plant surface microbiology.pdf

6.2 Biotic Factors
2 Root Colonisation Following Seed Inoculation 27
In potting soil, numbers of inoculated bacteria on the root system are usually
ten-fold lower. Decreased root colonisation is not only caused by competition
with soil-borne bacteria since numbers of inoculated bacteria on roots in
non-sterilised and gamma-irradiated soil are comparable.
Sometimes plant roots grown in potting soil are difficult to remove from
the glass colonisation tube. In such cases, a mixture of potting soil/sand (1:3
w/w) can be used as a compromise between the wish to use potting soil and
that to experimentally study colonisation.
When a fungal pathogen is included in the system, it is possible to determine
biocontrol abilities of strains under controlled conditions. In our lab,
bioassays with tomato and the fungal pathogens Fusarium oxysporum f. sp.
radicis-lycopersici (F.o.r.l.), Rhizoctonia solani, and Pythium ultimum systems
have been successfully employed to determine antifungal abilities of
pseudomonads and bacilli (Lagopodi et al. unpublished data) and to perform
microscopic analyses of rhizosphere interactions (see Chap. 23, Visualisation
of Rhizosphere Interactions of Pseudomonas and Bacillus Biocontrol Strains).
The pathogen can be introduced together with the biocontrol agent onto the
seed or mixed with the sand as a spore or mycelium suspension, depending on
the question under study. For the tomato-F.o.r.l. system, spores are collected
from a 3-day-old culture of F.o.r.l. grown in liquid Czapek-Dox medium.
Mycelium obtained from a PDA agar culture was used for inoculation of the
culture. Spores are collected after passage through a miracloth filter, washed
with water, and resuspended in PNS. Numbers of spores can be determined
using a haemocytometer. Finally, the spores are mixed through the sand to a
final concentration of 50 CFU/g sand.
P. ultimum is grown for 3–4 weeks in clarified V8-medium (20 % V8 vegetable
juice [Campbell Foods, Inc.], 25 mM CaCO 3,30mg/ml cholesterol).
Prior to use,V8 is clarified by sedimentation at 6000 rpm for 30 min. Alternatively,
the fungus can be cultured in hemp (Cannabis sp.) seed extract for
1–2 weeks. Oospores that are abundantly produced during incubation are collected
and freed from the mycelium. The fungal mycelium is washed three
times in sterile water and blended in 0.1 M sucrose for 1–2 min. The culture is
incubated for 2 h at 130 rpm at 28 °C. The suspension is sedimented by centrifugation
at 4000 rpm for 10 min., resuspended in 1 M sucrose, and incubated
at –20 °C for 12 h to kill the mycelium fragments. After washing with
water, the suspension is layered over 1 M sucrose and centrifuged at 2351 rpm
for 1 min. Consecutive washing steps remove most of the mycelium fragments.
Oospores are added to the sand to a final concentration of 3–24
oospores/g sand.
Plants are judged according to a fixed disease index based upon disease
symptoms (Table 2). The presence of the fungus on diseased plants can be
confirmed by dipping suspected diseased parts in 0.05 % household bleach