Aida Terezinha Santos Matsumura

Intraspecific genetic diversity of Drechslera tritici-repentis as detected by random amplified polymorphic DNA analysis

The phytopathogenic fungus Drechslera tritici-repentis causes tan spot, an important disease of wheat in the southern Brazilian state of Rio Grande do Sul. Twelve D. tritici-repentis isolates were obtained from wheat seeds from different locations in the state. Their colony morphology on potato dextrose agar and polymorphisms in genomic DNA by the random amplified polymorphic DNA (RAPD) method were investigated. For the RAPD method, 23 primers were tested of which nine were selected for use in the study of D. tritici-repentis polymorphisms. The degree of similarity between isolates was calculated using a simple matching coefficient and dendrograms constructed by the unweighted pair-group method with arithmetical averages (UPGMA). The morphological and RAPD analyses showed intraspecific polymorphisms within the isolates, but it was not possible to establish a relationship between these polymorphisms and the geographical regions from where the host seeds were collected.

Susceptibility of Nasutitermes ehrhardti (Isoptera: Termitidae) to Bacillus thuringiensis subspecies

O efeito de Bacillus thuringiensis sobre o cupim Nasutitermes ehrhardti (Isoptera, Termitidae) foi avaliado em condicoes laboratoriais. Ensaios in vivo com 55 cepas do patogeno, cedidas pelo Instituto Pasteur de Paris foram realizados em condicoes controladas onde sete destas foram consideradas patogenicas, sendo que B. thuringiensis subsp. yunnanensis, B. thuringiensis subsp. huazhongiensis, B. thuringiensis subsp. brasiliensis, B. thuringiensis subsp. colmeri, B. thuringiensis subsp. kurstaki, provocaram mortalidade inferior a 72% em isopteros. Os isolados B. thuringiensis subsp. sooncheon e B. thuringiensis subsp. roskildiensis causaram 100% de mortalidade ao setimo dia apos a aplicacao das bacterias. As CL50 para B. thuringiensis subsp. sooncheon corresponderam a 47x108; 66,2x106 e 5,1x105 celulas/ml. Os valores correspondem aos tres, cinco e sete dias, respectivamente. Para B. thuringiensis subsp. roskildiensis, no terceiro dia a CL50 correspondeu a 30,8x105, no quinto dia a 48,4x106 e no setimo dia a 16,8x104 celulas/ml. Os dados obtidos mostram que as duas subespecies com maior patogenicidade podem ser estudadas, considerando o controle do cupim N. ehrhardti.

Morphological characterization and genetic analysis of Drechslera teres isolates

Net blotch, caused by the phytopathogen Drechslera teres, is a common disease of barley (Hordeum vulgare L) and is responsible for large economic losses in some barley growing areas. In this study the morphology and genetic variability of eight D. teres isolates from different regions of the Brazilian state of Rio Grande do Sul were investigated. Colony morphology was studied on potato-dextrose-agar (PDA) and genetic variability investigated using the random amplified polymorphic-DNA (RAPD) technique. 27 commercially available primers were tested of which 16 were selected for use in polymorphic analysis due to their good resolution and reproducibility. Similarity coefficients were used to construct dendrograms based on colony morphology and RAPD data showing the relationship between the eight isolates studied. Colony morphology showed variability between the isolates while RAPD assays showed high similarity coefficients, but grouping of the isolates according to the geographic origins of the seeds from which they were isolated was not possible.

Virulência, atividade enzimática e padrões de isoesterases de isolados de Pyrenophora chaetomioides, agente etiológico da mancha de grãos e folhas de aveia

