. incognita, some recently described or redescribed species (M. floridensis, M. paranaensis, and M. ethiopica) plus numerous unidentified isolates. All mitotic parthenogenetic species, except for M. oryzae, were included in this clade. Other, less well supported clades included the amphimictic and facultative meiotic species M. hapla, M. microtyla, M. maritima and M. duytsi. One such clade comprised three meiotic parthenogens (M. exigua, M. graminicola and M. chitwoodi) and M. oryzae. This clade was moderately supported (77% bootstrap) but the relationships within this clade were poor. For mitochondrial DNA data, only the species in clade I from rDNA analysis, and M. hapla were analysed. These species formed a well supported clade (100% bootstrap) to the exclusion of M. mayaguensis and M. hapla. The addition of taxa and mtDNA data to publicly available records improved the discrimination sensitivity of species and atypical, non-identified, isolates.

Identification and genetic diversity of Meloidogyne spp. (Tylenchida: Meloidogynidae) on coffee from Brazil, Central America and Hawaii.

The present study was based on 18 populations of Meloidogyne spp. originating from different coffee fields in Brazil, Central America and the USA (Hawaii). The identification of the main species and an outline of the diversity of root-knot nematodes parasitising coffee in these countries with respect to esterase phenotypes, morphology and molecular polymorphism, are provided. With the present electrophoretic procedure, esterase phenotypes were demonstrated to be species-specific and constitute a good tool for identifying root-knot species from coffee, viz., M. incognita (Est I1, I2), M. paranaensis (Est P1, P2), M. arenaria (Est A2), M. arabicida (Est AR2), M. exigua (Est E1), M. mayaguensis (Est M2) and two unknown populations that probably represent new species (Est SA2, SA4). The perineal pattern is often an unreliable character when used alone for making diagnostic conclusions but, when used as a complementary tool together with enzyme characterisation, is essential for checking the morphological consistency of the identification. Male characters are important for confirming the diagnosis of some species, such as M. paranaensis, M. konaensis and M. incognita. The results showed that the RAPD markers produced are consistent with other approaches (esterase phenotypes and morphological features) for confirming species identification and for estimating genetic relationships among species and isolates. Phylogenetic analyses showed that M. mayaguensis and M. exigua are more closely related to one another than they are to the other species. This was also true for M. javanica, M. arenaria and Meloidogyne spp. Low levels of intraspecific polymorphism were detected in M. exigua (8.6%), M. incognita (11.2%) and M. paranaensis (20.3%). Conversely, M. arenaria and the two unknown Meloidogyne spp. exhibited higher levels of intra- or interspecific variability (34.9 and 29.9%, respectively).

Enzyme phenotypes and genetic diversity of root-knot nematodes parasitising Musa in Brazil

Esterase phenotypes were used to characterise 25 populations of Meloidogyne spp. collected on bananas from different banana producing areas in Brazil, using a simplified technique for routine analysis. Meloidogyne javanica, M. incognita, M. arenaria, and Meloidogyne spp. were detected at percentages of 61.7, 32.2, 4.3 and 1.8, respectively. The Meloidogyne populations represented mixed species in 80% of the samples, M. javanica and M. incognita prevailing under field conditions. Genetic analyses were conducted using RAPD markers which provided, alone or in combination, reliable polymorphisms both between and within species. Based on the presence or absence of bands, RAPD analysis of the data resulted in clustering of species and isolates congruent with esterase phenotype characterisation. The intraspecific variability in M. javanica, M. incognita and M. arenaria represented 29.1, 19.5 and 40.2% of the polymorphic amplified fragments, respectively. A general lack of correlation was observed between banana cultivar group, geographical origin and differentiation of the nematodes.

Meloidogyne ethiopica, a major root-knot nematode parasitising Vitis vinifera and other crops in Chile

Enzyme phenotypes, specifically esterases (EST) and malate dehydrogenase (MDH), were used to characterise different species of Meloidogyne from Chile. Esterase activity was highly polymorphic and was the most useful in the identification of the different species. Using this enzyme it is possible to characterise and identify M. ethiopica in about 80% of samples on grapevine, kiwi and tomatoes. Another three species, M. javanica, M. hapla and M. arenaria, were identified on tomatoes, kiwi and pomegranate with only one or a few populations. It was possible to detect minor atypical (unidentified) phenotypes, generally in mixed populations with M. ethiopica. Only the profiles N1 and H1 of MDH were detected. N1 was not specific and H1 allowed identification of M. hapla. Contaminated nursery stock has probably resulted in serious infestation by M. ethiopica in vineyards in various localities in Chile.

Resistance to Meloidogyne mayaguensis in Psidium spp. Accessions and their grafting compatibility with P. guajava cv. Paluma

Meloidogyne mayaguensis has been reported in some states of Brazil causing severe damage on commercial guava (Psidium guajava L.). Accessions of Psidium spp. were selected from a collection maintained in Embrapa Clima Temperado (Pelotas, Rio Grande do Sul State). Plants of different accessions were grown from seed in plastic bags and, when they reached 15-20 cm in height, were inoculated with 10,000 eggs/plant of M. mayaguensis. Eight months after inoculation, the different accessions were evaluated for resistance to M. mayaguensis. Three accessions of P. guajava were highly susceptible (RF=59.2) to this nematode. Psidium friedrichsthalianium was considered to be moderately resistant (RF=1.9). Three accessions of P. cattleyanum were immune to M. mayaguensis (RF = 0). When used as rootstocks P. cattleyanum and P. friedrichsthalianium were compatible with P. guajava cv. Paluma. Considering these results, the use of resistant rootstocks provides a promising control method for M. mayaguensis in commercial guava crop.

