Claudio Marcelo Gonçalves de Oliveira

Morphological and molecular diagnostics for plant-parasitic nematodes: working together to get the identification done

Nematodes are considered one of the most difficult organisms to identify due to their microscopic size, morphological similarity, limited number of distinguishable taxonomic characters and overlapping morphometric measurements. However, there are few trained nematode taxonomists remaining, primarily due to retirement without replacement and/or young scientists lack of interest in classical taxonomy. Due to the continuing decline in classical taxonomic expertise of many taxa including Nematoda, there is an increasing reliance on developing molecular-based diagnostic protocols to identify pests and pathogens. To improve the resolution and reliability of nematode phylogenetic and diagnostic studies, they should ideally combine morphological with molecular data. However, few of the published studies have compared phylogenetic trees derived from morphological/morphometric characters with those derived from molecular data. Furthermore, nematode sequences deposited in public databases should have an identification based on morphological and morphometrical characters ascertained by a trained taxonomist. Nevertheless, molecular diagnostics do offer new opportunities in Nematology, such as improving the capacity to deal with large numbers of samples or with species mixtures which have previously been impractical. In conclusion, a balanced molecular and morphological taxonomic approach is required as proposed by the integrative taxonomy concept. Classical taxonomy and molecular diagnostics should be considered as complementary.

Fauna de fitonematóides em áreas preservadas e cultivadas da floresta amazônica no Estado de Mato Grosso

The aim of this work was to study the diversity of the fauna of plant-parasitic nematodes in preserved areas of the Amazon forest, Mato Grosso state (MT), and to assess the effect of agricultural land use on plant-parasitic nematode communities. Soil and root samples were collected in each location in the late spring during the rainy season of 2005, in two areas of primary vegetation in Nova Maringa (Northwest) and Guaranta do Norte (North) and two adjacent areas planted with teak trees (Tectona grandis) and pasture (Brachiaria brizantha). Fourteen taxa of plant-parasitic nematodes were identified at species level: Discocriconemella degrissei, D. limitanea, Dolichodorus minor, Helicotylenchus erythrinae, H. pseudorobustus, Meloidogyne exigua, M. javanica, Mesocriconema ornata, Paratrichodorus minor, Pratylenchus loosi, P. zeae, Rotylenchus caudaphasmidius, Xiphinema ensiculiferum and X. luci (for the first report of this in Brazil) and five at genus level (Atalodera sp., Hemicriconemoides sp., Meloidogyne sp., Paratylenchus sp., and Trophotylenchulus sp). These taxa, mainly those from primary vegetation, belong to families with different parasitic behavior, probably due to great plant diversity in the Amazon forest. Comparison between the two preserved areas revealed low index of similarity, as a consequence of the endemic flora in the Amazon forest, and no similarity was observed between preserved native vegetation and adjacent cultivated areas, demonstrating the high influence of agricultural activity on the plant-parasitic nematode communities. There is evidence of recent introduction of plant-parasitic nematodes in these cultivated areas; therefore measures should be taken to prevent the loss of economic sustainability in Amazonian soils.

Pathogenicity of two Pratylenchus coffeae populations from Brazil on coffee plants

There is limited information on the influence of Pratylenchus coffeae on the growth and development of coffee plants, in spite of the widespread occurrence of this nematode in coffee plantations. In addition, populations of P. coffeae vary in morphological and molecular features, as well as reproductive fitness and pathological potential. The objective of the present study was to compare the pathogenicity of two Brazilian P. coffeae populations, K5 from Coffea arabica roots and M2 from Aglaonema sp. roots, in terms of their influence on the plant growth and photosynthesis of Arabian coffee (C. arabica). Five experiments were conducted in controlled conditions, and the results demonstrated that K5 is pathogenic on coffee, as it can reproduce and causes obvious damage on the plant. Moreover K5 proved to be very virulent on Arabian coffee, considering its effects on seedling mortality, plant growth and photosynthesis. By contrast, M2 was considered to be of low virulence, or even non-pathogenic, on coffee because it failed to reproduce. Thus, the results indicate that K5 and M2 may be distinct species, supporting the hypothesis of previous authors.

