Conidial suspensions of ALF 247 were adjusted to concentrations
of 1 104, 1 105, 1 106, 1 107 and 5 107 conidia mL1
in distilled water and sprayed to near run-off on cacao pods in the
field, using a 1-L sprayer-nozzle bottle. Considering the average
surface area of cacao pods, the volume of suspensions applied
per pod with this procedure were 40–50 mL. Four month-old pods
in trees of the cultivar ‘SIAL 70’ showing no evidence of injury or
disease were used for each suspension tested. To facilitate humidity
for spores germination and help prevent the applied suspensions
to be washed off by frequent rains in the area, pods were
covered with transparent plastic bags. These bags were applied
1 day before application of treatments and removed 24 h after
that, thereby allowing the experiment to be conducted under natural
field conditions. Seven days after the BCA applications, each
pod was challenged by a suspension of 2 105 zoospores mL1
of P. palmivora which was also sprayed to near run-off. To prevent
wash off by rain, to induce stomata opening that facilitate pathogen
penetration, and to obtain a homogeneous infection for all
inoculated treatments, pods were again covered with plastic bags
from 1 day before until 24 h after this inoculation, being removed
afterwards. After 13 days, disease symptoms were scored based
upon the following scale: ‘1’ = no sign of disease symptoms;
‘2’ = restricted lesions of approximately 2 mm in diameter or
5 mm in length; ‘3’ = expanding lesions with diameters from 0.2
to 2 cm; ‘4’ = lesions and/or necrosis of several sizes, covering up
to 25% of the pod surface; ‘5’ = extended lesions and necrosis over
25% of the pod surface. The whole experiment was repeated
1 month later. The experiments were conducted on a completely
randomized design, with each endophyte concentration (treatment)
being applied on 10 pods (replicates). For the analysis of variance,
the severity score was converted to their square root and the
means were compared by the Fisher’s Least Significant Difference
(LSD) test. A logarithmic regression analysis was performed based
on a linear scale for the disease scores (y axis) and a logarithmic
scale for the spore concentrations (x axis), using the data analysis
tool of the Microsoft Office Excel 2003.