The complexity of the population of Pyrenophora chaetomioides, the major causal agent of the oat (Avena sativa) leaf and kernel spot, is not well known in south Brazil. Therefore, studies on population diversity are useful to the breeding programs aimed at obtaining genetic resistance. In this work, eight isolates were selected from oat seeds harvested in the three southern Brazilian States. In order to test their virulence, the isolates were inoculated on six oat varieties and evaluated for disease severity and lesion type. All cultivars were susceptible to the isolates tested although variation in disease intensity among them was noticed. In another study, the isolates were evaluated in relation to their amilolytic, proteolytic and lipolytic activities on enzyme-specific media. Results from the inoculations on six oat cultivars showed a correlation between higher enzymatic activity and virulence of the isolates. Also, their a and b esterase patterns were compared by polyacrylamide gel electrophoresis. Seven distinct isoenzymatic patterns of a and b esterase were observed among the isolates, although they were not correlated with virulence on oat seedlings

Especificidade de Pyrenophora avenae aos tecidos da semente de Avena sativa e sua atividade enzimática

The presence of dark spots on oat (Avena sativa) kernels has been a limiting factor toward their commercialization in Brazil since they make affected kernels less acceptable to the industry. The mycelium localization of Pyrenophora avenae on the kernels and its enzymatic activity may be involved in the cause of the spots. The objectives of this work were to determine the localization of P. avenae mycelium on the caryopsis tissues, its enzymatic activity and its effects on lipids and protein contents. The localization of the mycelium in tissues was determinated by analysis of kernel slices under microscope and stereomicroscope after hydration. The evaluation of the enzymes lipase, protease, and amilase was performed on 18 isolates, selected from the most cultivated areas of southern Brazil, by growing the isolates on enzyme-specific media. Proteins and lipids were analyzed by Kjeldahl and Bligh & Dyer methods, respectively. The P. avenae mycelium is the main cause of kernel spots in oats and its growth is restricted to the cells of the pericarp. Pyrenophora avenae has good lipase and protease enzymatic activity but poor amylase activity. Spotted oat kernels did not differ in protein and lipid content when compared to healthy ones. In particular, their content was higher in the external tissues of the pericarp than in the rest of the grain, regardless of the presence of the spots. These results may explain the superficial growth of P. avenae mycelium in oat cariopsis and its association with the kernel spot.

Ocurrence of Rice stripe necrosis virus in rice crops from Rio Grande do Sul, Brazil

Ocurrence of Rice stripe necrosis virus in rice crops from Rio Grande do Sul, Brazil A viral disease in rice (Orysa sativa), caused by Rice stripe necrosis virus (RSNV), is reported for the first time in Rio Grande do Sul, Brazil. Na safra agricola 2001/02, constatou-se sintomas de plântulas mortas, listras cloroticas em folhas e retorcimento de folhas e paniculas (Figura 1A) em lavouras de arroz (Oryza sativa L.) irrigado, localizadas proximas ou no municipio de Dona Francisca, Regiao da Depressao Central do Rio Grande do Sul. Como hipotese, sugeriu-se como agente causal dos sintomas o Benyvirus denominado Rice stripe necrosis virus (RSNV) transmitido pelo protista Polymyxa graminis Ledingham, causador do “entorchamiento” ou “crinkling”. Para confirmar a hipotese, plantas sintomaticas dessa regiao foram analisadas para deteccao do virus e do vetor. Amostras dessas plantas com sintoma de virose foram enviadas ao Centro Internacional de Agricultura Tropical (CIAT), na Colombia, sendo, as raizes submetidas a coloracao com fucsina e analisadas em microscopio otico para visualizacao das estruturas do vetor, e os extratos foliares analisados em microscopio eletronico (JEOL 100SX) para visualizacao das particulas virais. No Brasil, apos purificacao parcial segundo o protocolo de Fauquet & Thouvenel, exceto as etapas de ultracentrifugacao (C. R. Acad. Sci. Paris 296:575. 1983), o virus foi inoculado em folhas de Nicotiana benthamiana Domin, especie indicadora e, como controle foi inoculado tampao fosfato de potassio (0,01 M) pH 7,2. Tambem realizou-se Double Antibody Sandwich–Enzyme-Linked Immunosorbent Assay (DASELISA) com anticorpo policlonal reativo contra RSNV (Morales et al., European Journal of Plant Pathology 5:643. 1999). Alem disso, 160 plantas da cultivar IRGA 417 foram cultivadas ate 42 dias apos a emergencia, 80 em vasos contendo solo coletado em areas com plantas sintomaticas e as outras 80 em solo de areas com plantas sem sintomas. Na analise dos extratos foliares observaram-se particulas virais caracteristicas de RSNV (Kanyuka et al., Molecular Plant Pathology 4:393. 2003) e nas raizes verificaram-se cistosoros de P. graminis (Figura 1B). Somente as plantas de N. benthamiana inoculadas com RSNV apresentaram necrose (Figura 1C). No DAS-ELISA todas as amostras de plantas com sintomas foram positivas para RSNV. Os sintomas foram observados apenas nas plantas cultivadas em solo de areas com plantas sintomaticas. Assim, conclui-se que o agente causal dos sintomas descritos e Rice stripe necrosis virus. FIG. 1 A. Planta de arroz (Oryza sativa) com sintoma de Rice stripe necrosis virus (RSNV); B. cistosoro de Polymyxa graminis em aumento de 100x; C. Folhas de Nicotiana benthamiana, a da esquerda inoculada com tampao fosfato de potassio (controle) e da direita inoculada com RSNV, parcialmente purificado.

