MODERN METHODS FOR IDENTIFYING BACTERIA

DAVID E. STEAD, JUDY HENNESSY and JUDITH WILSON

Central Science Laboratory, Sand Hutton, York, Y04 1L2, UK

1. INTRODUCTION

Bacteria found as contaminants of plant tissue cultures are diverse and belong to a range of
ecological groups. They include plant pathogens, epiphytes, endophytes and accidental
contaminants, e.g. from air or from humans during handling. Bacteria from all these broad
ecological niches can cause problems in plant tissue cultures. However, perhaps
paradoxically, plant endophytes and pathogens may often cause less obvious symptoms
than saprophytic and other contaminant bacteria not normally considered pathogens of
plants. The effects of antibiotics and nutrients in culture media also influence bacterial
growth. Thus, pathogenicity in plant tissue cultures is a complex issue. Identification of the
causal agent is often important since this may determine the appropriate control measures.

Consulting a standard text on diagnosis of plant pathogenic bacteria may be insufficient

since a much wider range of bacteria may be involved. Methods are required that give as
much taxonomic information as possible. In recent years there has been a move away from
the traditional nutritional and physiological methods to more cost-effective methods.
However, many of the more modern methods are designed to compare two bacteria and ask
the question - does this bacterium belong to species X or not?, rather than - to which
species/genus/family does this bacterium belong?

This paper reviews some of the modern methods available with particular reference to
contaminants of plant tissue cultures and takes into account accuracy, speed and cost. It
also reviews methods for which no data are presented.

2. MATERIALS AND METHODS

2.1. Bacterial cultures

All strains were either from the National Collection of Plant Pathogenic Bacteria (NCPPB)
housed at CSL or from the CSL diagnostic clinic, to which bacterial contaminants of plant

A.C. Cassells (ed.), Pathogen and Microbial Contamination Management in Micropropagation, 61-73.
© 1997 Kluwer Academic Publishers.
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tissue cultures are regularly submitted for identification. All strains were cultured on
appropriate media [1,2] and were maintained either by freeze drying or by storage at -80°C
[3].

2.2. Identification by fatty acid profiling

All strains were cultured on trypticase soy agar at 28°C for either 24 or 48 h. Fatty acid
profiles were prepared according to standard protocols [2] using the Microbial Identification
System based on software available from MIDI (Newark, DE, USA). Fatty acid profiles
were compared with libraries available commercially (MIDI) or self-generated using MIDI
library generation software.

2.3. Identification by protein profiles

All strains were cultured on nutrient agar at 28°C for either 24 or 48 h. Protein profiles
were prepared largely according to the methods outlined by Stead [4] with occasional small
unpublished modifications. In brief, after appropriate extraction and purification, proteins
were separated by polyacrylamide gel electrophoresis at 6°C on 1 mm thick gels for 3 h at
20 rnA per gel. Gels were stained with Coomassie blue before drying overnight on a simple
air drying frame. Gels were scanned either by laser densitometry or a flat bed scanner and
the gel images normalised according to calibration standards using Gelcompar software
(Applied Maths, Kortricht, Belgium). Normalisation accounted for inter- and intra-gel
differences in electrophoretic rates. Profiles were compared using a range of methods
available in the Gelcompar software package, primarily by unweighted pair group matching
analysis and principal component analysis.

2.4. Genetic fingerprints

Several PCR-based methods were used. Randomly amplified polymorphic DNA assays
(RAPD-PCR) were done using the primers and methods of Maki-Valkama and Karjalainen
[5]. All results presented are for Erwinia chrysanthemi.

Repetitive sequence PCR (rep-PCR) assays were based on the use of primers to REP, ERIC
and BOX repetitive DNA sequences according to the methods of Louws et aZ. [6] with
occasional as yet unpublished modifications depending on the primer set and genus of
bacterium used.

PCR products were separated by agarose gel electrophoresis and DNA bands stained by
ethidium bromide before visualising and photographing under ultraviolet light. Profiles
were scanned on a flat bed scanner and the gel images normalised and compared as for
proteins.