Maria Ritta A. Almeida

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).

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.

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.

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.

Meloidogyne izalcoensis n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitising coffee in El Salvador

A new root-knot nematode parasitising coffee in the region of the Izalco volcano, Sansonate, El Salvador, is described as Meloidogyne izalcoensis n. sp. The suggested common name is El Salvador coffee root-knot nematode. The perineal pattern is characterised by the moderately high to high, squareish to rounded, dorsal arch, striae coarse, smooth to wavy, sometimes zigzaggy, usually without a distinct whorl, and similar to that of M. incognita and M. paranaensis. The female head region set off from body, sometimes annulated, and the labial disc has two prominent elevations or bumps on the ventral side that are slightly raised above the medial lips. The female stylet is robust, 15.0-16.0 μm long and with the DGO at 4.5-6.0 μm posterior to the knobs. Males have a high, round, head cap continuous with the body contour and the labial disc is fused with the medial lips to form an elongated structure. The head region is never marked by incomplete annulations. The stylet is robust, 23.0-26.0 μm long, and has rounded, backwardly sloping, knobs with the DGO located at 4.0-7.0 μm posteriorly. The stylet of second-stage juveniles is 12.0-13.0 μm long and the DGO is located 3.0-4.0 μm posterior to the stylet base; the tail is 45-48 μm long, conoid, with a rounded terminus. Biochemically, the esterase phenotype I4 (= Sa4) (Rm: 0.86, 0.96, 1.24, 1.30) is unique and is the most useful character to differentiate M. izalcoensis n. sp. from all other species.

Reaction of coffee genotypes to different populations of Meloidogyne spp.: detection of a naturally virulent M. exigua population

The reaction of seven genotypes of Coffea arabica to 10 Meloidogyne spp. populations collected mainly from coffee plantations in Brazil and Costa Rica was evaluated under greenhouse conditions. The inoculum consisted of 10,000 eggs per plant. Evaluations were done 8 months after inoculations considering the root fresh weight, gall and egg mass indices, number of eggs per gram of root and reproduction factor (RF). The cultivars Obata IAC 1669-20, Sarchimor IAC 4361 and Tupi Amarelo IAC 5111 exhibited susceptibility to the four Brazilian M. exigua populations tested. However, cv. Tupi Vermelho IAC 1669-33 revealed resistance (RF value of 0.7) to the M. exigua population from Lavras, Minas Gerais State, Brazil. A population of M. exigua from Bom Jesus de Itabapoana, Rio de Janeiro State, Brazil, was highly virulent on cv. IAPAR 59 (RF= 165.7), bearing resistance gene Mex-1, and was also virulent on genotype Paraiso (H 419-5-4-5-2) (RF=396.2). A Meloidogyne sp. population on coffee from Garca, Sao Paulo State, Brazil, reproduced at low rates (RF ranging from 0.1 to 3.9) on all genotypes. All tested cultivars were susceptible to M. incognita and M. paranaensis. M. mayaguensis of guava from Parana State, Brazil, reproduced at low rates in all coffee genotypes; however, another population of coffee, from Costa Rica, was more aggressive and showed RF value that ranged from 0.8 to 12.4. Results of this study point for the first time to the ability of a naturally occurring M. exigua population to overcome the resistance conferred by the Mex-1 gene.

Detecção de Meloidogyne mayaguensis em goiabeira e mamoeiro no estado de Goiás, usando marcadores moleculares

Detection of Meloidogyne mayaguensis on guava and papaya in Goias State of Brazil using molecular markers Meloidogyne mayaguensis was reported for the first time in the State of Goias (Formosa and Luziânia), causing damage in oneyear old and 14 year-old commercial guava (Psidium guajava) cv. Paluma orchards. Plants infected by the nematode showed symptoms such as stunted growth, general chlorosis, nutrient deficiency and consequent decline in yield quality and quantity. Severely infested root systems were poorly developed, distorted by small and large multiple galls and devoid of fine roots. Plants of papaya cv. Formosa were cultivated in consortium with guava in the Formosa orchard and presented several galls in the root system, but no root-knot-nematode root-knot-nematode secondary symptoms were observed in the aerial part. The production of papaya fruits was high, evidencing tolerance of this cultivar to the symptoms were observed in the aerial part. The production of papaya fruits was high, evidencing tolerance of this cultivar to the were observed in the aerial part. The production of papaya fruits was high, evidencing tolerance of this cultivar to the nematode. The M2 phenotype (Rm: 0.7, 0.9) was detected for the isoenzyme esterase and M. mayaguensis was identified in both crops and orchards. The analyses with species-specific molecular markers using primers that amplify intergenic regions of the ribosomal DNA and the mitochondrial DNA confirmed the same diagnosis. Surveys carried out in other localities of the farm in Formosa detected the presence of Meloidogyne javanica javanica in low population, corroborating the idea that M. mayaguensis was introduced from the Petrolina nursery with the Petrolina nursery with

