2.8. In vivo trails
In vivo studies were carried out following two distinct
procedures.
2.8.1. Direct application of antagonists
Experiments were performed statistically as a completely
randomized design (CRD) with 10 replicates for each treatment.
After surface disinfestation (Zhou et al., 2001; He et al., 2003) of
fruit, wounding of fruit to the depth of 1 mm was performed with a
sterile needle upon which appropriate disk plugs were placed as
follows. For each isolate of Actinomycetes, four treatments were
designated: (A) 10 wounded apple fruit were treated with plain
disks of CGA medium, (B) 10 wounded apple fruit were inoculated
with 6 mm disks of well-grown culture of Actinomycete isolate on
wounded areas, (C) 10 wounded apple fruit were inoculate with
6 mm disks of well-grown culture of C. gloeosporioides, (D) 10 fruit
were inoculate with 6 mm disks of well-grown cultures (possessing
abundant conidia) of both antagonist and the pathogen,
consecutively. Inoculation of the antagonist and pathogen was
performed as follows: (1) All apple fruit were aseptically needlewounded
as described above, (2) 6 mm plugs of antagonist or
pathogen were placed on wounds and incubated for 12 h, (3) after
this period, disks in treatment 4 were removed and replaced with
6 mm disks of well-grown culture of C. gloeosporioides (possessing
abundant conidia). Replacements in treatment 1 were done with
plain agar disks and in treatment 2 with Actinomycete plugs. All
treatments were kept for an additional 12 h under sealed
incubation, (4) after this period, all disk plugs were removed from
all treated samples, (5) All treatments were packed in new,
separate, sealed, plastic bags and incubated at 28 C up to 16 days,
(6) Progress of the disease was monitored at 4 days intervals up to
the 16th day by measuring the diameter of rotten areas for
statistical analysis.