Entomopathogenic nematodes in agricultural areas in Brazil

Entomopathogenic nematodes (EPNs) (Steinernematidae and Heterorhabditidae) can control pests due to the mutualistic association with bacteria that kill the host by septicemia and make the environment favorable for EPNs development and reproduction. The diversity of EPNs in Brazilian soils requires further study. The identification of EPNs, adapted to environmental and climatic conditions of cultivated areas is important for sustainable pest suppression in integrated management programs in agricultural areas of Brazil. The objective was to identify EPNs isolated from agricultural soils with annual, fruit and forest crops in Brazil. Soil samples were collected and stored in 250u2009ml glass vials. The nematodes were isolated from these samples with live bait traps ([Galleria mellonella L. (Lepidoptera: Pyralidae) larvae]. Infective juveniles were collected with White traps and identified by DNA barcoding procedures by sequencing the D2/D3 expansion of the 28S rDNA region by PCR. EPNs identified in agricultural areas in Brazil were Heterorhabditis amazonensis, Metarhabditis rainai, Oscheios tipulae and Steinernema rarum. These species should be considered pest biocontrol agents in Brazilian agricultural areas.

Variabilidade in vitro, in vivo e molecular de isolados de Cercospora coffeicola

Neste estudo, sete isolados de Cercospora coffeicola, agente causal da cercosporiose em cafeeiros, foram estudados com relacao ao crescimento micelial em diferentes temperaturas, comparacao das sequencias das regioes ITS1 e ITS2 do DNA ribossomico e de parte do gene da calmodulina, e com relacao a patogenicidade a oito cultivares de cafeeiro (Catuai Vermelho IAC 81, Catuai SH3, Tupi IAC 1669 - 33, Tupi RN IAC 1669 - 13, Icatu Vermelho IAC 4045, Ouro Verde IAC H 5010-5, Geisha IAC 1137 e Mundo Novo IAC 376-4). As temperaturas de maior crescimento micelial foram de 23,8, 23,7 e 23,9oC para os isolados IBLF277 (Franca, SP), IBLF280 (Sao Sebastiao do Paraiso, MG) e IBLF379 (Sao Joao do Manhuacu, MG), respectivamente; de 24,6oC e de 25,1oC para os isolados IBL199 (Patos de Minas, MG) e IBLF270 (Boa Esperanca, MG); e de 27,6 e 27,8oC, para os isolados IBLF004 (Campinas, SP) e IBLF206 (Patrocinio, MG). Na analise filogenetica os isolados IBLF270 e IBLF379 formaram um subgrupo unico, mas as sequencias do gene da calmodulina de todos os isolados apresentaram 100% de identidade. Os isolados IBLF277, IBLF270, IBLF379 e IBLF199 foram os mais patogenicos as mudas de cafeeiro das diferentes cultivares. A cultivar Ouro Verde IAC H 5010-5 foi a mais resistente a cercosporiose e as cultivares Geisha IAC 1137, Mundo Novo IAC 376-4 e Tupi IAC 1669-33 as mais suscetiveis, entretanto, no segundo experimento, houve interacao entre cultivares e isolados.

Patogenicidade de Pratylenchus coffeae em plântulas de cafeeiro cv. Mundo Novo

The pathogenicity of two isolates of Pratylenchus coffeae from Brazil on coffee (Coffea arabica) seedlings cv. Mundo Novo was evaluated in two greenhouse experiments. In the first, the effect of initial population densities (Pi = 0; 333; 1,000; 3,000 and 9,000 nematodes per plant) was studied using an isolate of P. coffeae from Marilia, SP, Brazil (host: coffee). The data were adjusted for the Seinhorst non linear model Y = m + (1-m).Z Pi-T. At the end of the experiment (270 days after inoculation), all plants infested with 9000 nematodes and most of those infested with 3000 were dead. Growth and photosynthesis were reduced, starting at 333 and 1000 nematodes, respectively. In the second experiment, two isolates of P. coffeae from Marilia and Rio de Janeiro (host: Aglaonema sp.) were compared with respect to their pathogenicity, inoculating Pi = 8,000 nematodes per plant in coffee seedlings with two pairs of true leaves. Although photosynthesis was similarly reduced by both isolates, the Marilia isolate caused intense darkening of the roots, leaf chlorosis and stronger reduction of root and shoot growth. In both experiments, multiplication factors of the isolates were low, indicating that coffee is a poor host for both isolates of P. coffeae. The differential pathogenicity observed in the second experiment supports previous results in the literature showing differences between these isolates concerning morphological features and host ranges.

