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.

Endophytic and pathogenic Phyllosticta species, with reference to those associated with Citrus Black Spot.

We investigated the identity and genetic diversity of more than 100 isolates belonging to Phyllosticta (teleomorph Guignardia), with particular emphasis on Phyllosticta citricarpa and Guignardia mangiferae s.l. occurring on Citrus. Phyllosticta citricarpa is the causal agent of Citrus Black Spot and is subject to phytosanitary legislation in the EU. This species is frequently confused with a taxon generally referred to as G. mangiferae, the presumed teleomorph of P. capitalensis, which is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts. DNA sequence analysis of the nrDNA internal transcribed spacer region (ITS1, 5.8S nrDNA, ITS2) and partial translation elongation factor 1-alpha (TEF1), actin and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes resolved nine clades correlating to seven known, and two apparently undescribed species. Phyllosticta citribraziliensis is newly described as an endophytic species occurring on Citrus in Brazil. An epitype is designated for P. citricarpa from material newly collected in Australia, which is distinct from P. citriasiana, presently only known on C. maxima from Asia. Phyllosticta bifrenariae is newly described for a species causing leaf and bulb spots on Bifrenaria harrisoniae (Orchidaceae) in Brazil. It is morphologically distinct from P. capitalensis, which was originally described from Stanhopea (Orchidaceae) in Brazil; an epitype is designated here. Guignardia mangiferae, which was originally described from Mangifera indica (Anacardiaceae) in India, is distinguished from the non-pathogenic endophyte, P. brazilianiae sp. nov., which is common on M. indica in Brazil. Furthermore, a combined phylogenetic tree revealed the P. capitalensis s.l. clade to be genetically distinct from the reference isolate of G. mangiferae. Several names are available for this clade, the oldest being P. capitalensis. These results suggest that endophytic, non-pathogenic isolates occurring on a wide host range would be more correctly referred to as P. capitalensis. However, more genes need to be analysed to fully resolve the morphological variation still observed within this clade.

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.