Morphology, molecular phylogeny and biomass evaluation of Desmodesmus abundans (Scenedesmaceae-Chlorophyceae) from Brazil.

The biotechnological potential of microalgae has been the target of a range of research aimed at using its potential to produce macromolecules with high added value. Particular focus has been given to biofuels' production, such as biohydrogen, biodiesel, and bioethanol from lipids and carbohydrates extracted from microalgal biomass. Bioprospecting and accurate identification of microalgae from the environment are important in the search for strains with better performance. Methodologies that combine morphology and molecular techniques allow more precise knowledge of species. Thereby, this work aimed to identify the new strain LGMM0013 collected at Iraí Reservoir, located in Paraná state, Brazil, and to evaluate the production of biomass, carbohydrates, and lipids from this new microalgal strain. Based on morphology and phylogenetic tree from internal transcribed spacer (ITS), strain LGMM0013 was identified as Desmodesmus abundans. D. abundans accumulated 1500 mg L-1 of dried biomass after 22 days of cultivation in autotrophic conditions, 50% higher than Tetradesmus obliquus (LGMM0001) (Scenedesmaceae-Chlorophyceae), usually grown in photobioreactors located at NPDEAS at the Federal University of Paraná (UFPR) to produce biomass. Analysis of the D. abundans biomass from showed an accumulation of 673.39 mg L-1 of carbohydrates, 130% higher than T. obliquus (LGMM0001). Lipid production was 259.7 mg L-1, equivalent to that of T. obliquus. Nitrogen deprivation increased the production of biomass and carbohydrates in D. abundans LGMM0013, indicating this new strain greater biomass production capacity.

Genetic Diversity of Colletotrichum spp. an Endophytic Fungi in a Medicinal Plant, Brazilian Pepper Tree.

In this study, we reported thirty-nine endophytic fungi identified as Colletotrichum spp. associated with Brazilian pepper tree or aroeira (Schinus terebinthifolius Raddi. Anacardiaceae) in Paraná state, Brazil. These endophytes were identified by morphological and molecular methods, using PCR taxon-specific with CaInt/ITS4, CgInt/ITS4, and Col1/ITS4 primers, which amplify specific bands in C. acutatum, C. gloeosporioides lato sensu, and Colletotrichum boninensis, respectively, and by DNA sequence analysis of the nrDNA internal transcribed spacer region (ITS1, 5.8S, ITS2). We also assayed the presence of dsRNA particles in Colletotrichum spp. isolates. Combining both morphological characters and molecular data, we identified the species C. gloeosporioides, C. boninense, and C. simmondsii. However, we found a high genetic variability intraspecific in C. gloeosporioides which suggests the existence of several other species. Bands of double-stranded RNA (dsRNA) were detected in three of thirty-nine isolates. Identity of these bands was confirmed by RNAse, DNAse, and S1 nuclease treatments for the isolates LGMF633, LGMF726, and LGMF729. This is the first study reporting these particles of dsRNA in C. gloeosporioides.

Agrobacterium tumefaciens-mediated transformation of Guignardia citricarpa.

Guignardia citricarpa, the causal agent of Citrus Black Spot, was successfully transformed via Agrobacterium tumefaciens with cassettes for gfp and bar expression. Transformation is essential to understand the role of genes during interaction between plants and its pathogens. Using a binary plasmid vector based in the pPZP201BK, both germinated conidia and physically fragmented hyphae of G. citricarpa were transformed. Eight independent transformants of G. citricarpa resistant to ammonium glifosinate displayed GFP fluorescence. The majority (93.75%) of the G. citricarpa transformants was mitotically stable and contained a single T-DNA copy ectopically integrated to the chromosome. This is the first report of G. citricarpa transformation and will allow future work on virulence determinants of the fungus and possibly its control.

