✽ To whom correspondence should be addressed. (Tel) 55-19-3429-4251; (Fax) 55-19-3433-6706 (E-mail) wlaraujo@esalq.usp.br The Journal of Microbiology, February 2007, p. 11-14 Copyright ⓒ 2007, The Microbiological Society of Korea Vol. 45, No. 1 Evaluation of Endophytic Colonization of Citrus sinensis and Catharanthus roseus Seedlings by Endophytic Bacteria Paulo Teixeira Lacava 1 , Welington Luiz Arau ˊ jo 1,2, * , and Joa ˜ o Lu ˊ cio Azevedo 1,2 1 Department of Genetics, Escola Superior de Agricultura ‘Luiz de Queiroz’, University of São Paulo, Piracicaba, SP, Brazil 2 Núcleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil (Received April 19, 2006 / Accepted January 12, 2007) Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus roseus using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endo- phytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa. Keywords: endophytic bacteria, diazotrophic endophyte, green fluorescent protein, Citrus sinensis, Catharanthus roseus Endophytic bacteria have been defined as bacteria that can be isolated from the disinfected surfaces of plant tissues or that can be extracted from within the plant, and as bacteria that do not cause visible harm to the host (Hallmann et al., 1997) and external visible structures (Azevedo et al., 2000). They can promote the growth of many field crops by pro- ducing plant growth-promoting substances and by fixing ni- trogen from the atmosphere (Sturz et al., 2000; Lodewyckx et al., 2002). They also have the potential to act as biocontrol agents against phytopathogens (Sturz et al., 1998) and insects (Azevedo et al., 2000). The bacterium Klebsiella sp. is a common endophyte found in maize (Zea mays) (Fisher et al., 1992; McInroy and Klopper, 1995; Chelius and Triplett, 2001), red clover (Sturz et al., 1998), grapevine (Bell et al., 1995), rice (Elbeltagy et al., 2000), sweet potato (Paula et al., 1993; Adachi et al., 2002), alfalfa (Dong et al., 2003a) and soy- bean (Kuklinsky-Sobral et al., 2004), where it may improve plant growth via nitrogen fixation, as demonstrated by the dinitrogenase reductase protein of K. pneumoniae found within the roots of maize (Chelius and Triplett, 2000). Nitrogen (N)-fixing bacteria that inhabit the interior of plants without causing any disease are called diazotrophic endophytes (Iniguez et al., 2004). The K. pneumoniae 342 (Kp342) strain is able to produce the NifH protein in maize (Chelius and Triplett, 2000) and wheat (Iniguez et al., 2004). The citrus culture in Brazil is an economically important crop, and special attention has recently been given to the study of endophytic bacteria from C. sinensis living in xylem vessels, with emphasis on the biological control of citrus disease and the understanding of the interaction between endophytes and phytopathogens that co-habit the same niche (Araújo et al., 2001; Araújo et al., 2002; Andreote et al., 2004; Lacava et al., 2004). This interaction among the endophytic bacteria Xylella fastidiosa and their host plants has been studied; however, plant studies are time consuming primarily due to the disease characteristics, which take at least six months to develop into symptoms in citrus plants. Since the bacterium X. fastidiosa demonstrates the ability to colonize different hosts while expressing similar symptoms in each host (Lopes et al., 2000, 2003; Monteiro et al., 2001), model plants, such as C. roseus and Nicotiana spp., have been evaluated to study the interaction between this fastidious bacterium and endophytes. Catharanthus roseus has been used as a plant model in the study of important citrus diseases such as citrus variegated chlorosis (CVC) (Monteiro et al., 2001; Andreote et al., 2006). Andreote et al. (2006) described C. roseus as a model for studying the endophytic bacteria from citrus. Furthermore, green fluorescent protein (GFP) is a useful tool for studying the microbial colonization of plants, par- ticularly endophytic bacteria, in time and space without dis- turbing the bacterium or the host tissue. The use of GFP allows the integrity of the plant structures and the bacteria residing within them to be preserved (Gage et al., 1996; Bloemberg et al., 1997; Tombolini et al., 1999; Chelius and Triplett, 2000). Therefore, the objective of the present study