Vol.7, No. 4 2007.pmd 
Artículo Científico                                                                                                                                                                                                         Biotecnología Vegetal Vol. 7, No. 4: 211 - 214, octubre - diciembre, 2007
ISSN 1609-1841 (Versión impresa)
 ISSN 2074-8647   (Versión electrónica)

 

Ochrobactrum anthropi, contaminants of in vitro culture of sugarcane cells and tissues

Yelenys Alvarado-Capó*, Nayanci Portal González, Leyanis García-Aguila, Marisol Freire-Seijo, Yudith Martínez, Rafael G. Kosky. *Corresponding author

Instituto de Biotecnología de las Plantas. Universidad Central Marta Abreu de Las Villas. Carretera a Camajuaní km 5.5, Santa Clara, Villa Clara, Cuba. CP 54 830. e.mail : yelenys@ibp.co.cu

ABSTRACT

The purpose of this paper was to report, for first time, the presence of Ochrobactrum anthropi as contaminant of in vitro cell and tissue culture of sugarcane. Bacteria contaminant of in vitro plants were isolated directly from visibly contaminated plant culture on Tryptone soy agar medium and some drops of sugarcane cell suspension contaminated were inoculated on tubes with 3ml of Tryptone Soy Broth (TSB). Isolated bacteria were examined microscopically (shape, motility) and some biochemical test (Gram´s stain (Hucker´s modification), oxidase, catalase, O/F (oxidation/fermentation), were performed. Additional tests, according to Bergey´s Manual of Determinative Bacteriology, were performed. Identification was complemented by fatty acid analysis. Different bacteria can be contaminating plant cell and tissue culture. However, references about the presence of Ochrobactrum anthropi as contaminant of in vitro culture of plant cell and tissue have not been reached. For controlling microbial contaminants, is necessary to identify it.

Key words: bacterial contamination,cell suspension, identification, in vitro plants, Saccharum

RESUMEN

El propósito de este trabajo fue informar, por primera vez, la presencia de Ochrobactrum anthropi como contaminante del cultivo in vitro de células y tejidos de caña de azúcar. La bacteria contaminante de las plantas in vitro se aisló directamente en el medio de cultivo Agar Triptona soya a partir de plantas visiblemente contaminadas y algunas gotas de suspensiones celulares contaminadas se inocularon en tubos de ensayo con 3ml de Caldo Triptona soya. Las bacterias aisladas se examinaron microscópicamente (forma, motilidad) y mediante prueba bioquímicas (tinción de Gram; modificación de Hucker), oxidasa, catalasa, O/F (oxidación/fermentación). Otras pruebas adicionales fueron realizadas de acuerdo con lo descrito en el Manual de Bergey. La identificación se complementó por análisis de ácidos grasos. Diferentes bacterias pueden aparecer contaminando el cultivo in vitro de células y tejidos vegetales, sin embargo, no se han encontrado referencias sobre la presencia de Ochrobactrum anthropi como contaminante del cultivo in vitro de caña de azúcar. Para controlar los contaminantes bacterianos es necesario primero identificarlos.

Palabras clave: contaminación bacteriana, identificación, plantas in vitro, Saccharum, suspensiones celulares

INTRODUCTION

Different bacteria can be contaminating plant cell and tissue culture. Species of the genera Pseudomonas, Bacillus, Enterobacter, Klebsiella, Staphylococcus, Lactobacillus, etc. (Leifert and Cassells, 2001) have been identified frequently.

Ochrobactrum, formerly known as CDC group Vd, is an oxidase-positive, nonfermenting, gram-negative bacillus that was first described by Holmes et al. (1988) with a single species, Ochrobactrum anthropi. Hospital environments (Bizet and Bizet, 1995), activated sludge (Danilo et al., 1996), seawater samples (Croci et al., 2001), plant rhizosphere (Tripathi et al., 2002) or root nodules (Ngom et al., 2004) have been frequently places to be found. However, references about the presence of this bacterial specie as a contaminant of in vitro culture of plant cell and tissue have not been reported.

Development of in vitro techniques in sugarcane has resulted in more efficient and effective means for international exchange of germoplasm (Taylor and Dukic, 1993;). According to Dookun (1998) and Lakshmanan et al. (2005) the application of biotechnology to sugarcane has a major role to play in increasing the productivity of this crop. Bacteria constitute the most common and troublesome kind of contaminating microorganism in plant tissue culture (George, 1993), however, only few authors (Taylor and Dukic; 1993; Moutia and Dookun, 1999) described the problems of bacterial contamination on sugarcane tissue culture. For controlling microbial contaminants, is necessary to identify it.