Fungicide resistance and genetic variability in plant pathogenic strains of Guignardia citricarpa / Resistência a fungicidas e variabilidade genética em linhagens patogênicas de plantas Guignardiacitricarpa

Citrus black spot (CBS) is a plant disease of worldwide occurrence, affecting crops in Africa, Oceania, and South America. In Brazil, climate provides favorable conditions and CBS has spread to the Southeast and South regions. CBS is caused by the fungus Guignardia citricarpa (anamorph: Phyllosticta citricarpa) and its control is based on the use of fungicides, such as benzimidazoles. In South Africa, the disease was kept under control for 10 years with benomyl, until cases of resistance to high concentrations of this fungicide were reported from all citrus-producing areas. Azoxystrobin (a strobilurin) has been found effective in controlling phytopathogens, including CBS, in a wide range of economically important crops. The present study investigated in vitro the effects of the fungicides benomyl and azoxystrobin on 10 strains of G. citricarpa isolated from lesions in citrus plants from Brazil and South Africa. Benomyl at 0.5 µg/mL inhibited mycelial growth in all strains except PC3C, of African origin, which exhibited resistance to concentrations of up to 100.0 µg/mL. The spontaneous mutation frequency for resistance to benomyl was 1.25 ï 10-7. Azoxystrobin, even at high concentrations, did not inhibit mycelial growth in any of the strains, but significantly reduced sporulation rates, by as much as 100%, at a concentration of 5.0 µg/mL. Variations in sensitivity across strains, particularly to the strobilurin azoxystrobin, are possibly related to genetic variability in G. citricarpa isolates.

A Mancha Preta dos Citros (MPC) tem ocorrência mundial afetando a produção de citros na África, Oceania e América do Sul. No Brasil, onde o clima é favorável ao seu desenvolvimento, a doença está espalhada nas regiões Sul e Sudeste. O controle da MPC, causada pelo fungo Guignardia citricarpa (anamorfo: Phyllosticta citricarpa) é baseado na aplicação de fungicidas, como os benzimidazóis. Na África do Sul, após 10 anos de controle da doença com o fungicida benomil, os casos de resistência a altas concentrações deste fungicida atingiram todas as áreas produtoras. O fungicida estrolilurina chamado azoxistrobina tem se mostrado eficiente no controle dos fitopatógenos de uma grande variedade de culturas economicamente importantes, incluindo a MPC. Neste trabalho foram investigados os efeitos in vitro dos fungicidas benomil e azoxistrobina em 10 linhagens de G. citricarpa isoladas de lesões em plantas cítricas no Brasil e na África do Sul. Houve inibição do crescimento micelial a 0,5 µg/mL do fungicida benomil entre as linhagens testadas, com exceção de PC3C de origem sul-africana, que apresentou resistência até a concentração de 100,0 µg/mL de benomil. A freqüência de mutação espontânea para resistência ao benomil foi de 1,25 ï 10-7. A estrobilurina azoxistrobina, mesmo em altas concentrações, não inibiu o crescimento micelial dos isolados, entretanto reduziu significativamente a produção de esporos, chegando a 100% de inibição em concentrações de 5,0 µg/mL de azoxistrobina. A variação na sensibilidade das linhagens, principalmente com a estrobilurina azoxistrobina, possivelmente está relacionada com a variabilidade genética dos isolados de G. citricarpa.

Fungicide resistance and genetic variability in plant pathogenic strains of Guignardia citricarpa.

Citrus black spot (CBS) is a plant disease of worldwide occurrence, affecting crops in Africa, Oceania, and South America. In Brazil, climate provides favorable conditions and CBS has spread to the Southeast and South regions. CBS is caused by the fungus Guignardia citricarpa (anamorph: Phyllosticta citricarpa) and its control is based on the use of fungicides, such as benzimidazoles. In South Africa, the disease was kept under control for 10 years with benomyl, until cases of resistance to high concentrations of this fungicide were reported from all citrus-producing areas. Azoxystrobin (a strobilurin) has been found effective in controlling phytopathogens, including CBS, in a wide range of economically important crops. The present study investigated in vitro the effects of the fungicides benomyl and azoxystrobin on 10 strains of G. citricarpa isolated from lesions in citrus plants from Brazil and South Africa. Benomyl at 0.5 µg/mL inhibited mycelial growth in all strains except PC3C, of African origin, which exhibited resistance to concentrations of up to 100.0 µg/mL. The spontaneous mutation frequency for resistance to benomyl was 1.25 × 10(-7). Azoxystrobin, even at high concentrations, did not inhibit mycelial growth in any of the strains, but significantly reduced sporulation rates, by as much as 100%, at a concentration of 5.0 µg/mL. Variations in sensitivity across strains, particularly to the strobilurin azoxystrobin, are possibly related to genetic variability in G. citricarpa isolates.