plant surface microbiology.pdf

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Thomas F.C. Chin-A-Woeng and Ben J. J. Lugtenberg
ity since the experiments start with a homogenous set of seedlings and this
eliminates problems associated with irregular seed germination. The use of a
sterile system not only ensures more reproducible bacterial numbers on the
root system, but also results in higher numbers on the root due to the absence
of competition by indigenous soil bacteria.
Various environmental conditions influence root colonisation efficiency in
the gnotobiotic sand system. The effect of a number of biotic and abiotic factors
on colonisation was determined in a tomato-P. fluorescens WCS365 system.
These factors include growth substrate, temperature, soil humidity, pH,
and the presence of (competing) indigenous bacteria. Usually, ten times lower
bacterial numbers are found on the tomato root system when experiments are
performed in non-sterile potting soil instead of sterile quartz sand, which
might be explained by the presence of indigenous competing organisms. The
choice of material to sustain growth of seedlings is mainly determined by the
system of interest. The use of chemically clean sand ensures a reliable experimental
approach, but cannot be applied for studies requiring field conditions.
Sand can be replaced by potting or field soil and the soil can be practically
freed from indigenous organisms by gamma irradiation. Rockwool drained in
plant nutrient solution also supports plant growth and bacterial colonisation.
For more compact soil systems, such as clay-containing soils, the soil can be
amended with sand to facilitate the recovery of roots from the system. The
gnotobiotic system has been tested for tomato, radish, potato, cucumber,
grass, and wheat, and may well be suitable for growth of other plant species.
Although our seedlings in the gnotobiotic sand system are normally grown
for 7 days, they can be grown for up to 14 days without watering.
To determine the influence of a number of abiotic factors on colonisation
in the gnotobiotic system, P. fluorescens WCS365 was marked with a b-glucuronidase
reporter and singly inoculated on tomato seedlings. The overall
bacterial distribution of the marked bacteria was determined using dilution
plating and visualised using root prints (unpublished data). Increasing fluid
content from 10 up to 20 % (v/w) in sand results in an overall increase of bacterial
numbers on the tomato root tip. The increased colonisation may be due
to increased motility or passive transport of bacteria down the root. Utilisation
of 5 % (v/w) nutrient solution severely limits plant growth and consequently,
bacterial numbers are lower. Temperatures at which plants are grown
need to be selected depending on the plant species. Although we grow tomato
seedlings at an intermediate temperature of 19 °C, growth is significantly
enhanced at higher temperatures (e.g. 28 °C). This is also reflected in the number
of bacteria sustained by the plant root system, possibly due to the effect of
increased root exudation on bacterial growth.