Diversity of Meloidogyne arenaria using morphological, cytological and molecular approaches

Thirteen Meloidogyne arenaria isolates representing two cytological types (3n = 51-56, 2n = 42-48) and four enzymatic phenotypes (esterase and malate dehydrogenase: A3N1, A2N1, A1N1, A2N3) were studied using different approaches. The analysis of molecular markers showed a high level of polymorphism among the isolates. The trees obtained with RAPD or ISSR polymorphisms showed concordant results and agree with morphological studies. By considering morphometrical and morphological features, it was possible to conclude that the isolate with enzymatic phenotype A2N3 race 1 was the M. arenaria described in 1949 by Chitwood and appearing clearly separated in the trees, as well as in the outgroups. The seven isolates with phenotype A2N1 from different localities and isolate A1N1 can be considered morphometrically typical of M. arenaria race 2 and they were apparently clustered by geographical origin. Morphologically, they differed from isolate A2N3 race 1. The two isolates with phenotype A3N1 appeared to be closely related to the isolate of M. morocciensis and, considering all of the features described for this species, were identified as such. The two isolates A2N3 race 2 were identified either as an atypical M. arenaria or an unidentified species (females and males having atypical stylets), and clustered together and separated from other M. arenaria isolates with high bootstrap support. The same M. arenaria isolates were tested with the species-specific molecular marker, type SCAR. A fragment of 420 bp was obtained for ten isolates of M. arenaria, including the atypical A2N3 race 2 and M. morocciensis. This fragment was not amplified for three typical A2N1 isolates of M. arenaria.

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.

Validation of reference genes aiming accurate normalization of qPCR data in soybean upon nematode parasitism and insect attack.

BackgroundSoybean pathogens and pests reduce grain production worldwide. Biotic interaction cause extensive changes in plant gene expression profile and the data produced by functional genomics studies need validation, usually done by quantitative PCR. Nevertheless, this technique relies on accurate normalization which, in turn, depends upon the proper selection of stable reference genes for each experimental condition. To date, only a few studies were performed to validate reference genes in soybean subjected to biotic stress. Here, we report reference genes validation in soybean during root-knot nematode (Meloidogyne incognita) parasitism and velvetbean caterpillar (Anticarsia gemmatalis) attack.FindingsThe expression stability of nine classical reference genes (GmCYP2, GmELF1A, GmELF1B, GmACT11, GmTUB, GmTUA5, GmG6PD, GmUBC2 and GmUBC4) was evaluated using twenty-four experimental samples including different organs, developmental stages, roots infected with M. incognita and leaves attacked by A. gemmatalis. Two different algorithms (geNorm and NormFinder) were used to determine expression stability. GmCYP2 and GmUBC4 are the most stable in different organs. Considering the developmental stages, GmELF1A and GmELF1B genes are the most stable. For spatial and temporal gene expression studies, normalization may be performed using GmUBC4, GmUBC2, GmCYP2 and GmACT11 as reference genes. Our data indicate that both GmELF1A and GmTUA5 are the most stable reference genes for data normalization obtained from soybean roots infected with M. incognita, and GmCYP2 and GmELF1A are the most stable in soybean leaves infested with A. gemmatalis.ConclusionsFuture expression studies using nematode infection and caterpilar infestation in soybean plant may utilize the reference gene sets reported here.

Cultural and morphological characterization of Pochonia chlamydosporia and Lecanicillium psalliotae isolated from Meloidogyne mayaguensis eggs in Brazil

The root-knot nematode, Meloidogyne mayaguensis, causes serious economic loss to guava plantation in Brazil. Fungi associated with eggs of M. mayaguensis in Brazilian soil have not been studied yet, but this knowledge could form the basis for future field studies on biological control of this nematode. The egg parasitic fungi, Pochonia chlamydosporia var. chlamydosporia, P.chlamydosporia var. catenulata and Lecanicillium psalliotae, were isolated from M. mayaguensis eggs and analyzed based on their cultural and morphological characteristics. The optimum temperature for growth and spore production ranged between 24 and 28oC for P. chlamydosporia isolates. For the isolate of L. psalliotae, the best growth was at 30oC. In solid state fermentation, P. chlamydosporia isolates produced between 3.5 and 5.2 x 106 chlamydospores per g of colonized substrate, whereas L. psalliotae isolate produced 8.5 x 108 conidia per g of colonized substrate. Future studies are required to clarify the potential of these fungi as biological control agents of M. mayaguensis.

Taxonomy of Coffee-Parasitic Root-Knot Nematodes, Meloidogyne spp.

Meloidogyne species are characterized primarily on morphological features of females, males and second-stage juveniles. Based on these characters, identifying the 17 coffee-parasitic Meloidogyne species is a difficult task even for well-qualified taxonomists. This chapter outlines the most diagnostic morphological and morphometric features for Meloidogyne taxonomy, and presents the useful characters for identification of those 17 species. In recent years, esterase phenotyping has become a practical and reliable taxonomic tool for this genus. Unfortunately, only 12 out of the 17 coffee-parasitic species have had their phenotypes characterized; M. africana, M. decalineata, M. kikuiensis, M. megadora and M. oteifae can only be identified by morphological features. Recently, a new identification tool has been developed: the multiplex PCR (SCAR primers) allows unambiguous differentiation of M. exigua, M. incognita and M. paranaensis from Brazil, with prospects for extending this method to other species. This chapter concludes by outlining studies and initiatives that should be undertaken to facilitate and improve the reliability of coffee-related Meloidogyne taxonomy.