Cultural, morphological and molecular diversity of Ustilaginoidea virens (Cooke) Tak. isolates causing false smut of rice in different agroclimatic regions of Karnataka

The ascomycete fungal pathogen Ustilaginoidea virens (Cooke) Takahshi cause false smut in rice and considerable yield loss. In this study, we collected isolates of U. virens from the rice growing ecosystems of Karnataka and characterized for cultural, morphological and molecular characters. The isolates of the fungus on Potato Sucrose Agar media exhibited distinct colonies with colony growth ranging from 21.50 mm (Uv-20) to 70.00 mm (Uv-15). The colony colour appeared as whitish to yellowish with varied growth pattern from flat, raised flat to fluffy and raised fluffy colonies with sectoring in Uv-1, Uv-3, Uv-6 and Uv-9 isolates. The isolates of U. virens also showed variation in the morphology of spores, where the conidia were globose, irregularly round to elliptical and warty on the surface with spore radius ranging from 2.91 to 5.36 ?m. The scanning electron microscopy revealed hyaline globose to irregularly rounded ornamented chlamydospores with prominent spines. Besides cultural and morphological characters, molecular identification of false smut isolates was confirmed through ITS sequencing which showed 91 to 99 per cent identity with U. virens in NCBI-BLAST analysis. Dendrogram constructed using ITS sequence data broadly separated the isolates into two major clusters with divergence among clusters. This ITS (internal transcribed spacer) sequencing of isolates should help better understanding of the phylogenetic relationships among these isolates.

Predatory activity of chlamydospores of the fungus Pochonia chlamydosporia on Toxocara canis eggs under laboratory conditions / Atividade predatória de clamidósporos do fungo Pochonia chlamydosporia sobre ovos de Toxocara canis em condições laboratoriais

The objective of this study was to use chlamydospores of the fungus Pochonia chlamydosporia (isolates VC1 and VC4) against Toxocara canis eggs in a 15-day in vitro assay. One thousand T. canis eggs were placed in Petri dishes containing 2% water agar medium with different concentrations of chlamydospores (1,000, 10,000 or 100,000) of each fungal isolate of P. chlamydosporia (treated groups) and 1,000 eggs in Petri dishes without fungus (control group). Egg counts were performed to determine the ovicidal activity, which was classified as three effect levels: type 1, type 2 and type 3. Significant differences (P < 0.01) in egg destruction were found in comparison with the control group. The highest percentage of egg destruction was found in plates containing 100,000 chlamydospores (68.5% for VC1 and 70.5% for VC4). Chlamydospores of P. chlamydosporia were effective in destroying T. canis eggs and may contribute in the future towards combating the eggs of this parasite.

O objetivo do trabalho foi utilizar clamidósporos do fungo Pochonia chlamydosporia (isolados VC1 e VC4) na destruição de ovos de Toxocara canis, num ensaio in vitro, realizado no intervalo de 15 dias. Em cada placa de Petri com ágar-água 2% foram vertidos 1.000 ovos de T. canis em 1.000, 10.000 ou 100.000 clamidósporos de cada isolado do fungo (grupos tratados). Foram realizadas as contagens para verificar a atividade ovicida, classificada em três níveis de efeito: tipo 1, tipo 2 e tipo 3. Os resultados demonstraram que houve diferença significativa (P < 0,01) na destruição dos ovos em relação aos ovos observados nas placas do grupo controle. O maior percentual de ovos destruídos foi observado nas placas contendo 100.000 clamidósporos (68,5% para VC1 e 70,5% para VC4). Clamidósporos do fungo P. chlamydosporia foram efetivos na destruição dos ovos de T. canis podendo contribuir no futuro para o combate aos ovos deste parasito.