Additional information on Meloidogyne inornata Lordello, 1956 (Tylenchida: Meloidogynidae) and its characterisation as a valid species

A root-knot nematode parasitising yakon (Polymia sonchifolia) in Sao Paulo State, Brazil, is identified as Meloidogyne inornata. The species is redescribed from this material and compared with the original description of M. inornata. The female perineal patterns have a distinct, high, dorsal arch composed of smooth to wavy striae, similar to Meloidogyne incognita. The female stylet is 15.0-17.0 ?m long with the cone generally slightly curved dorsally and with well developed knobs. DGO is 3.5-4.5 ?m. Males have a high, rounded, head cap that is continuous with the body contour and has a large, round, centrally concave, labial disc raised above the medial lips. The head region is never marked by incomplete annulations and the stylet is robust, 20.0-25.0 ?m long, with a straight cone, cylindrical shaft with several small projections and pear-shaped, backwardly sloping knobs. The stylet length of second-stage juveniles is 10.0-13.0 ?m, DGO is 2.5-3.5 ?m, tail length is 35.0-58.0 ?m and c = 6.7-13.9. Biochemically, the esterase phenotype I3 (= Y3) is species-specific and is the most useful character for differentiating M. inornata from other Meloidogyne species. Reproduction is by mitotic parthenogenesis, 3n = 54-58. In a soybean test, cv. Abura was susceptible and cv. LA411219 was highly resistant. As the type material is lost, a neotype female is formally designated.

Meloidogyne luci n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitising different crops in Brazil, Chile and Iran

A new root-knot nematode parasitising vegetables, flowers and fruits in Brazil, Iran and Chile, is described as Meloidogyne luci n. sp. The female has an oval to squarish perineal pattern with a low to moderately high dorsal arc and without shoulders, similar to M. ethiopica. The female stylet is robust and 15-16 µm long; the distance from the dorsal pharyngeal gland orifice to the stylet base (DGO) is 3-4 µm. Males have a high, rounded head cap continuous with the body contour. The labial disc is fused with the medial lips to form an elongated lip structure. The head region is not marked by incomplete annulations. Male stylet robust, 20.8-23.0 µm long with rounded knobs; the DGO is 2.5-4.5 µm. The stylet of second-stage juveniles (J2) is 12.0-13.5 µm long and the DGO to the stylet base is 2.3-3.3 µm. The J2 tail is conoid with finely rounded terminus and is 40.0-48.5 µm long. Biochemically, the esterase phenotype L3 (Rm: 1.05, 1.10, 1.25) is unique and is the most useful character to differentiate M. luci n. sp. from all other Meloidogyne species. Reproduction is by mitotic parthenogenesis (2n = 42-46 chromosomes). In a differential host test, the population from Lavandula spica, Caxias do Sul, RS State, Brazil, reproduced on tomato cv. Rutgers, tobacco cv. NC95 and pepper cv. California Wonder. No reproduction occurred on watermelon cv. Charleston Gray, cotton cv. Deltapine 61 or peanut cv. Florunner. In Neighbour-Joining analyses of ITS and D2-D3 rRNA sequences, populations of M. luci n. sp. from Brazil, Chile and Iran clustered together and were clearly separated from other Meloidogyne spp., thus confirming that all three populations are very similar and conspecific.

Meloidogyne spp. populations from native Cerrado and soybean cultivated areas: genetic variability and aggressiveness

A significant portion of the Cerrado (Brazilian savanna) has been replaced by major crops such as soybean. This may reveal populations of nematodes with different genetic backgrounds compared to cultivated fields. The objectives of this study were to evaluate the genetic variability and aggressiveness of isolates of Meloidogyne spp., contrasting nematodes from preserved areas of the Cerrado with those originating from cultivated soybean fields. Cluster analysis separated isolates of Meloidogyne spp. and isolates from Cerrado and soybean but did not separate an aggressive Meloidogyne morocciensis isolate. The aggressiveness of six selected populations of Meloidogyne spp. from Cerrado and soybean against soybean cultivars was evaluated. Results showed that populations of M. javanica and M. incognita from Cerrado and soybean showed similar aggressiveness. However, for M. morocciensis , the population from soybean was much more aggressive than the one from Cerrado. Aggressiveness is a very intriguing subject that needs special attention for future research in nematology.