Isolation and characterization of bacteria associated with rice grain discolouration

In Italy, in spite the widespread use of fungicides to control blast and brown spot epidemics, the incidence of discoloured rice grains has recently increased. Widely grown rice varieties like ‘Selenio’, ‘Loto’, and ‘Balilla’ are highly susceptible to grain discolouration. Disease incidence is highest in early sown crops and it is negatively correlated to panicle brown spot incidence and severity. These observations together with lack of fungicides efficacy, suggest that bacteria may cause the disease, as it occurs in tropical countries. To have a better insight into grain discolouration aetiology in Italy we analysed the bacterial population associated with discoloured grains of cv Selenio (N=43) and other varieties (N=8). More than 400 bacterial isolates were obtained. DNA Repetitive Extragenic Palindromic (REP) sequences were amplified for each isolate, using the primer BOXA1R. More than 200 isolates had unique BOX-fingerprint and were further char-acterized with molecular and physiological analyses. About 80% of the bacterial population was composed by Gram-negative species: 60% of these isolates were fermenting, and the majority was Pantoea spp., while 40% resulted strictly aerobic. About 70 isolates were identified as Pseudomonas (sensu stricto) and cluster analysis of ITS1-RFLP data showed 9 major groups. Partial sequences of 16S rDNA of representative isolates were aligned and matched with the following species: Ps. fluorescens, Ps. stutzeri, Ps. fulva and Ps. putida. Acidovorax avenae sub. sp. avenae, a seed-transmitted and highly destructive bacterium in tropical countries, was found in 25% of the rice samples (N=21). Pathogenicity tests provided additional evidence that rice grain discolouration in Italy is due to bacteria.