In-vitro predatory activity of nematophagous fungi from Costa Rica with potential use for controlling sheep and goat parasitic nematodes

In tropical and subtropical regions of the world, parasitic diseases are a main cause of losses in livestock productivity. The increased acquired resistence to anthelmintics by gastrointestinal nematodes, requires biological control be considered as a potential feasible and effective alternative. The most effective natural soil enemies of nematodes are nematophagous fungi. In order to collect and identify predator nematophagous fungi (PNF), samples were obtained from 51 farms distributed throughout the seven provinces of Costa Rica. The origin samples included: soil from different crops (potatoes, tomatoes, bananas, ornamental plants, squash and coffee); animal feces (cattle, sheep, goat and horse); soil and fallen leaves from forest; and plants with signs of nematode infection. Each sample was processed using three techniques for the extraction of fungi from soil: sprinkling technique, soil dilution and humidity chamber. Twenty four strains of nematophagous fungi were found in 19 farms; 83.3% of the fungi were isolated by sprinkling technique. The following fungi were idenified: Arthrobotrys oligospora (n=13); Candelabrella musiformis (n=9); and for the first time there was isolation of A. conoides (n=1) and A. dactyloides (n=1) in the country. Moreover, 16 strains from Trichoderma (n=13), Beauveria (n=1), Clonostachys (n=1) and Lecanicillium (n=1) were obtained. In addition, pH of each possible fungal isolation source was measured, and it varied from 5.2 to 9.9, however PNF isolates fell within the range of 5.6 to 7.5. The PNF strains were cultivated in four different media for the production of chhlamydospores: potato dextrose agar (PDA); corn meal agar (CMA); malt extract agar (MEA) and potato carrot agar (PCA). Out of these cultures, 95.8% of the strains formed chlamydospores primarily in the PCA. Of these strains, the profilic spore producers were subjected to ruminant artificial gastrointestinal conditions. A total of 14 fungi were tested, out of which 42.9% survived the digestive analysis. Neither A. conoides nor A. dactyloides were viable following the in vitro gastrointestinal test. The PNF isolated in this study demostrated an action against ovine and caprine gastrointestinal nematodes and are candidates for use in biological control of these organisms. Among these microorganisms, Candelabrella musiformis appears to be the most promising fungi for use as a biological control agent in Costa Rica. Rev. Biol. Trop. 59 (1): 37-52. Epub 2011 March 01.

El control biológico es en la actualidad una alternativa para el control de los nematodos gastrointestinales que desarrollaron resistencia a los principales grupos de antihelmínticos. Para el aislamiento e identificación de hongos nematófagos depredadores, se tomaron muestras de 51 fincas distribuidas entre todas las provincias de Costa Rica. La naturaleza de las muestras incluyó: suelos de diferentes sembradíos (papa, tomate, banano, plantas ornamentales, chayote y café), heces de animales (bovinos, ovinos, caprinos y equinos), suelo y hojarasca de bosques y plantas con signos de enfermedad causada por nematodos. Las muestras se procesaron mediante 3 técnicas diferentes para la extracción de hongos a partir del suelo: espolvoreado en placa, dilución de suelos y cámara húmeda. Veinticuatro cepas de hongos nematófagos fueron aisladas de 19 fincas; el 83.3% de éstos fueron aislados mediante las técnica de espolvoreado en placa. Los hongos fueron identificados como: Arthrobotrys oligospora (n=13), Candelabrella musiformis (n=9) y por primera vez se reporta el aislamiento de A. conoides (n=1) y A. dactyloides (n=1) en el país. Asimismo, se aislaron 16 cepas de hongos de los géneros Trichoderma (n=13), Beauveria (n=1), Clonostachys (n=1) y Lecanicillium (n=1). Adicionalmente se les midió el pH, el cual varió entre 5.2-9.9, ubicándose los HND dentro de un rango entre 5.6-7.5. Las cepas de HND fueron cultivadas en 4 medios diferentes para la producción de clamidosporas: papa dextrosa agar, harina de maíz, extracto de malta y agar papa-zanahoria. El 95.8% de las cepas aisladas produjeron clamidosporas, principalmente en el medio agar papa-zanahoria. De estas cepas, se escogieron las de mayor producción para ser sometidas a la prueba de digestibilidad in vitro. Un total de 14 cepas fueron sometidos a esta prueba, de las cuales el 42.9% resultaron viables; de éstas, las cepas de A. conoides y A. dactyloides no sobrevivieron a la prueba de digestibilidad in vitro. De los microorganismos aislados, Candelabrella musiformis se considera el más promisorio de los hongos como agente biológico en Costa Rica.