2.3.2. Endophyte evaluationDue to t

2.3.2. Endophyte evaluation
Due to the large number of plant samples that needed to be
surface-sterilized and plated onto Petri dishes, processing for the
endophyte evaluation required three consecutive days; therefore,
six of the 12 blocks were evaluated for endophytic colonization by
the fungal entomopathogens 7–9 days post-inoculation and the
remaining six blocks were evaluated 47–49 days post-inoculation.
For the first evaluation, the two longest bud roots were removed
from each plant and gently washed under running tap water for
approximately 30 s to remove soil particles. From each of these
roots, two 60 mm pieces were taken from both the proximal and distal
ends of each root (i.e., there were four 60 mm pieces of root for
each plant). The root pieces were pre-washed in 0.05% Triton X-
100 for three minutes and then surface-sterilized by immersing in
0.5% NaOCl (diluted in 0.05% Triton X-100) for three minutes, ethanol
(70%) for one minute and then rinsing three times in sterile distilled
water for 15 s each rinse. The bulk surface-sterilization system
described by Greenfield et al. (2015) was used in all surfacesterilizations.
To confirm that surface-sterilization was effective,
eight root pieces were randomly selected from each block (with each
block containing 24 root pieces in total) to make imprints on 75%
PDA (in 100 mm 15 mm Petri dishes) by gently pressing the root
piece onto the surface of the agar (Schulz et al., 1998). Each root
piece was then dissected into three 8 mm length sections (discarding
the ends) and placed onto individual 60 mm 15 mm Petri
dishes containing PDA (75%) supplemented with antibiotics (0.1 g
penicillin, 0.2 g streptomycin and 0.05 g tetracycline/L). All Petri
dishes were incubated at 25 ± 2 C in darkness and were inspected
for 30 days for the presence of B. bassiana or M. anisopliae. Other fungal
endophytes were also recorded and assigned morphotype codes.
The proportion of root parts colonized was calculated for each plant
as the number of root sections exhibiting fungal growth divided by
the total number of root sections plated. The imprints were also
incubated and monitored for at least 14 days for presence of fungi
and if any fungi were found on an imprint, the corresponding block
was discarded from the dataset.