Effects of host resistance on the isozymatic patterns of Bipolaris sorokiniana (Dematiaceae, Moniliales)

Nine isolates of Bipolaris sorokiniana were inoculated on three cultivars of wheat plants (susceptible, moderately resistant, resistant). Eight days after the inoculation, the isolates were recovered (27 isolates) and the following isozymatic patterns were analyzed: esterase, alkaline phosphatase, acid phosphatase, malate dehydrogenase, and superoxide dismutase. The esterase system was the most polymorphic, and the isolates recovered from the susceptible cultivar showed the highest variability. This is evidence that this cultivar exerts low selection pressure on the pathogen

Técnica de obtenção de filtrado de cultura de Alternaria solani

This work was developed to adapt culture filtrates of Alternaria solani to be used in vitro selection of resistant potato. Three isolates of A. solani (I1 and I2) from Eldorado do Sul and Rio Pardo were used. Two liquid media, V8 and Czapek, were used to grow each of the fungal isolate, giving six culture filtrates (I1V8, I2V8, I3V8, I1Cz, I2Cz and I3Cz). Two sterilization forms, Millipore and autoclave were tested. There was no difference in these two sterilization forms. Tissue culture and toxic filtrates of A. solani have a potential to reduce the time in selection of resistant potato.

Biocontrol of Botrytis cinerea and Calonectria gracilis by eucalypts growth promoters Bacillus spp.

The clonal Eucalyptus plants are commonly obtained by vegetative propagation under a protected environment. This system improves the Botrytis cinerea and Calonectria spp infection on the young eucalypts plantings, resulting gray mold and cutting rot respectively. Currently, the unique available control method is based on chemicals. As alternative, novel methods to manage plant diseases, endophytic microorganisms could be an interesting alternative. Thus, we aimed to evaluate endophytic Bacillus isolated from eucalypts as a biocontrol agent against Botrytis cinerea and Calonectria gracilis, important fungal pathogens in the greenhouse, using clonal plantlets of E. urograndis. Eight endophytic strains of Bacillus, previously described as eucalyptus growth promoters, were evaluated in vitro and in vivo against Botrytis cinerea and Calonectria gracilis. The diffusible metabolites assay showed the potential of endophytic Bacillus to decrease the growth of both pathogens. Differences in the susceptibility of the pathogens to bacterial volatile metabolites were observed, B. cinerea showed more susceptible than Calonectria gracilis. In vivo assays, Bacillus amyloliquefaciens EUCB 10 demonstrated better overall reductions in these diseases. Based on the results obtained from the in vitro and in vivo analyses, we suggest that the endophytic B. amyloliquefaciens strain EUCB 10 constitutes a promising biocontrol agent against B. cinerea and Calonectria gracilis. Furthermore, this is the first reporting of B. amyloliquefaciens previously describe as plant growth promoter and also as potential control agent of B. cinerea and Calonectria gracilis to eucalyptus.