Rosângela D.L. Oliveira

Ditylenchus gallaeformans sp. n. (Tylenchida: Anguinidae) – a neotropical nematode with biocontrol potential against weedy Melastomataceae

Ditylenchus gallaeformans sp. n. was found on several hosts at numerous locations in Brazil and Costa Rica. In its native habitats it attacks several genera in the Melastomataceae, including two species ranked as among the worst invasive weeds of Pacific island forests, namely Miconia calvescens and Clidemia hirta. The new species causes a severe disease on infected plants involving the formation of gall-like structures on infected leaves, inflorescences and stems, and may cause significant impact on its hosts. Morphological study using light and scanning electron microscopy and analysis of the partial 18S rRNA, the D2-D3 expansion fragments of 28S rRNA and the ITS rRNA gene sequences showed little variations between populations from different hosts or geographical origins. The molecular study revealed that the new species is related to D. drepanocercus, which was recently found in association with M. calvescens but causing angular leaf spots on this host. Ditylenchus gallaeformans sp. n. is distinguished from D. drepanocercus by having a bursa reaching the tail tip (vs covering around 50% of tail in D. drepanocercus) and a conical tail, regularly tapering towards a variable tip (vs tail with a distinctive apical falciform appendage in both sexes in D. drepanocercus). PCR with species-specific primers was developed for diagnostics of both Ditylenchus species. Ditylenchus gallaeformans sp. n. deserves further investigation as a potential biocontrol agent against M. calvescens and C. hirta.

Effect of neem seed extracts on the development of the Soybean Cysts Nematode

The effect produced by neem seed extracts and a neem seed concentrate (NSC) on the mortality and development of Heterodera glycines was evaluated. Second-stage juvenile (J2) mortality produced by the extracts and NSC was determined. Soybean seedlings treated with each of the extracts (aqueous, methanolic and hexane) were inoculated with J2 and the number of females and eggs per root system determined after 30 days. Both the hexane extracts were ineffective as no statistical difference (P>0.05) in J2 mortality was observed between them and the control. No statistical difference (P>0.05) in J2 mortality (>98%) was obtained between the water extracts and methanol extract at 1000 mg L-1 treatments. The J2 percentage of mortality exponentially increased with the NSC dosages (R2=0.94). LD50 was 42.6 mg L-1 for NSC on H. glycines. In the greenhouse experiment, the highest reduction in the number of females was 84%, obtained with the aqueous (41.6 mg L-1) and methanolic extracts (1000 mg L-1). In the same way, the highest reduction in the number of eggs, of about 90%, was obtained with the same extracts. Thus, using aqueous extracts is more suitable than the remaining extracts in field conditions. The activities of the methanol and NSC extracts were attributed to the seven tetranortritepenoids (azadirachtin H, azadirachtin A, azadirachtin B, desacetylnimbin, desacetylsalannin, nimbin and salannin) identified by reverse phase high performance liquid chromatography.

