OCCURRENCE OF BACTERIA INSIDE THE CELLS OF VETIVERIA ZIZANIOIDES (L.) NASH ROOTS

The species Vetiveria zizanioides (L.) Nash is a perennial graminaceous plant native to north India. Recently the vetiver has been introduced in Europe as sterile domesticated genotype selected from the wild type plant. Since ancientness vetiver is known, and rural people have used it for centuries both for the essential oil from its roots and for the leaves. Vetiver oil is one of the most complex mixtures of sesquiterpene alcohols and hydrocarbons and for this reason it is difficult to synthesize in laboratory. Electron microscope analysis of the root cells of Vetiveria zizanioides (L.) Stapf (Viano et al., 1991) highlighted the presence of bacteria into lysigen lacunae, which are peculiar cells of the vetiver roots in which the essential oil is stored (Maffei 2002). The close relationship between bacteria and the essential oil stimulated the idea of a direct involvement of those symbiotic bacteria in the essential oil metabolism. These results raised the question whether the occurrence of bacteria in the roots of Vetiveria zizanioides (L.) Stapf was a casual event specific for this vetiver species cultivated in different stations on the Reunion Islands, or it is a cellular property of roots of all Vetiveria zizanioides species. The aim of the present work is to verify by Transmission Electron Microscope (TEM) analysis the presence of bacteria in the cortical parenchymatous cells of roots of the species Vetiveria zizanioides (L.) Nash cultivated in the Campania Region, Italy. Planting in Campania Region of the species Vetiveria zizanioides (L.) Nash native to Thainlandia was performed in the spring 2002 using vetiver culms with short roots and leaves of approximately 20 cm. Roots samples for electron microscope analysis were collected either on April 2003 or on April 2004, respectively. For electron microscopy, apices of primary and secondary roots were fixed simultaneously with 2% glutaraldehyde in cacodilate buffer 0,1 M for 1 h, then washed in the same buffer and postfixed with 1% OsO4 in DDW for 30’. After a quick rinse in DDW samples were dehydrated in a graded ethanol series, transferred to propylenoxide and embedded in Epon 812. Ultrathin section were cut using a Laica Ultracut UCT ultramicrotome, stained with uranyl acetate (4% for 15’) and lead citrate (0,3% for 1’) and viewed in a Philips (Eindhoven, The Netherlands) EM 404 Trasmission Electron Microscopy.Our electron microscopy observations revealed the occurrence of bacteria as well as of electrondense crystals of essential oils in the external layers of cortical parenchymatous cell up to those close to the endodermata. Bacteria seem to be more abundant in the roots grown in the second year, especially in their apical part. Our results support the hypothesis of the symbiotic presence of bacteria as a characteristic of cortical parenchymatous cells of roots of the species Vetiveria zizanioides, and of their involvement in the metabolism of essential oil. The identity of the symbiotic bacteria will be established by culture-based and culture-independent (rDNA amplification and sequencing) methods.