ISOLATION AND CHARACTERIZATION OF ENDOPHYTIC BACTERIA IN VETIVERIA ZIZANIOIDES (L.) NASH ROOTS

Vetiver grass is a perennial graminaceous plant (Gramineae) native to India, growing wild, half wild or cultivated in many tropical and subtropical areas. In particular, selected germlines of the species Vetiveria zizanioides (L.) Nash have long been cultivated for their odorous roots that contain the essential oil of Vetiver, used extensively in perfumery and cosmetics (Maffei, 2002). Root tissues contain oil-producing cells, responsible for its characteristic odor. These secretory cells are localized in the first cortical layer outside the endodermis of mature roots. Essential oil can be detected in the inner bark within the cortical layer. In the latter, lysigen lacunae were also observed, which are a true storage for the essential oil of the Vetiver root. Electron microscope analysis of the root cells of Vetiveria zizanioides (L.) Nash revealed the occurrence of bacteria as well as of electrodense crystals of essential oils in the external layers of cortical parenchymatous cell up to those close to the endodermata (Massardo et al. 2004). The close relationship between bacteria and the essential oil stimulated the idea of a direct involvement of those endophytic bacteria in the essential oil metabolism. The aim of the present work is the isolation and characterization of the microbial community inside the cells of Vetiveria zizanioides (L.) Nash roots. Planting in Campania Region of the species Vetiveria zizanioides (L.) Nash native to Thailand was performed in the spring 2002 using vetiver culms with short roots and leaves of approximately 20 cm. Root samples for isolating endophytic bacteria were collected during April-September 2004. The isolation of the pure bacterial cultures was performed by: 1. Preliminary treatment of the samples: the roots were initially washed five-ten times with distilled sterilized water, than treated four-five times with 70% EtOH. The root surface was checked for the presence of bacteria. 2. Taking the sample from inside of the roots: the root surfaces were cutted with scalpel and samples were taken with bacterial needle suspending it in 1 ml sterile water. 3. Isolation of single bacterial colony: aliquots of suspensions (previous step 2) were plated on solid rich medium and incubated 48h at 30°C. The control of purity of microbial cultures and bacterial identification were performed according both microscopic observations and standard physiological and biochemical tests. Besides, independent bacterial isolates from the microbial community inside cell roots were molecularly controlled by analyzing the differences in ribosomal 16S DNA. The bona fide endophytic bacteria species of Vetiveria zizanioides (L.) Nash roots included the cultivable microorganisms Pseudomonas putida, Pseudomonas sp. WDL5, Pseudomonas sp. Fa2, Pseudomonas corrugata, Pseudomonas thivervalensis, Serratia grimesii, Duganella violaceusniger and non-cultivable bacterial strains including an alpha-, two beta- and one delta Proteobacteria. Work is in progress to analyze the capacity of those bacterial isolates to degrade the vetiver essential oil.