Distribution of Meloidogyne species in carrot in Brazil

ABSTRACT: Root-knot nematodes (RKN - Meloidogyne spp.) are one of the most serious threats to carrot production worldwide. In Brazil, carrots are grown throughout the year, and economic losses due to RKN are reported. Since little is known on the distribution of RKN species in carrot fields in Brazil, we collected plant and soil samples from 35 fields across six states. Based on the morphology of perineal patterns, esterase phenotypes and species-specific PCR, three Meloidogyne species were identified: 60% of the fields were infested with Meloidogyne incognita, M. javanica was reported in 42.9% of the areas, whereas M. hapla was detected in 17.1% of carrot fields. Mixed populations were reported in 20% of the areas with a predominance of M. incognita + M. javanica. The combination of morphological, biochemical, and molecular techniques is a useful approach to identify RKN species.

Integrative taxonomy of Meloidogyne graminicola populations with different esterase phenotypes parasitising rice in Brazil

Summary The rice root-knot nematode, Meloidogyne graminicola, has been reported in Southeast Asia, China, India, South Africa, USA, Brazil, and other countries. Recent surveys in Southern Brazil showed that M. graminicola was widespread in irrigated rice in Rio Grande do Sul, Santa Catarina and Paraná states, and the presence of a species complex with a predominance of M. graminicola (Est VS1 = G1) and other variants showing similar esterase phenotypes (Est G2 = R2, G3 = R3). Meloidogyne oryzae (Est O1) and M. ottersoni (Est Ot0) were also part of this complex and were recently re-described and detected on rice. The present study provides an integrative taxonomy approach of the typical and atypical populations of M. graminicola on the basis of morphological, morphometric and molecular data. Considering morphological and morphometric features, the two atypical populations (Est G2 and G3) are in close agreement with the description of M. graminicola. Based on the molecular characterisation, populations G1, G2 and G3 were successfully amplified by M. graminicola SCAR markers, although the specificity of these markers was questioned. Phylogenetic relationships complemented and confirmed the other studies. In maximum likelihood analysis of ITS, D2-D3 rRNA and COXII-16S rRNA sequences, all populations of M. graminicola from different esterase phenotypes clustered together with other M. graminicola populations, thus confirming that these enzyme phenotypes (G1, G2 and G3) are related to the same species. A high level of intraspecific variability was detected among all populations, but no correlation between genetic variability and geographic origins occurred.

Integrative taxonomy of Meloidogyne paranaensis populations belonging to different esterase phenotypes

Summary Meloidogyne paranaensis is one of the most destructive root-knot nematode species affecting coffee cultivation. This species presents different esterase phenotypes (Est): P1, P2 and P2a, previous studies showing that Est P2 and P2a populations were more aggressive to susceptible coffee cultivars than populations with Est P1, and local producers have even asked if they may be described as other species. The objective of this study was to characterise M. paranaensis populations of different esterase phenotypes (Est P1, P2 and P2a), regarding morphological, morphometric and phylogenetic relationships in distinct regions of ribosomal DNA (rDNA), mitochondrial gene cytochrome c oxidase II (COII) and nuclear protein coding gene HSP90. All populations were identified by esterase phenotype and SCAR-specific markers. Regarding morphology/morphometrics, the three populations were very similar to the description of the species, differing only in the morphology of the male stylet and second-stage juvenile hyaline tail length. Based on the phylogenetic analysis, a low intraspecific variability was detected among M. paranaensis Est P1 and Est P2 populations from Brazil; the Guatemalan population Est P2a, however, showed a genetic differentiation from the Brazilian populations, confirming the geographic genetic distance of this aggressive population. According to this multi-source approach study, in spite of the intraspecific variation, the phylogenetic position of M. paranaensis is absolute, regardless of the enzymatic phenotype and SCAR markers.

