2.1.3. Test of pathogenicity of Fusarium isolates
To assess the pathogenicity and aggressiveness of the isolated
Fusarium species, clean colonies of the pathogen were subcultured
onto fresh PDA plates and allowed to grow for up to
21 days. Thereafter the plates were flooded with sterile water
and the mycelia were scraped off the PDA media using a sterile
spatula into new sterile water. The resulting slurry was then filtered
through a double layer of muslin cloth. The inoculum solution
was adjusted using a haemocytometer and used to inoculate
a previously sterilized (121 C; 30 min) soil mix (2:3) at a rate of
3000 conidia/g of soil (Abawi and Pastor Corrales, 1990).
Common bean seeds from a susceptible line (K132) were surface
sterilized with 3% sodium hypochlorite for 1 min, rinsed three
times in sterile distilled water and planted in 3 l pot filled with the
inoculated soil. Soil without inoculum was used as control. The
trial was replicated three times. The pathogenicity was assessed
28 days after planting based on symptoms development on the
bean roots and stem on a 1–9 CIAT rating scale (Abawi and
Pastor Corrales, 1990) where: 1 = no visible symptoms; 3 = light
discoloration either without necrotic lesions or with approximately
10% of the hypocotyl and root tissues covered with lesions;
5 = approximately 25% of the hypocotyl and root tissues covered
with lesions but tissues remain firm with deterioration of the root
system; 7 = approximately 50% of the hypocotyl and root tissues
covered with lesions combined with considerable softening, rotting
and reduction of root system; and 9 = approximately 75% or
more of the hypocotyl and root tissues affected with advanced
stages of rotting combined with severe reduction in the root system.
On the basis of these criteria, the most pathogenic Fusarium
isolate was selected and progressed for molecular identification
and susceptibility studies.