Detection, isolation, and preliminary characterization of bacteria contaminating plant tissue cultures

Monika Kałużna, Artur Mikiciński, Piotr Sobiczewski, Marta Zawadzka, Elżbieta Zenkteler, Teresa Orlikowska


In order to limit the contamination problem in plant tissue cultures experiments on selection of media suitable for detection and isolation of bacteria contaminating plant tissue explants, and preliminary characterization of isolates were made. In the first experiment aiming at detection of bacteria in plant explants four strains representing genera most often occurring at our survey of plant tissue cultures, and earlier isolated and identified (Bacillus, Methylobacterium, Pseudomonas and Xanthomonas) were streaked on five bacteriological media (NA, King B, K, R2A and 523) and on the medium used for plant culture initiation – ½ MS with milk albumin (IM). All strains grew on all media but on K and IM at the slowest rate and on 523 medium at the fastest. The IM medium proved to be useful for immediate bacteria detection at the initial stage of culture. In the second experiment, aiming at characterization of isolates on the basis of colony growth and morphology 14 strains (Agrobacterium, Bacillus, Curtobacterium, Flavobacterium, Lactobacillus, Methylobacterium – 2 strains Mycobacterium, Paenibacillus, Plantibacterium, Pseudomonas, Stenotrophomonas, Xanthomonas, and species Serratia marcescens) were streaked on five microbiological media: KB, NBY, YDC, YNA and YPGA. All strains grew on all those media but at different rates. The only exception was the strain of Lactobacillus spp., which did not grow on King B medium. This medium allowed the detection of such characteristic traits as fluorescence (Pseudomonas) and secretion of inclusions (Stenotrophomonas). The third experiment was focussed on assessment of the sensitivity of detection of specific bacteria in pure cultures and in plant tis- sue cultures using standard PCR and BIO-PCR techniques with genus specific primers and 2 methods of DNA isolation. Results showed that the use of Genomic Mini kit enabled an increase of the sensitivity by 100 times as compared to extraction of DNA by boiling. Moreover, the application of BIO-PCR increased sensitivity of detection from 102 to 105 times over the standard PCR. If looking for unknown cultivable bacteria more effective detection seems to be use of microbiological method enabling detection on bacteriological media single cells in the fragments of explants or in wash liquids, in which fragmented explants were shaken.


bacterial contamination; plant tissue culture; bacteria detection; PCR; BIO-PCR

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Holland MA, Polacco JC. PPFMs and other covert contaminants: is there more to plant physiology than just plant? Annu Rev Plant Physiol Plant Mol Biol. 1994; 45: 197–209.

Orlikowska T, Zawadzka M. Bakterie w kulturach tkanek roślinnych in vitro/Bacteria in plant tissue culture. Biotechnologia. 2006; 4(75): 64–77 (in Polish with English abstract).

Orlikowska T, Sobiczewski P, Zawadzka M, Zenkteler E. Kontrola i zwalczanie zakażeń i zanieczyszczeń bakteryjnych w kulturach roślinnych in vitro/The control and eradication of bacterial infections and contaminations in plant tissue culture. Biotechnologia. 2010; 2(89): 57–71 (in Polish with English abstract).

Leifert C, Ritchie JY, Waites WM. Contaminants of plant-tissue and cell cultures. World J Microbiol Biotechnol. 1991; 71: 452–469.

Leifert C, Waites WM, Nicholas JR. Bacterial contaminants of micropropagated plant cultures. J Appl Bacteriol. 1989; 67: 353–361.

Thomas P. In vitro decline in plant cultures: detection of a legion of covert bacteria as the cause for degeneration of long-term micropropagated triploid watermelon cultures. Plant Cell Tiss Organ Cult. 2004; 77: 173–179.

Ivanova E, Doronina N, Trotsenko Y. Hansschlegelia plantiphila gen. nov. sp. nov., a new aerobic restricted facultative methylotrophic bacterium associated with plants. System Appl Microbiol. 2007; 30: 444–452.

Reasoner DJ, Blannon JC, Geldreich EE. Rapid seven-hour fecal coliform test. Appl Environ Microbiol. 1979; 38: 229–236.