Molecular identification and phylogenetic analysis of Metarhabditis blumi (Nematoda: Rhabditida)

The species Metarhabditis blumi was diagnosed for the first time in Brazil using a molecular assay. Parasites isolated from two cattle herds from the state of Minas Gerais, southeastern Brazil, were classified as M. blumi according to their D2/D3 expansion fragments of the 28S rDNA sequence, which had a high degree of homology (100% similarity) with a M. blumi sequence deposited in GenBank (EU195965.1). The phylogenetic tree comparison also confirmed grouping of the populations evaluated in the current study as M. blumi.

Longidorids from Minas Gerais State, Brazil, with focus on the morphometric variability of Xiphinema krugi (Nematoda: Longidoridae) populations

The occurrence and distribution of Longidorid nematodes in the state of Minas Gerais, Brazil, were assessed in 126 soil samples collected at different habitats in 12 municipalities across the state. The nematodes were extracted from 1xa0kg of soil and morphometric measures and identification of species were conducted using a light microscope with the aid of a camera lucida. Eight species of Xiphinema were identified (X. brasiliense, X. diffusum, X. elongatum, X. ensiculiferum, X. krugi, X. variegatum, X. setariae/vulgare complex and X. surinamense) and also two species of Xiphidorus (Xiphidorus amazonensis and an undescribed Xiphidorus species). Xiphinema diffusum, X. variegatum, X. ensiculiferum and Xiphidorus amazonensis constitute new reports for Minas Gerais. Populations of X. krugi, X. elongatum and X. ensiculiferum showed variation in morphometric measures and a principal component analysis separated X. krugi nematodes in two groups.

Characterization of Brazilian populations of Pratylenchus brachyurus using morphological and molecular analyses

In Brazil, Pratylenchus brachyurus damages economic important crops and it is managed with non-chemical methods such as crop rotation or resistant cultivars. However, the variability in reproduction and damaging effects observed among P. brachyurus populations could limit the efficacy and long lasting effect of nematode control practices. Twelve populations of P. brachyurus from different geographical areas of Brazil were analyzed morphologically and molecularly. The morphological and morphometric characters of the populations studied were variable, but fitted those of P. brachyurus. Assemblages of populations with different host preferences and reproductive capabilities were revealed by PCA of these populations using selected morphological characters. Phylogenetic analysis using ITS-rDNA sequences distinguished statistically supported groups of sequences that matched those of other populations of P. brachyurus deposited in the GenBank, confirming the identity of populations here studied. Intraspecific sequence diversity was observed among the populations. These variant sequences suggest the presence of microheterogeneity between ITS-1 copies in individuals. The findings of this study did not provide evidence of cryptic species in the P. brachyurus populations studied. The variability within and among populations of P. brachyurus observed in this work must be taken into consideration in breeding programs for resistance to this nematode.

Nematoides fitoparasitas associados à mandioca na Amazônia brasileira

A cultura da mandioca (Manihot esculenta) tem importância socioeconomica no Brasil, no entanto ha ainda uma escassez de informacoes sobre a associacao de nematoides fitoparasitas, bem como ao comportamento de cultivares de mandioca ao parasitismo desses agentes. O objetivo do presente trabalho foi relatar a distribuicao geografica e novas ocorrencias das principais especies de nematoides encontradas associadas a mandioca em diferentes municipios dos estados do Acre, Amapa, Para e Rondonia. Alem disso, estudou-se, em casa de vegetacao, a reacao de algumas cultivares de mandioca ao parasitismo de Meloidogyne incognita raca 3, Pratylenchus brachyurus e P. zeae. Nematoides foram extraidos de 10 g de raizes ou 250 cm3 de solo e identificados sob microscopio de luz. A especie mais prevalente foi P. brachyurus, presente em 37,1% das amostras analisadas (n=35), seguida de M. incognita, encontrada em de 14,2% das amostras. Constituiram novas ocorrencias para mandioca no Brasil: Helicotylenchus erythrinae, M. enterolobii e Xiphinema longicaudatum. As cultivares de mandioca provenientes do Amapa (Amazonas, Colonia Matapi, Manteiga, Pretinha e Po-da-China) e as cultivares de mandioca Colonial e Caipora, provenientes do Acre, comportaram-se como suscetiveis a M. incognita raca 3. Por outro lado, as cultivares Caipora e Colonial comportaram-se como resistentes a P. brachyurus (fator de reproducao = 0,5 e 0,9, respectivamente) e imunes a P. zeae. Informacoes geradas neste estudo podem ser uteis para o manejo de areas cultivadas com mandioca e infestadas pelos nematoides das lesoes, P. brachyurus e/ou P. zeae.