plant surface microbiology.pdf

23 Visualisation of Rhizosphere Interactions 433
used either as an alternative to, or in combination with chemicals to reduce
the dose of these chemicals. Pseudomonas and Bacillus spp. are often abundantly
present in the rhizosphere and surrounding soil of many crop plants.
Many of these species produce secondary metabolites that inhibit growth of,
or kill, soil-borne phytopathogens. These antagonistic bacteria can either be
isolated from the rhizosphere or from the soil in which plants have been
grown.
In the following isolation procedure, tomato plants harvested at the end of
the growing season were picked randomly. Plant roots (0.3–0.4 g fresh weight)
were vigorously shaken in phosphate buffer saline (PBS) for 1 h to detach the
rhizosphere bacteria from the roots. The resulting bacterial suspensions from
individual root systems were diluted and plated on one tenth strength tryptic
soy agar (TSA) supplemented with the fungicide cycloheximide (50 mg/ml).
The use of a nutrient-poor medium was reported to yield the highest numbers
of isolates.After an incubation period of 2–7 days at 28 °C, a large variety
of colonies with different morphologies were observed.
The number of fluorescent pseudomonads found in the rhizosphere is very
often variable. In some studies they were reported to be a dominant group,
whereas other studies report that their numbers did not exceed 1 % of the
total rhizosphere population isolated. The variations may be due to differences
in plant species or cultivars, soil type, age of the plant roots, or the isolation
method. Recently, it was also found that the percentage of antagonistic
pseudomonads from a maize rhizosphere grown without chemical pesticides
in Totontepec, Oaxaca State, Mexico, was 20 times higher than that from a rhizosphere
grown in a commercial tomato field treated with chemicals in
Andalusia, Spain (van den Broek et al., unpubl. data). No single medium is
definitely suited for an unbiased selection of all culturable rhizosphere bacteria.
Pseudomonas isolation (PI) agar can be used to specifically favour the
growth of pseudomonads. One should also keep in mind that only the culturable
part of the rhizosphere population will be obtained.
Putative Bacillus strains are isolated by heating root samples at 80 °C for
10 min prior to washing the bacteria from the roots. The bacterial solution is
plated on Luria-Bertani (LB) agar plates supplemented with cycloheximide
(50 mg/ml) and incubated for 2–5 days at 28 °C. Colonies with a Bacillus-like
morphology are then compared to Bacillus-type strains. To determine
whether one is dealing with Gram-positive or Gram-negative organisms, a
first identification of colonies can be performed by determining the ability to
form mucoid threads after pulling a toothpick out of a bacterial suspension in
3 % KOH, which is indicative for Gram-negative organisms. A definite determination
requires a standard Gram stain. Further characterisation methods
include the use of Biolog, which is based on the ability of a strain to oxidise
particular carbon sources, amplified ribosomal DNA restriction analysis
(ARDRA), or PCR amplification of 16S ribosomal DNA fragments with specific
primers followed by nucleotide sequencing and homology studies. In the