Luiz Gonzaga Chitarra

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 103-107 CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 103-106 CFU/mL and immuno-FCM from 104-106 CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds.

Cottonseeds treatment with fungicides for the control of seedling damping-off pathogens

The cotton seedling damping-off is a worldwide problem caused by a complex of soil-borne and seed-borne fungi, occurring separately or in combination, and cause pre- or post-emergence damping-off. The main etiological agents causing the damping-off are Rhizoctonia solani Khun, Colletotrichum gossypii South and Colletotrichum gossypii var. cephalosporioides. The aim of this work was to evaluate the effectiveness of many fungicides that have been used on the treatment of cottonseeds to control pathogens associated with seeds and/or presents in the soil. Cottonseeds free of pathogens, inoculated and non-inoculated with Colletotrichum gossypii var. cephalosporioides (Cgc), were treated with tolylfluanid + pencycuron + triadimenol; carboxin + thiram and fluazinam + thiophanate methyl. On the Growing on Test, the non-treated seeds inoculated with Cgc showed a lower level of emergence. The higher level of emergence occurred on non-inoculated seeds treated with tolylfluanid + pencycuron + triadimenol. The most efficient treatment in the control of post-emergence damping-off in substrates containing Rhizoctonia solani, was obtained with the mixture of tolylfluanid + pencycuron + triadimenol, followed by carboxin + thiram. In the field, the higher severity of ramulosis, caused by Cgc, occurred in plants from non-treated seeds inocculated with Cgc. None of the fungicides tested showed phytotoxity to cotton.