Viss PR, Brooks EM, Driver JA. A simplified method for the control of bacterial contamination in woody plant tissue culture. In Vitro Cell Dev Biol Plant 1991; 27P: 42.

Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant. 1962; 15:473–497.

Lloyd G, McCown B. Commercially-feasible micropropagation of mountain laurer, Kalmia latifolia, by use of shoot tip culture. Comb Proc Int Plant Propag Soc. 1980; 30: 421–427.

Lelliot RA, Stead DE. Methods for the diagnosis of bacterial diseases of plants. Blackwell Scientific Pub. 1987; 216

Schaad NW, Jones JB, Chun W. Laboratory guide for identification of plant pathogenic bacteria. APS Press, 3rd Ed., St. Paul, MN, USA; 2001.

Hansen BM, Leser TD, Hendriksen NB. Polymerase chain reaction assay for the detection of Bacillus cereus group cells. FEMS Microbiol. Lett. 2001; 202: 209–213.

Nishio T, Yoshikura T, Itoh H. Detection of Methylobacterium species by 16S rRNA gene-targeted PCR. Appl. Environm. Microbiol. 1997; 63: 1594–1597.

Widmer F, Seidler RJ., Gillevet PM, Watrud LS, Di Giovanni GD. A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (Sensu Stricto) in environmental samples. Appl Environ Microbiol. 1998; 64: 2545–2553.

Weisburg WG, Barns SM, Pellettier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991; 173: 697–703.

Cassels AC, Tahmatsidou V. The influence of local plant growth conditions on non-fastidious bacterial contamination of meristem-tips of Hydrangea cultured in vitro. Plant Cell Tiss Organ Cult. 1996; 47: 15–26.

Pirttilä AM, Laukkanen H, Pospiech H, Myllylä R, Hohtola A. Detection of intracellular bacteria in the buds of schotch pine (Pinus sylvestris L.) by in situ hybridization. Appl Environm Microbiol. 2000; 66: 3073–3077.

Norman DJ, Alvarez AM. Latent infections of in vitro anthurium caused by Xanthomonas campestris pv. dieffenbachiae. Plant Cell Tiss Organ Cult. 1994; 39: 55–61.

Wang ZK, Comstock JC, Hatziloukas E, Schaad N.W. Comparison of PCR, BIO-PCR, DIA, ELISA and isolation on semiselective medium for detection of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane Plant Pathol. 1999; 48: 245–252.

Akhavan M, Bahar G, Saeidi, Lak M. Comparison of different methods for detection of Xanthomonas axonopodis pv. phaseoli in bean seeds Iran. J Plant Path. 2009; 45: 1–3.

Schaad NW, Berthier-Schaad Y, Knorr D. A high throughput membrane BIO-PCR technique for ultra-sensitive detection of Pseudomonas syringae pv.phaseolicola. Plant Pathol. 2007; 56: 1–8.

Puławska J, Sobiczewski P. Detection of Erwinia amylovora in and on apple tissue using PCR. Acta Hortic. 2002; 590: 163–166.

López MM, Gorris MT, Llop P, Cubero J, Vicedo B, Cambra M. Selective enrichment improves isolation, serological and molecular detection of plant pathogenic bacteria. [In:] Dehne H.W., Adam G., Diekmann M., Frahm J., Mauler-Machnik A, van Halteren P. (eds). Diagnosis and identification of plant pathogens. Kluwer Academic Publishers, Dordrecht, The Netherlands; 1997.

Puławska J, Sobiczewski P. Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil. J Appl Microbiol. 2005; 98: 710–721.

Thomas P, Swarna GK, Patil P, Rawal RD. Ubiquitos presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue culture. Plant Cell Tiss Organ Cult. 2008; 93: 39–54.

Ulrich K, Ulrich A, Ewald D. Diversity of endophytic bacterial communities in poplar grown under field conditions. FEMS Microbiol. Ecol. 2008; 63: 169–180.

Chelius MK, Triplett EW. The diversity of Archaea and Bacteria in association with the roots of Zea mays L. Microbial Ecology. 2001; 41: 252–263.

Bulgari D, Casati P, Brusetti L, Quaglino F, Brasca M, Daffonchio D, Bianco PA. Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogenity-PCR. J Microbiol. 2009; 47: 393–401.


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