Twenty five isolates of <i>Burkholderia gladioli</i>, the causal agent of a bacterial disease recently reported on saffron (<i>Crocus sativus</i> L.) grown in central Sardinia (Italy), were characterized using different approaches. The characteristic symptoms of the disease on saffron plants were rot of emerging shoots and leaves and spots on leaves and corms. In the field, the disease was destructive and reduced flowering by about 80%. Two types of colonies of bacteria cultured from affected plants were selected on the basis of their characteristic morphology and pigment production on nutrient-glucose-agar. One type was round, wrinkled, and producing yellowish pigment; while the second was round, smooth and without pigment. All 25 selected isolates were pathogenic on saffron leaves and corms. Ten were pathogenic on gladiolus and lily leaves. None of the tested isolates was pathogenic on onion plants. The isolates were characterized by conventional tests, Biolog, PCR and PCR-RFLP analysis. Conventional tests and PCR identified all isolates as <i>B. gladioli</i>. PCR-RFLP analysis of 16S rDNA products digested with the three restriction enzymes <i>Alu</i> I, <i>Dde</i> I and <i>Bss</i> KI, identified ten of the isolates as <i>B. gladioli</i> pv. <i>gladioli</i>. Sequencing and comparison of the 16S rDNA PCR products confirmed that ten of of the isolates were <i>B. gladioli</i> and the remaining 15 were an unidentified <i>Burkholderia</i> species. Sequencing the gene encoding for b-subunit polypeptide of DNA gyrase (<i>gyrB</i>) did not assist identification of these isolates. This study suggests that other <i>Burkholderia</i> species are involved with bacterial softrot of saffron in Sardinia, and further studies are in progress to verify this hypothesis
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