Resumo -O objetivo deste trabalho foi avaliar a resistência de Guignardia citricarpa aos fungicidas carbendazim e piraclostrobina, por meio de avaliação do crescimento em meio de cultura, decomposição de tecido foliar e produção de corpos de frutificação. Para isso, o fungo G. citricarpa foi isolado de lesões de frutos de laranja (Citrus sinensis), produzidos em área com intensa aplicação de fungicida. Os isolados obtidos foram avaliados quanto à sensibilidade aos fungicidas piraclostrobina e carbendazim, nas dosagens de 0,5, 1 e 2 µg mL -1 de i.a., para se verificar o efeito da pressão de seleção causada pelo uso destes compostos em áreas citrícolas. Embora tenha sido observada redução efetiva no número de estruturas reprodutivas e na decomposição de folhas e frutos infectados com G. citricarpa, após a aplicação dos fungicidas, 7,5% dos isolados avaliados sobre meio de cultura apresentaram resistência a esse fungicida, o que indica que pode ocorrer seleção de isolados resistentes no campo. Para a piraclostrobina não foi observada resistência, o que indica que pode ser um composto alternativo para ser utilizado de forma alternada com carbendazim, para diminuir as chances de ocorrência de resistência do patógeno.Termos para indexação: mancha-preta dos citros, pinta-preta, tolerância a fungicidas, citricultura. Guignardia citricarpa resistance to benzimidazolesAbstract -The objective of this work was to evaluate the resistance of Guignardia citricarpa to pyraclostrobine and carbendazim fungicides, through: growth analysis in culture media amended with the fungicides; leaf decomposition; and production of reproductive structures on leaves naturally infected with these fungi. G. citricarpa was isolated from symptomatic fruits of sweet orange (Citrus sinensis) cultivated in area with intense fungicide application. The sensibility to fungicides of G. citricarpa isolates was evaluated with pyraclostrobine and carbendazim, in the doses 0.5, 1 e 2 µg mL -1 a.i., to verify the effect of selection pressure caused by continuous use of these compounds. Although, reduction on leaf decomposition and on the number of reproductive structures were observed after application of pyraclostrobine and carbendazim, 7.5% of the isolates, evaluated in culture media, exhibited resistance to carbendazim, suggesting that the use of this agrochemical must be combined to other active principles in a consortium to reduce the chances of resistance occurrence. No resistance to pyraclostrobine was observed among tested isolates, what indicates that this could be an alternative compound to be used in combination to carbendazim, to minimize the chance of resistance occurrence.Index terms: citrus black-spot, fungicide resistance, citriculture. IntroduçãoA mancha-preta dos citros (MPC), cujo agente causal é o fungo Guignardia citricarpa Kiely (anamorfo Phyllosticta citricarpa McAlpine 1973), é caracterizada por lesões na casca de frutos de laranjas doces, limões, tangerinas e seus híbridos, o que os torna depreciados para o mercado de fruta fresca. Q...

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

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