RESUMO Fungos fitopatogênicos durante o processo de penetração e colonização são capazes de secretar uma variedade de enzimas, as quais os tornam capazes de infectar o tecido vivo do hospedeiro, atuando na degradação de ceras, cutícula e das paredes celulares. A capacidade de um agente patogênico em produzir ou não enzimas pode determinar o grau de severidade da doença. Foram avaliados 33 isolados de Colletotrichum gloeosporioides relacionados à antracnose e mancha manteigosa do cafeeiro, quanto a sua capacidade de produção de enzimas hidrolíticas (amilase, lipase, protease, lacase, pectinase e celulase) e enzimas específicas degradadoras da parede celular “EDPC” (poligalacturonase, polimetilgalacturonase e pectinaliase), e sua relação com a patogenicidade/agressividade dos isolados. Para todos os isolados de C. gloeosporioides foi possível a detecção de enzimas extracelulares, com exceção de celulases. Os isolados I-9 e I-24 apresentaram os maiores índices de produção de enzimas extracelulares, e assim como de EDPCs. Os mesmos obtiveram os maiores índices de intensidade de doença, sugerindo a relação entre estas e a agressividade dos isolados.

ABSTRACT Phytopathogenic fungi during the penetration and colonization process are capable of secreting several enzymes, which enable infection of the host live tissue, acting on the degradation of wax, cuticle and cell walls. The ability of a pathogenic agent to produce enzymes or not can determine the severity degree of a disease. In this study, 33 isolates of Colletotrichum gloeosporioides related to anthracnose and blister spot on coffee trees were evaluated for their ability to produce hydrolytic enzymes (amylase, lipase, protease, laccase, pectinase and cellulase) and specific cell wall degrading enzymes “CWDEs” (polygalacturonase, polymethylgalacturonase and pectin-lyase), as well as their relationship with the pathogenicity/aggressiveness of isolates. For all isolates of C. gloeosporioides, extracellular enzymes could be detected, except cellulases. Isolates I-9 and I-24 produced the highest levels of extracellular enzymes, as well as CWDEs. They also had the highest disease intensity indexes, suggesting a relationship between enzymes and aggressiveness of the isolates.

Colletotrichum gloeosporioides no Brasil está associado a um complexo de sintomas na cultura do café. Apesar da observação e da identificação da patogenicidade deste, o patógeno ainda tem sua importância questionada, devido às várias formas endofíticas do mesmo, levantando dúvidas sobre a real importância do patossistema. O presente trabalho teve como objetivo demonstrar por meio da utilização de um isolado transformado com o gene gfp a capacidade de infecção e colonização de C. gloeosporioides em plântulas de café. Pode-se observar após o quarto dia da inoculação a exteriorização de sintomas como necrose pontuais as quais progrediram durante o período de avaliação, culminando na morte das plântulas. Por meio de microscopia de epifluorescencia pode-se confirmar a presença do patógeno nas plântulas, assim como visualizar a colonização interna do tecido, a formação de acérvulos e produção de conídios, confirmando que o mesmo foi o responsável pelos sintomas observados.

In Brazil, Colletotrichum gloeosporioides is associated with a complex of symptoms in coffee culture. Although this pathogen had its pathogenesis observed and identified, its importance has still been questioned due to its several endophytic forms, raising doubts as to the real importance of the pathosystem. The aim of this study was to demonstrate, by using an isolate transformed with the gene gfp, the infection and colonization capability of C. gloeosporioides in coffee seedlings. After the fourth day of inoculation, manifestation of symptoms as punctual necrosis could be observed, which progressed during the evaluation period, culminating in the death of seedlings. Epifluorescence microscopy confirmed the presence of the pathogen in the seedlings, as well as the visualization of internal colonization of tissues, acervulus formation and conidium production, confirming that it was responsible for the observed symptoms.

Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host x pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.