The purpose of this study was to report for first time the presence of Ochrobactrum anthropi as contaminant of sugarcane cell and tissue culture.

MATERIAL AND METHODS

Plant material

In vitro sugarcane (Saccharum spp. hybrid) plants, variety C 87-51 in multiplication stage (on culture medium containing Murashige and Skoog salts (Murashige and Skoog, 1962) supplemented with 0.3mg.l-16-benzylaminopurine, 1.0mg.l-1 tiamine, 30g.l-1 sucrose and solidified with 7g. l-1 agar) visibly contaminated by bacteria were used. Besides, sugarcane cell suspension, cultured as indicated by Freire et al. (2002) and previously detected as contaminated by microscopically observation, were employed.

Isolation and identification

Bacteria contaminants of in vitro plants were detected as whitish exudates around the base of in vitro plants. They were isolated directly from visibly contaminated plant culture by streaking of bacterial growth on Tryptone soy agar (TSA) medium. Plates were incubated at 30°C for 24-72h. Final purification was obtained by restreaking single colonies on similar medium and were maintained by transferring them to TSA slants for short-term storage and by preparing the inocula for further identification test.

Some drops of contaminated sugarcane cell suspension were inoculated on tubes with 3ml of Tryptone Soy Broth (TSB). Tubes were incubated in static conditions at 30°C for 24-72h during a week. Purification was developed by the same protocol those bacterial isolates from in vitro plants.

Cultural characteristics of bacterial growth in bacteriological media (colony shape, colour, borders, consistency, optical and surface characteristics) were recorded.

Isolated bacteria were examined microscopically (shape, motility) and some biochemical test (Gram´s stain (Hucker´s modification), oxidase, catalase, O/F (oxidation/fermentation), were performed according to Lelliot and Stead (1987) and Klement et al. (1990).

Additional test were performed according to Bergey´s Manual of Determinative Bacteriology (Krieg and Holt, 1994), and identification was complemented by fatty acid analysis, following the protocol described by manufacturer (MIDI, Sherlock, Microbial Identification System, version 3.0, 1999). Fatty acid profiles were compared using MIDI library software.

RESULTS AND DISCUSSION

Two typical contaminants were isolated from each type of plant material (strain CCIBP-M29 from in vitro plant and strain CCIBP-Sp108 from cell suspensions).

Both strains produced colonies on TSA whitish, circular, convex, smooth and translucent with entire edges.

Microscopic observation showed Gram negative rods, singly and motile, forming in the cell characteristics described in Bergey´s Manual (Krieg and Holt, 1994). Results of other performed test are listed in table 1. Two strains were different for their growth at 42ºC, and fatty acid composition. Identification by the analysis of cultural and biochemical characteristics was not possible.

Fatty acid analysis, however, was conclusive and the strains CCIBP-M29 and CCIBP-Sp108 were identified as most closely related to Ochrobactrum anthropi (similarity index: 0.658 and 0.896, respectively). Table 2 shows fatty acid profiles from both.

The specie Ochrobactrum anthropi found in sugarcane cell suspensions and in vitro plants were not known to be contaminant in plant cell and tissue culture. In this crop, Moutia and Dookun (1999) identified Bacillus cereus, B. megaterium, B. pumilus, Xanthomonas maltophilia, Burkholderia gladioli and some members of Enterobacteriacea family as contaminants of buds culture using fatty acid analysis of isolates.

According to Leifert and Woodward (1998), very few bacterial species produced characteristic symptoms or growth in plant tissue cultures. For this reason, different bacterial genera can not be separated based on their colonies morphology. Methods that give as much taxonomic information as possible are required (Stead et al., 1998).

Holmes et al. (1988) created genus Ochrobactrum. It included the species O. anthropi (Holmes et al. 1988), O. intermedium (Velasco et al. 1998), O. tritici and O. grignonense (Lebuhn et al., 2000) and O. gallinifaecis (Kämpfer et al., 2003).

Bacteria found as contaminants of plant tissue culture are diverse and belong to a range of ecological groups. They include plant pathogens, epiphytes, endophytes and accidental contaminants (Stead et al., 1998). Identification of O. anthropi as contaminant of sugarcane in vitro culture indicated the capacity of this bacterium for growing together with plants and cells in controlled condition.

O.anthropi has been isolated, besides, from internal tissue of sweet corn (Zea mays) (Mclnroy and Kloepper, 1995), rhizosphere of different plants (Tripathi et al., 2002) and soil (Lebuhn et al., 2000).Since the presence of this specie on sugarcane whole plants, not been reported, the source of it could be related with initial explants because appear.

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