Esterase phenotype S1 in Meloidogyne incognita in Brazilian coffee plantations

Esterase phenotype S1 in Meloidogyne incognita in Brazilian coffee plantations The rare esterase phenotype S1 of Meloidogyne incognita is reported here for the first time in Brazil. The root-knot nematode studied population was detected in a coffee (Coffea arabica) field in the state of Sao Paulo. The affected plants had typical symptoms of the M. incognita parasitism. Em 2003, no municipio de Garca SP, amostras de solo e raiz foram coletadas em cafezais que exibiam quadro sintomatologico caracteristico do ataque de Meloidogyne incognita (Kofoid & White) Chitwood. Femeas retiradas das raizes de cada amostra foram submetidas a analise dos fenotipos de esterase em gel de poliacrilamida. Em uma das propriedades foi encontrada uma populacao de Meloidogyne sp. com perfil de esterase diferente dos ja detectados em nematoides das galhas de cafeeiro (Coffea arabica L.). A populacao apresentou o fenotipo S1 (Figura 1A), que se caracteriza pela presenca de uma banda de menor mobilidade em relacao aquela primeira banda de M. javanica (Treub) Chitwood, utilizada como padrao de comparacao, e ao fenotipo I1, tipico de M. incognita. Para dirimir duvidas referentes ao diagnostico dessa populacao atipica foram analisados perfis de outras isoenzimas, configuracao da regiao perineal, estrutura do estilete de femeas, caracteristicas da regiao labial de machos e teste de hospedeiros diferenciadores da Universidade Estadual da Carolina do Norte. A populacao apresentou os fenotipos N1, I2 e N1 para as enzimas malato desidrogenase, superoxido dismutase e glutamatooxaloacetato transaminase, respectivamente. As configuracoes perineais das femeas avaliadas apresentaram arco dorsal alto e trapezoidal, estrias onduladas e bifurcacoes na regiao dos campos laterais (Figura 1B). O estilete das femeas possuia o cone curvo na extremidade, com aumento gradual do diâmetro da haste em direcao a base (Figura 1C). Os discos labiais dos machos mostraram-se planos a concavos. O tomateiro (Lycopersicon esculentum Mill.) ‘Rutgers’, a melancia (Citrullus vulgaris Schrad.) ‘Charleston Gray’ e o pimentao (Capsicum annuum L.) ‘Early California Wonder’ se mostraram hospedeiros da populacao. A analise conjunta de todas as caracteristicas confirmou que a populacao pertencia a especie M. incognita, raca 1. Este e o primeiro relato do fenotipo S1 em M. incognita infectando o cafeeiro no Brasil, fato relevante nao so para a diagnose da doenca como para os trabalhos de melhoramento visando resistencia a esse patogeno. FIG. 1 Fenotipo isoenzimatico e caracteristicas morfologicas de uma populacao de Meloidogyne incognita, proveniente de cafeeiro (Coffea arabica): A fenotipo S1 de esterase (Mj = M. javanica, utilizado como padrao de comparacao); B configuracao perineal; C estilete da femea. Mj Mj S1

Morphological and molecular characterisation of Aphelenchoides besseyi and A. fujianensis (Nematoda: Aphelenchoididae) from rice and forage grass seeds in Brazil

Morphologically similar Aphelenchoides spp. populations extracted from rice and forage grass seeds from different geographical regions in Brazil were morphologically and molecularly characterised. Overall, the populations studied separated into two groups based on morphological and phylogenetic analyses, referred to herein as ‘Group-rice’ and ‘Group-forage’. Bayesian phylogenetic analyses of SSU, LSU and mtCOI regions strongly supported the presence of two dichotomous groups with Group-rice and Group-forage populations genetically similar to A. besseyi and A. fujianensis , respectively. This study reports the presence of a morphologically similar species to A. besseyi associated with seeds of grasses, but genetically distinct based on three genomic regions, which our results strongly suggest to be A. fujianensis , this being a new geographical record for Brazil. Additional information regarding spicule morphology of male A. besseyi is also reported.

Meloidogyne paranaensis attacking coffee trees in Espirito Santo State, Brazil

In 2009, Meloidogyne paranaensis was detected in coffee trees (Coffea arabica var. Catucaí), in the municipality of Baixo Guandu, Espirito Santo State, Brazil. Based on disease symptoms, morphological characteristics of the nematode and isozyme profiles, M. paranaensis was identified. This is the first report of M. paranaensis in Espirito Santo State.

Variabilidade fisiológica em populações de Meloidogyne paranaensis

Physiological variability of two populations of Meloidogyne paranaensis Two populations of Meloidogyne paranaensis, one from soybean plants (Mp-s) and another from coffee plants (Mp-c) were studied to compare their ability in reproducing on different hosts. Mp-s was able to reproduce more than Mp-c on tomato plants and on two soybean cultivars, but Mp-c showed a higher reproduction factor on coffee plants. On tomato ‘Santa Clara’, both populations reproduced significantly more than on other hosts, but no difference was detected between the soybean cultivars ‘MS/BR 34’ and ‘Fepagro RS 10’. However, a larger number of populations should be studied. Additional keywords: Glycine max, host, virulence of population, root-knot nematode.