Diagnostic methods for identification of root-knot nematodes species from Brazil

ABSTRACT: The accurate identification of root-knot nematode (RKN) species (Meloidogyne spp.) is essential for implementing management strategies. Methods based on the morphology of adults, isozymes phenotypes and DNA analysis can be used for the diagnosis of RKN. Traditionally, RKN species are identified by the analysis of the perineal patterns and esterase phenotypes. For both procedures, mature females are required. Over the last few decades, accurate and rapid molecular techniques have been validated for RKN diagnosis, including eggs, juveniles and adults as DNA sources. Here, we emphasized the methods used for diagnosis of RKN, including emerging molecular techniques, focusing on the major species reported in Brazil.

Characterisation of a Meloidogyne species complex parasitising rice in southern Brazil

Root-knot nematodes (RKN) are important plant pathogens affecting rice in South-East Asia and southern Brazil in irrigated rice fields. In order to investigate the specific diversity of RKN associated with irrigated rice in southern Brazil, Meloidogyne spp. from Rio Grande do Sul (RS) and Santa Catarina (SC) States were characterised biochemically by esterase (Est) and malate dehydrogenase (Mdh) phenotypes. Fifty-six Meloidogyne spp. populations were detected in 48% of rice samples, and a total of five esterase phenotypes were identified, four of which presented as drawn-out bands in different positions. In RS State, M. graminicola (Est VS1), Meloidogyne sp. 2 (Est R2) and Meloidogyne sp. 3 (Est R3) were identified, which corresponded to ca 80, 40 and 10% of samples, respectively. In SC State, M. graminicola, M. javanica (Est J3), Meloidogyne sp. 1 (Est R1), Meloidogyne sp. 2 and Meloidogyne sp. 3 accounted for ca 93.75, 12.50, 62.50, 12.25 and 6.25% of samples, respectively. The esterase phenotypes R1, R2 and R3 are new, never having been detected on rice before. Meloidogyne javanica showed a N1 Mdh phenotype (Rm: 1.0), while four other populations exhibited a N1a (Rm: 1.4) phenotype. All populations were tested with two SCAR markers specific to M. graminicola, which confirmed that, but no specificity was obtained with both markers in relation to the atypical populations analysed. Sequencing and phylogenetic analyses of internal transcribed spacer-rRNA (ITS) were performed to infer the phylogenetic relationship of these atypical Meloidogyne spp. populations. Meloidogyne sp. 1 grouped with the mitotic parthenogenetic species, while the two others (Meloidogyne sp. 2 and sp. 3) clustered with M. graminicola and other meiotic parthenogenetic species. Taken together, these data highlight the unprecedented specific diversity of RKN associated with irrigated rice in southern Brazil. Further morphological and phylogenetic studies involving these atypical isolates will be carried out to identify this complex of species.

Meloidogyne luci, a new infecting nematode species on common bean fields at Paraná State, Brazil

SummaryCommon bean diseased plants with symptoms of decline and root knots were collected in two growing areas in the municipality of Araucária, Paraná State (Brazil). Morphological (perineal patterns), biochemical (esterase phenotypes) and molecular (ITS1 sequences) studies allowed us to identify the infecting nematode as Meloidogyne luci. To our knowledge, this is the first formal record of M. luci parasitizing common bean in Paraná State, Brazil.

Root-Knot Disease Caused by Meloidogyne arenaria and M. javanica in Teak in São Paulo State, Brazil

