Transmission, colonization and molecular detection of Fusarium oxysporum f. sp. phaseoli in common bean seeds

Abstract

The common bean (Phaseolus vulgaris L.) is a crop of great economic and social importance in Brazil and one of the basic diets of the Brazilian population. Several diseases occur in this crop, causing yield losses and/or decreases in seed quality, such as Fusarium wilt, caused by Fusarium oxysporum f. sp. phaseoli (Fop). This organism can be spread by seeds and it is classified as a Regulated Non-Quarantine Pest in Brazil. The current known methods for its detection and identification in seeds are blotter tests and semi-selective agar medium, followed by a pathogenicity test. The goals in this work were to extend knowledge on seed transmission of Fop in common bean as well as to investigate the close interaction between that fungus in infected seeds through GFP technique associated to scanning electron microscopy and to establish a protocol for detection of this fungus in seeds by real time PCR (qPCR). In paper 1, transmission rates of the pathogen from artificially and naturally Fop-contaminated seeds to emerged plants were tested. Two strains of Fop, two genotypes of bean, two environment temperatures and four inoculum potentials were used in the experiments with artificially inoculated seeds, in order to assess the symptomatic and asymptomatic plants. The frequence of symptomatic plants was lower than 5% but the transmission rates of those plants were 100%. The transmission rates of asymptomatic plants were 57% and 49.7% for BRSMG Majestoso and Ouro Negro, respectively. In respect to comparison between temperatures, the rates were 54.4% at 20 ºC and 52.3% at 25 ºC. For Fop strains, the transmission rates were 83.6% and 94.2% for FOP005 and FOP014. The mean rate at P3 was 64.4% and 58% at P1. From the assays with naturally Fop- contaminated seeds, transmission rates were lower than those determined for inoculated seeds, ranging from 8.1% to 16.7%. In paper 2, Fop was transformed by green fluorescent proteins (GFP) containing the resistance gene of hygromycin-B. Seed infection by the transformed Fop was visualized in the embryo, including the plumule, and in the endosperm. A large amount of fluorescent mycelium was observed externally on bean seedling roots, which presented vascular discoloration, which is the typical symptom of Fusarium wilt disease. In paper 3, the results of the experiments on molecular detection of Fop in common bean seed samples showed that the specific primers and probe used as part of the qPCR protocol in this study were viable to detect Fop in infected seeds with high sensitivity, at 0.25% of Fop incidence. TaqMan assays provided more reliable, sensitive, effective and quicker results than SYBR Green assays, which confirm previous reports for other pathosystems. Analysis of naturally Fop- contaminated seeds by qPCR correlated with results of the blotter test but further studies are needed to optimize sampling and subsampling of seed health testing using PCR-based assays.