plant surface microbiology.pdf

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Thomas F.C. Chin-A-Woeng and Ben J. J. Lugtenberg
abiotic factors, such as growth substrate, soil humidity, soil and rhizosphere
pH, and temperature heavily influence root colonisation. The study of the
molecular mechanism of root colonisation of a host plant by one or more bacterial
strains is complicated due to many biotic and abiotic field-soil variables
which can be difficult to control. The use of a gnotobiotic system limits the
biological variation and results in more reliable and reproducible experimental
data. However, since the purpose of colonisation studies is to learn about
the processes which occur under realistic conditions, we always test interesting
gnotobiotic results in field or potting soil. With only one exception, the
gnotobiotic results also appear to be the case in soil.
Various visualisation systems, including light and electron microscopy and
confocal laser scanning microscopy (CLSM) combined with reporter systems
such as those using genes for autofluorescent proteins, b-glucuronidase, and
b-galactosidase allow us to determine numbers of bacteria on the root and
follow the fate of inoculant bacteria in the spermosphere after seed inoculation
and along the root system after growth.
In this chapter, we will also focus on the genetic and metabolic burdens in
the rhizosphere as a consequence of genetic modification of the organisms
required to enable the marking, tracking, recovery, and selection of bacteria
in and from the rhizosphere. The gnotobiotic system provides a reproducible
method to study root colonisation in terms of strategies and competition.
Afterwards, the data should be verified under more natural conditions as
emphasised before. Various growth substrates including sand, potting soil,
field soil, and stonewool have been successfully used in the root colonisation
system presented in this chapter. The system has been extended by introducing
soil-borne pathogens, which allows the study of interactions between
pathogen, microbes, and host plants at the cellular level which may be important
for applications such as biocontrol.
3 Analysis of Tomato Root Tip Colonisation After Seed
Inoculation Using a Gnotobiotic Assay
3.1 Description of the Gnotobiotic System
To assay colonisation, a gnotobiotic sand system comprised of two glass tubes
is used. A silicone ring of 15 mm, cut from a silicone tube (25x35 mm, Rubber
BV, Hilversum, The Netherlands), is placed around the top tube (outer diameter
25 mm, inner diameter 21 mm, length 200 mm) at 5 cm from the end
(Fig. 1). The same end is closed with gauze using a rubber band. This end is
placed in a bottom tube (outer diameter 40 mm, inner diameter 35 mm, height
95 mm) that contains 3 ml of water to prevent the tube content from desiccation.
Subsequently, high quality quartz sand (quartz sand 0.1–0.3 mm;
Wessem BV, Wessem, The Netherlands) is moisturised with plant nutrient