Colletotrichum gloeosporioides : transmissibilidade em sementes e mecanismos de defesa de mudas micropropagadas de cafeeiro (Coffea arabica L.)

Abstract

No complexo Colletotrichum x cafeeiro, poucos são os estudos no que se referem transmissibilidade do patógeno em plantas infectadas e aos mecanismos de defesa o que dificulta a elucidação de aspectos importantes da doença na cultura. Neste contexto, o presente trabalho foi realizado com o objetivo de acompanhar via microscopia de epifluorescência, a de transmissibilidade do patógeno semente-plântulas, os efeitos da presença do patógeno na semente sobre parâmetros de qualidade destas e os mecanismos de defesa que cafeeiros utilizam em reação a presença do patógeno e dessa maneira esclarecer pontos importantes do patossistema Colletotrichum x cafeeiro. O efeito de C. gloeosporioides na germinação, na viabilidade da semente e no estabelecimento de plântulas foi realizado com um isolado não transformado (I2) e um transformado pela técnica da GFP (I2-T) inoculados em sementes da cultivar Catuaí Vermelho (sementes de plantas com (PCS) e sem (PSS) sintomas de mancha-manteigosa) submetidas por diferentes potenciais de inóculo ( P0 – P5), pelo contato direto com a colônia em placas de Petri. Foram consideradas as variáveis, sanidade, germinação, índice de velocidade de emergência, tetrazólio e transmissibilidade. Verificou-se que, com o aumento do potencial de inóculo, houve aumento da incidência nas sementes para os dois materiais estudados. A porcentagem máxima de infecção nas sementes foi de 6% para PSS (I2) e 26,66% para PCS (I2-T). Em relação à variável germinação, só foi possível observar interação significativa (p<0,05) entre material genético x potencial de inóculo. Pelo teste do IVE ficou evidenciado que houve decréscimo de vigor, à medida que se aumentou o potencial de inóculo em ambos os materiais estudados. Em relação ao estande inicial e final, foi possível observar diminuição de valores dessas variáveis, tanto em MOPSS como em MOPCS para todos os tratamentos. O teste de tetrazólio mostrou que os prejuízos fisiológicos relacionados à elevação do potencial de inóculo apresentaram a mesma tendência que no teste de germinação, sendo possível observar em PSS, que a viabilidade das sementes foi superior a PCS. Através do teste de isolamento em placas com MEA ficou evidenciado que C. gloeosporioides é capaz de ser transmitido de semente – plântula, confirmando desta forma a capacidade infectiva do isolado transformado. Para identificar reações de defesa de cafeeiro em resposta à invasão por C. gloeosporioides o experimento foi conduzido em esquema fatorial 3 x 4 x 2 {(2 isolados de C. gloeosporioides –I2 e I2-T + 1 testemunha), 4 períodos de exposição das mudas aos isolados (6, 12, 24 e 48 horas) – coletas e 2 tipos de mudas: MOPCS e MOPSC } em DBC com 4 repetições. Para as análises enzimáticas foram plotadas curvas de progresso da atividade das enzimas por tempo de coleta e para os teores de fenóis solúveis totais o teste de Scott-Knott. Ficou evidenciado que C. gloeosporioides transformado geneticamente não perde sua capacidade de se manifestar no hospedeiro e ser percebido pelo mesmo, ativando desta forma os mecanismos de defesa da planta observados pelos picos de atividades da peroxidase e polifenoloxidase e do aumento dos teores de fenóis solúveis totais no caso de MOPSC.

In the Colletotrichum x coffee complex, there are few studies which refer to the pathogen transmissibility in infected plants and defense mechanisms which became hard the elucidation of important aspects of this complex in the coffee plants. In this context, this work was carried out with objective of accompanying, through epifluorescence microscopy, the transmissibility of the pathogen seed-seedlings, the effects of the pathogen on quality seeds parameters and to study the defense mechanisms that coffee plants use in reaction to the presence of the pathogen and this way to explain important points in the Colletotrichum x coffee plant pathosystem The effect of C. gloeosporioides on seed germination, the seed viability and seedling establishment were conducted with the isolates I2 and I2- inoculated in seeds of Catuaí Vermelho (seeds with (PCS) and without (PSS), buttery spot symptoms), and the seeds submitted to the inoculation at 48, 96, 120 and 144 hours of exposure to the fungus by direct contact with the colony in a Petri dish. After this procedure all variables were evaluated: sanity test, germination test, emergence speed index, tetrazolium test and transmissibility. It was found that with increasing time of exposure to the fungus, there was an increased incidence of the fungus in the seeds for both materials studied. The maximum percentage of infected seeds was 6% for PSS at 144 hours after inoculation with isolate I2 and 26.66% for PCS also at 144 hours after inoculation with isolate I2-T. The independent variables, the evaluation period and exposure time showed a significant interaction for seed germination of both materials (seedlings from plants with (MOPCS) and without (MOPSS) buttery spot symptoms). The highest percentages of MOPSS was verified within 96 hours of exposure of seeds to pathogens, causing a decrease in these values as they increased the exposure period of both I2- T and I2 seeds. The emergence speed index test gave evidence that there was a decrease in plant vigor, as the exposure time of the seed to the pathogen increased in both materials studied. In MOPSS the highest rates were observed until 120 hours of exposure in both I2 and I2-T isolates, with a slight drop from this time onward. In the case of MOPCS increases were observed in emergence speed index only up to 96 hours of exposure as well as in both isolates I2 I2-T and it decreased after this. Regarding the initial and final plant stands, we observed decreased values of these variables in MOPSS and MOPCS for all treatments. The tetrazolium test showed that the physiological damage associated to an increased period of exposure of seeds to C. gloeosporioides showed the same trend as in the germination test. It was, possible to see that in PSS, the viability of seeds was higher than in PCS after the exposure time to the pathogen. The isolation test on plates with MEA culture medium evidenced that C. gloeosporioides can be transmitted from seed to seedling, thus confirming the infective capacity of the transformed and non-transformed unprocessed isolates. To identify the coffee defense reactions in response to invasion by C. gloeosporioides the experiment was conducted in a 3 x 4 x 2 factorial {(2 C. gloeosporioides isolates - I2 and I2- T + 1 control), 4 seedling exposure periods to C. gloeosporioides isolates (6, 12, 24 and 48 hours) - collections and two types of seedlings: MOPCS in DBC and MOPSC} with 4 repetitions. For the enzymatic analysis, enzyme activity progress curves were plotted by collection time and for the the total soluble phenol levels the Scott-Knott test was used. It was demonstrated that genetically transformed C. gloeosporioides does not lose its ability to be expressed in the host crop and be perceived by it, thus activating the plant defense mechanisms observed through the peroxidase and polyphenoloxidase activity peaks and increased total soluble phenols levels in the case of plant seedlings without buttery spot symptoms.