Genotypic and pathogenic characterization of Pseudomonas and Pantoea species isolated from discoloured rice in Italy

Grain discolouration can lower rice quality determining relevant economic impact: disease incidence for early sown rice and for susceptible varieties can be more than 30%. The fungal pathogen Bipolaris oryzae, which causes brown spot, seems to have a secondary role in grain discolouration etiology both in Italy and in other countries. In fact, fungicides controlling brown spot epidemics resulted not effective in reducing grain discolouration incidence, which was not correlated with brown spot incidence (Pizzatti et al., 2004a). Our attention was therefore focused on bacteria, which were isolated from discoloured rice samples in Italy. Bacterial microflora resulted mostly composed by strains of the genera Pseudomonas and Pantoea (Pizzatti, et al., 2004b). The objectives of this study were i) to analyze genetic diversity of the Pseudomonas and Pantoea populations, and ii) to assess the pathogenicity of strains within the two genera. Within the Pseudomonas population, analysis of 16S rDNA sequences of 17 type strains revealed the presence of six species: P. straminea, P. fulva, P. putida, P. psycrotolerans, P. stutzeri and P. fluorescens. The three species P. straminea, P. fulva and P. putida grouped together in a phylogenetic tree, and within this group the Amplified Ribosomal DNA Restriction Analysis (ARDRA) differentiated 7 different restriction patterns, while the BOX-fingerprints 13 haplotypes. The species P. psycrotolerans, P. stutzeri and P. fluorescens resulted phylogenetically distant from P. fulva, P. putida, P. straminea. Within these species 8 ARDRA restriction patterns and 17 BOX-fingerprints haplotypes were found. Twentythree Pseudomonas strains were inoculated by injection into the emerging panicles of the cv. Selenio. Two strains of the P. fulva – P. putida group and one strain identified as P. stutzeri induced more than 6% grain discolouration incidence, significantly higher than the control. Within the P. psychrotolerans species, 4 strains producing a dark brown pigment induced 2-5% disease incidence in field pathogenicity tests when spayed at flowering stage on cv Loto, although inoculations in vitro of grains of cv. Selenio resulted in 100% disease incidence. Within the Pantoea population, the pathogenic bacterium P. ananatis, the causal agent of palea browning, was identified using the specific primers Sn2b/As2c. The BOX-fingerprints analysis of the Pantoea strains showed that the population is subdivided in few main clusters. The pathogenicity tests demonstrated that two strains of Pantoea caused 6-7% grain discolouration incidence when injected into the emerging panicles of cv. Selenio. Two P. ananatis strains, sprayed on rice of two cultivars at the flowering stage, induced the typical symptoms of palea browning and grain discolouration incidence reached 4-5% on cv. Loto. This study shows that some strains of Pseudomonas and Pantoea species, known as saprophytic components of rice microflora, are involved in grain discolouration etiology.