Genetic Diversity of Bacterial Communities of Serpentine Soil and of Rhizosphere of the Nickel-Hyperaccumulator Plant Alyssum bertolonii A. Mengoni 1 , E. Grassi 1 , R. Barzanti 1,2 , E.G. Biondi 1 , C. Gonnelli 2 , C.K. Kim 3 and M. Bazzicalupo 1 (1) Dipartimento di Biologia Animale e Genetica, Universita ` di Firenze, via Romana 17, I-50125 Firenze, Italy (2) Dipartimento di Biologia Vegetale, Laboratorio di Fisiologia Vegetale, Universita ` di Firenze, via Micheli 1, I-50121 Firenze, Italy (3) Department of Microbiology, Chungbuk National University, Cheongju, Korea Received: 11 July 2003 / Accepted: 18 December 2003 / Online publication: 16 June 2004 Abstract Serpentine soils are characterized by high levels of heavy metals (Ni, Co, Cr), and low levels of important plant nutrients (P, Ca, N). Because of these inhospitable edaphic conditions, serpentine soils are typically home to a very specialized flora including endemic species as the nickel hyperaccumulator Alyssum bertolonii. Although much is known about the serpentine flora, few researches have investigated the bacterial communities of serpentine areas. In the present study bacterial communities were sampled at various distances from A. bertolonii roots in three different serpentine areas and their genetic diversity was assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The obtained results indicated the occurrence of a high genetic diversity and heterogeneity of the bacterial communities present in the different serpentine areas. Moreover, TRFs (terminal restriction fragments) common to all the investigated A. bertolonii rhizosphere samples were found. A new cloning strategy was applied to 27 TRFs that were sequenced and taxonomically interpreted as mainly belonging to Gram- positive and a-Proteobacteria representatives. In partic- ular, cloned TRFs which discriminated between rhizo- sphere and soil samples were mainly interpreted as belonging to Proteobacteria representatives. Introduction Serpentine (ultramafic) outcrops are distributed all over the world and, for their natural geological origin, are characterized by high levels of cobalt, chromium, and especially nickel [5]. The vegetation adapted to survive in these soils [22] can include the so-called nickel-hyper- accumulating plants [3] that concentrate metal in stems and leaves to levels higher than the substrate concentra- tion and far in excess from any physiological requirement (more than 1000 lgg )1 shoot dry matter). Alyssum bertolonii Desv. (Brassicaceae) is a nickel hyperaccumu- lator, endemic to the serpentine outcrops of Central Italy [51], belonging to a genus which recently has stirred new and increasing attention due to its practical application for phytoremediation [8, 41]. The study of metal-hyperaccumulating plant effects on soil microorganisms is an important topic. Microor- ganisms can have a great impact on the performances of revegetation of polluted soils [37]. Studies on serpentine soils may provide new insight into bacterial diversity under unfavorable conditions, new isolates, and probably new genetic information on heavy-metal resistance, which could be exploited. Bacteria present in serpentine soils and their interaction with hyperaccumulating plants have focused the attention of several investigators in past years [9, 15, 16, 26, 31, 36, 39, 44, 52, 53]. These authors found that serpentine bacterial communities tolerated spiking of metals, such as nickel, more than those from unpolluted soils and that the presence of hyperaccu- mulating plants as Sebertia acuminata, Thlaspi cae- rulescens, and Alyssum bertolonii led to an increase in metal-resistant bacteria proportion in the soil samples collected near the plants. Moreover, metal-resistant bacteria present in the plant rhizosphere may play an important role in regulating the availability of metal for the plant [46, 53]. Nickel-resistant rhizosphere bacteria have recently been shown to increase nickel uptake into the shoots of the nickel hyperaccumulator Alyssum murale [1]. Correspondence to: M. Bazzicalupo; E-mail: marcobazzi@dbag.unifi.it DOI: 10.1007/s00248-003-0149-1 d Volume 48, 209–217 (2004) d Ó Springer Science+Business Media, Inc. 2004 209