The objectives of this work were to evaluate the genetic variability of Meloidogyne enterolobii by molecular markers, and develop species-specific molecular markers for application in detection. Sixteen M. enterolobii isolates from different geographical regions (Brazil and other countries) and hosts were used in this study. The identification and purification of the populations were carried out based on isoenzyme phenotype. The DNA amplification of the intergenic region (IGS) of the rDNA and of the region between the cytochrome oxidase subunit II (COII) and 16S rRNA genes (mtDNA) produced specific fragments of the expected size for this nematode, i.e. 780 and 705 bp, respectively. Intraspecific variability among the isolates was evaluated with three different neutral molecular markers: AFLP, ISSR and RAPD. The results showed a low level of diversity among the isolates tested, indicating that M. enterolobii is a genetically homogeneous root-knot nematode species. The RAPD method allowed the identification of a species-specific RAPD fragment for M. enterolobii. This fragment was cloned and sequenced, and from the sequence obtained, a set of primers was designed and tested. The amplification of a 520-bp-long fragment occurred only for the 16 isolates of M. enterolobii and not for the 10 other Meloidogyne species tested. In addition, positive detection was achieved in a single individual female, egg-mass and second stage juvenile of this nematode. This SCAR species-specific marker for M. enterolobii represents a new molecular tool to be used in the detection of this nematode from field samples and as a routine diagnostic test for quarantine devices.

A phylogenetic analysis of the 5.8S rDNA and internal transcribed spacer (ITS1 and ITS2) sequences from some entomogenous Paecilomyces species supports the polyphyly of the genus and showed the existence of cryptic species. In the Eurotiales, anamorphs Paecilomyces variotii and Paecilomyces leycettanus were related to the teleomorphs Talaromyces and Thermoascus. In the Hypocreales, three major ITS subgroups were found, one of which included Paecilomyces viridis, Paecilomyces penicillatus, Paecilomyces carneus and isolates identified as Paecilomyces lilacinus and Paecilomyces marquandii. However, the majority of the P. lilacinus and P. marquandii isolates formed a distinct and distantly related subgroup, while the other major subgroup contained Paecilomyces farinosus, Paecilomyces amoeneroseus, Paecilomyces fumosoroseus and Paecilomyces tenuipes.

Twenty three isolates of

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Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n040-46). Three M. hispanica isolates (Hi3/N1, 2n032-36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n040-44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200 bp, 955 bp and 399 bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650 bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M.

Summary -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.

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RESUMOMeloidogyne mayaguensis foi detectado pela primeira vez no estado de Goiás, em duas propriedades (Formosa e Luziânia), causando dano em pomares comerciais de goiaba (Psidium guajava) cv. Paluma de um ano de idade e de 14 anos, respectivamente. Plantas infectadas pelo nematóide mostraram redução de crescimento, clorose generalizada, deficiência nutricional e redução qualitativa e quantitativa de produção. As raízes severamente infestadas apresentaram-se pouco desenvolvidas e deformadas pela presença de múltiplas galhas de tamanho variado. Mamoeiros (Carica papaya) cv. Formosa, plantados em consórcio com as goiabeiras na propriedade de Formosa, apresentaram numerosas galhas no sistema radicular, embora, nenhum sintoma secundário de meloidoginose tenha sido observado na parte aérea. A produção de frutos dos mamoeiros foi alta, evidenciando tolerância dessa cultivar ao nematóide. O fenótipo M2 para a isoenzima esterase (Rm: 0,7, 0,9) foi detectado e M. mayaguensis identificado em ambas as culturas e propriedades. As análises com marcadores moleculares espécie-específica usando primers que amplificam regiões intergênicas do DNA ribossomal e do DNA mitocondrial também confirmaram esse diagnóstico. Levantamento realizado, em outras localidades da fazenda em Formosa mostrou a presença de Meloidogyne javanica em baixa população, corroborando a idéia de introdução de M. mayaguensis na área, através do plantio de mudas infectadas, oriundas da região de Petrolina. Na propriedade em Luziânia, o nematóide é provavelmente de ocorrência natural, considerando-se a idade das plantas e o número reduzido de goiabeiras infectadas. Palavras-chave: fenótipo de esterase, DNA ribossômico, DNA mitocondrial, identificação, nematóide das galhas. ABSTRACT Detection of Meloidogyne mayaguensis on guava and papaya in Goiás State of Brazil using molecular markersMeloidogyne mayaguensis was reported for the first time in the State of Goiás (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...