Re: Contamination

[Agricell@AOL.COM]

Latent microbial contamination of plant tissue cultures has been described
since 1992, particularly by C. Leifert at the University of Aberdeen. The
problem is that plant tissue culture medium is not a particulary good growth=

medium for many microorganisms. They can persist for years without clouding
the medium or producing symptoms. The commonly used method for determining
whether cultures are "clean" - holding them up to the light to look for
cloudiness - is worthless, if the aim is to achieve an axenic culture. All
"eyeballing"does is tell you that whatever contaminants are present do not
produce visible growth in the medium that you are using, with the explant
that you are growing, under the culture conditions that you are using. I
wince whenever I read a paper that claims to deal with an axenic or clean
culture when the author has used nothing but an "eyeball" test for
contamination.

The following are two recent Agricell Report articles on latent contamination
that may be of interest in the contamination discussion:

Ed Herman, Editor
Agricell Report

New Method for Detecting Latent Contaminants in Plant Tissues

(SEPT 98, Agricell Report) It is now well established that microorganisms can
survive in plant tissue cultures for many years, through multiple
subcultures, with no visible indication of contamination. Such latent
contaminants are often difficult or impossible to detect and isolate by means
of standard bacteriological methods, but may be harmful to plants in vitro
and to plants regenerated from such contaminated cultures. What is even more
dangerous is that latent plant pathogens may be inadvertantly spread by
micropropagated plants contaminated with latent pathogens.
    At the ISHS Conference: Methods and Markers for Quality Assurance in
Micropropagation, held 24-27 August 1999 in Cork, Ireland, University of
London scientists G. Tsoktouridis, G. Tasiamis and S. Mantel described a
PCR-based strategy and protocols that permit rapid detection and isolation of
low numbers of latent bacteria in tissue culture-derived plants of Bilbergia..
The technique involves amplifying a fragment of the 16S rDNA fragment of
bacteria without amplifying the pure plant DNA. This PCR product can be used
to classify bacteria by cloning and sequencing.
    According to the authors, Use of any combinations of primers and
subsequent sequence analysis of any amplified fragment allows the rapid
detection and molecular identification of bacteria associated with plants.
One combination of primers may detect eucaryotic bacteria . . . present as
contaminants . . . and other combinations of primers would even have the
potential to allow rapid detection of unculturable bacteria.
    Tsoktouridis et al. suggest that their "molecular diagnostic tool
" canyield more reliable and consistent information about latent contaminants than
has hitherto been possible. They add that the potential benefits of the
technique offer "an extremely attractive opportunity in those cases where
high numbers of samples require rapid analysis and appropriate economies of
scale."

For further information:
G. Tsoktouridis, Dept. of Agric. & Hortic., Wye Coll., Univ. of London, Wye,
Ashford, Kent TN25 5AH, U.K.

Persistence of Pathogens in Plant Tissue Cultures

OCT 99, Agricell Report) Because autotrophic plant tissue culture does not
require the presence of added sugar, it is believed to be less vulnerable to
microbial contamination than heterotrophic culture. At the National
University of Ireland, however, S.M. Rafferty and associates have found that
human food poisoning pathogens can persist in autotrophic plant tissue
cultures despite normal disinfestation procedures and can also persist in
regenerated plants. Rafferty et al. inoculated cabbage seedlings with E.
coli, S. marcescens and enterococci. The seedlings were disinfested by
placing them in 80% ethanol for 45 seconds, 2% stericol for 30 minutes, then
washed in sterile distilled water three times. After disinfestation, some of
the plantlets were homogenized and the homogenate was plated onto an
appropriate bacterial culture medium and incubated.
    The authors reported at the meeting that bacterial isolates could be
recovered both from direct culture of seedlings and from homogenates. The
bacteria persisted during autotrophic culture, apparently using plant
exudates as a carbon source, and could be repeatedly re-isolated through
serial subcultures. Although some plants were asymptomatic, in others the
bacteria became vitropathic (pathogenic in vitro). When seedlings from these
cultures were transferred to greenhouse or field conditions, bacteria could
be isolated endophytically initially and epiphytically for long periods of
time, both from the plants and from the substrates upon which they were grown.
    It thus appears that, despite normal disinfestation procedures, in vitro
culture has the potential of spreading pathogenic microorganisms, even when
the culture is performed under autotrophic conditions.

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