Château-Musa - Bioversity International

to obtain unlimited access to the roots. Roots
of the same developmental stage were used
for inoculation.
Fusarium oxysporum f. sp. cubense (Foc)
race 4 isolate was obtained from infected
banana plants growing in a field in Kiepersol,
South Africa. Inoculum was prepared in
Armstrong’s liquid medium according to the
method described by Mohammed et al. (1999).
An incision was made in the root, 20 to 25 cm
from the root ball, in the region where lateral
root development had already taken place.
The root was then cut off 2 mm below the
incision. A 15-µl drop of spore suspension
was immediately pipetted on the wounded
root tip. The plants did not receive irrigation
for at least 2 to 3 hours after inoculation. The
number of roots infected and the length of root
colonization from the point of inoculation was
determined after 14 days. To determine the
effectiveness of the inoculation method and
root colonization, the inoculated roots were
removed from the plant, 20 cm above the site
of inoculation. Lateral roots were removed
and the main root sprayed with 70% ethanol.
The roots were left to dry on sterile blotting
paper. Once dry, the roots were aseptically
cut into 1-cm sections and placed on potato
dextrose agar (PDA) amended with 200 mg
chloramphenicol. The number of infected
root sections were determined 14 days after
incubation on PDA. A total of 50 roots per
treatment were sampled.
R. similis was initially isolated from infected
banana plants growing in a field in Hectorspruit,
Mpumalanga, South Africa, and subsequently
cultured on carrot disks (Speijer and De Waele
1997). To infect the roots with R. similis the
following two techniques were developed.
• Roots were infected with R. similis by
suspending the nematodes in liquid Gelrite
(4%) after cooling to 30°C. Eighteen-µl drops
of the mixture (9 µl of R. similis in water and 9
µl of gel 4%) were pipetted onto the roots and
left to set for several hours before irrigation
began.
• Plants were gently removed from the
polystyrene lid and placed in a 350-ml glass
bottle. The roots of the plants were then
covered with 2000 R. similis suspended in
4% Gelrite. The glass bottle was covered
with tin foil to prevent light penetration to the
roots. The plants remained in the bottle for
3 days after which the plants were placed
again in the polystyrene lid.
After 3 days, infected roots were removed and
stained according to the technique developed
by Byrd et al. (1983).
Results and discussion
Fusarium oxysporum f. sp. cubense
The effectiveness of root infection was as high
as 96%. An average of 11 to 13 cm of colonized
root tissue was recovered after 2 weeks, with
a maximum length of root colonisation of more
than 15 cm.
Radopholus similis
In the first technique, the use of Gelrite
prevented the droplets that contained R. similis
from drying out before the nematodes could
infect the roots. In the second technique, the
Gelrite in the bottles prevented nematodes
from precipitating thus making infection more
efficient.
An incubation period of 3 days was
necessary for successful root infection. The
incubation period might be shorter for more
pathogenic R. similis populations (Fallas et
al. 1995, Stoffelen 2000). Large numbers of
nematodes are still needed for infection, as
the initial infection percentage is low. In the
droplet technique successful, infection was
obtained when 100 or more nematodes per
drop were used. In greenhouse trials, 1000,
or more R. similis per plant are often applied
(Speijer and De Waele 1997). It was further
possible to determine whether infection was
successful after 3 days by staining selected
roots or root fragments without disturbing the
remaining roots. Staining after 28 days showed
that R. similis had multiplied successfully, as
numbers of nematodes in roots had increased
significantly (Figure 3). Eggs were visible
inside the roots (Figure 4) confirming that
reproduction of R. similis had taken place.
The droplet technique can further be used for
histological and histochemical investigations
(Valette et al. 1998), as specific sites can be
infected without damaging or disturbing the
rest of the root.
Conclusion
The aeroponic system provides a simple
and non-destructive method to study root
infection and colonisation by F. oxysporum f.
sp. cubense and R. similis. The system also
makes possible histological and histochemical
studies as the site of inoculation and rate of
infection can be manipulated.
Acknowledgements
The authors would like to thank Katrien
Beullens and Annelies Hauwermeiren from
the KULeuven, Belgium, for their technical
assistance and DuRoi Laboratories for the
plant material.
InfoMusa - Vol 12 - No.1 23
Figure 2. Root development of banana tissue
culture plants after 3 weeks under aeroponic
cultivation.
Figure 3. Nematode with eggs clearly visible in
the root, one month after infection.