Teak (Tectona grandis Linn. F.) is one of the most important forest crops in Brazil, occupying areas in different regions, such as Goiás, Mato Grosso, Paraná, and São Paulo states. Teak wood is used for many purposes such as shipbuilding, rolling and plywood, firewood, and charcoal. In May 2011, teak symptomatic feeder root samples, exhibiting inconspicuous, small galls, were collected in the municipality of Piracicaba, São Paulo State, Brazil (22°41′46.90″S, 47°38′36.84″W). Specimens were identified through perineal patterns and esterase phenotypes of 20 adult females (1,2). Perineal patterns and esterase phenotypes were consistent with those described for Meloidogyne arenaria (Neal, 1889) Chitwood, 1949 and M. javanica (Treub, 1885) Chitwood, 1949. Perineal patterns of M. arenaria showed a low dorsal arch, compressed dorsolaterally, with lateral field marked by some forked and broken striae; no punctate markings between anus and tail terminus were observed. Perineal patterns of M. javanica were rounded, with low dorsal arch, striae smooth, lateral field distinct, clearly demarcated from striae by parallel lines. From the esterase electrophoresis we obtained A2 (Rm:1.2;1.3) and J3 (Rm:1.0;1.25;1.4) phenotypes, typical from M. arenaria and M. javanica, respectively. To our knowledge, this is the first report of M. arenaria parasitizing teak roots in Brazil and elsewhere (new host) and the first report of M. javanica infecting teak in the State of São Paulo. Previously, M. javanica was reported to be infecting teak-growing areas in the State of Mato Grosso (3). This finding has a great importance, not only by the inclusion of these parasites in teak pathological scenario, but also for predicting possible damage in plant species used in teak-based intercropping systems. References: (1) P. R. Esbenshade and A. C. Triantaphyllou. J. Nematol. 22:10, 1990. (2) K. M. Hartman and J. N. Sasser. 1985. Page 115 in: An Advanced Treatise on Meloidogyne. Volume II, Methodology. K. R. Barker et al., eds. North Carolina State University Graphics, Raleigh,1985. (3) R. A. Silva et al. Nematol. Bras. 27:261, 2003.

Diversity of Meloidogyne exigua (Tylenchida: Meloidogynidae) populations from coffee and rubber tree

Isozymes (esterase and malate dehydrogenase), SCAR and RAPD-PCR were studied in 15 populations of three races of Meloidogyne exigua collected in coffee-producing areas in Brazil, Bolivia and Costa Rica and one population from rubber tree plantations in Brazil. This study revealed four esterase phenotypes (E1, E2, E2a, E3) and three malate dehydrogenase phenotypes (N1, N1a, N2) for M. exigua populations. The most common multi-enzyme phenotype was E2N1. The enzymatic phenotypes do not separate M. exigua races. Sixteen populations of M. exigua were tested in Multiplex PCR using SCAR primers ex-D15F/R that allowed the identification of all M. exigua populations. Phylogenetic analyses showed high intraspecific polymorphism (25.9-59.6%) for all M. exigua studied. However, all populations clustered together with 100% bootstrap support, thereby demonstrating the consistency of species identification. In general, no correlation was found between enzymatic profile, race and genetic polymorphism of the studied populations.

Characterisation of Meloidogyne spp. (Tylenchida: Meloidogynidae) from coffee plantations in Central America and Brazil

Isozymes (Esterases, MDH, SOD, GOT) and perineal patterns were studied in 29 isolates of Meloidogyne spp. collected on coffee (Coffea arabica) plantations in four Central American countries and on one isolate collected in Brazil. Five species were clearly diagnosed and six new multi-enzyme phenotypes were also revealed corresponding to within-species diversity or possible new species. Meloidogyne exigua was found in Costa Rica, Nicaragua and Honduras, M. arenaria in El Salvador and M. incognita ('M1a' esterase phenotype) in Brazil. Meloidogyne arabicida was found in Costa Rica and has a new esterase phenotype, 'M1F1b'. Nematodes with the 'F1' esterase phenotype were found in Guatemala and their specific status is discussed. Two isolates from El Salvador displayed unknown esterase phenotypes ('M1F1a' and 'Sa4'). One isolate from northern Guatemala was clearly identified as Meloidogyne hapla and another from the same area was related to M. enterolobii or M. mayaguensis. Neither of these latter isolates was able to develop in coffee roots under our growing conditions. The diversity of root-knot nematodes parasitising coffee roots in this region is discussed.

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).