A Rapid Diagnostic for Detection of Aphelenchoides besseyi and A. fujianensis Based on Real-Time PCR

Aphelenchoides besseyi and A. fujianensis have been frequently found in mixed populations associated with forage grass seed in Brazil. The morphological similarity between both species has previously led A. fujianensis to be erroneously identified as A. besseyi. A. besseyi is a quarantine pest in many countries that import Brazilian forage seed; however, there is no current evidence suggesting that A. fujianensis is a plant-parasitic species. Two real-time polymerase chain reaction (qPCR) diagnostics were developed to detect each species and an operational envelope was established. A set of primers and hydrolysis probes for each species was designed targeting the large subunit (LSU) region. To assess their specificity, primers and probes sets were tested with samples of nontarget Aphelenchoides and Paraphelenchus sp. also frequently associated with forage seed. Experiments using dilutions of purified plasmid standards underpinned the sensitivity of the qPCR assays, which detected as few as 10 copies of target nematode ribosomal DNA. Thus, the developed diagnostics were sufficiently sensitive to detect DNA extracted from a fragment of a single target nematode. There was a positive correlation between copy number of the target species and nematode abundance, suggesting the potential of this method for quantification. Evidence of intra-individual variability among cloned sequences of the LSU region in a single A. besseyi population is also reported.

Potential of soil fumigation with mustard essential oil to substitute biofumigation by cruciferous plant species

Soil fumigation with the synthetic essential oil of mustard (93% allyl isothiocyanate) (EOM) was evaluated as a substitute of bio-fumigation with cruciferous plant species, using Sclerotium rolfsii and Sclerotinia sclerotiorum as test pathogens, together with its non-target effect on the general population of soil microorganisms. The mortality of the sclerotia of both fungi was dependent upon the concentration of EOM and the exposure period. Exposure to the EOM vapors delayed in vitro germination of the sclerotia.Total germination of the control sclerotia of S. rolfsii, after 120 h incubation, was 94%, of which 88% germinated within 48 h. In contrast, to the total germination of 77% or 47%, less than 3% germinated in 48 h when exposed for 4 or 7 days to 50 µl EOM/L. Exposure to 100 µl/l for 4 or 7 days resulted in the mortality of 50 and 100% sclerotia, respectively. The tendency for delayed germination and mortality was similar for the sclerotia of S. sclerotiorum. No viable sclerotia of either fungus were detected to the depth of 10 cm in the air dried, moist or wet soil fumigated for 7 days with the EOM at the rate of 150 µl/L. In the field plots, no viable sclerotia of S. rolfsii were detected after 7-day exposure to the EOM applied to the upper soil layer at the rate of 9, 12, or 18 ml/m2 and covered with a plastic sheet, while 73, 50 and 15%, respectively, were recovered from uncovered plots. In field plots, the fluorescein diacetate hydrolysis decreased by EOM treatment. However, the colony forming units of actinomycetes and bacteria increased, but those of fungi decreased significantly. Soil fumigation with EOM can be used with several advantages, as a substitute of bio-fumigation with cruciferous plant species.

Preservação da capacidade reprodutiva de Meloidogyne exigua em mudas de pimentão

Coffee is the host-type of Meloidogyne exigua and significant inoculum production on this plant takes a long time. The objective of this study was to evaluate the reproduction of five M. exigua populations on coffee comparatively with reproduction on pepper, and the possible occurrence of physiological selectivity after successive generations on pepper. In the selectivity test, one population was maintained on coffee and pepper for 30 months and reproduction was evaluated 10 times, at 90 day intervals. The number of galls and eggs was always higher in pepper roots than in coffee ones. The reproductive rate in pepper was four times higher than in coffee. There was no difference in nematode reproduction in coffee between the two inoculum sources, coffee and pepper, during 30 months. Pepper proved to be a better host than coffee for the rearing of M. exigua, including those populations unable to reproduce in tomato roots, since pepper plants are easy to manage under green-house conditions and nematode reproduction is faster than in coffee. Therefore, pepper should be used to rear M. exigua since the nematode does not lose its ability to infect coffee.