Diversidad y control biológico de insectos - CyberTesis UACh ...

were estimated using a dilution plating method on agar-oat-dodine selective media (Chase
et al., 1986).
Spore persistence in soil. Eight soil cores (9 cm diameter, 10 cm deep) were extracted at
random in each plot assigned to B. bassiana treatment. They were pooled and put in a
plastic bag to get ca. 1-1.5 Kg of soil and cooled (5-10° C) prior to analysis. In the
laboratory, the soil was sieved and litter and roots were removed. Fifteen grams of fresh
soil were added to a flask with 25 ml of sterilized distilled water and drops of Tween-20 as
surfactant. In parallel, three samples of 100 g of soil were dried in a stove to measure the
soil water content and express the number of colonies on a dry soil basis. The mix was
shaken by hand for 5 min. An aliquot (2.5 ml) was transferred to a second tube and then
sterilized water and Tween-20 were added to complete 25 ml. The new tube was treated as
above and when all the dilutions were available (10 -1 to 10 -3 ), we transferred 150 µl of
suspension, using a pipette, to each plate with selective media (3 plates per dilution). The
plates were cultivated for ten days (no light, 20° C) and colony forming units (CFU) were
recorded at the end of this period. A proportion of the colonies was sampled and correct
identification was confirmed by microscopic exam. Counts were corrected by water content
to express the spore numbers on a dry soil basis. Spore number was estimated five times in
the study: before and 1, 5, 15 and 66 days after spraying.
Persistence of spores on leaves. Foliage samples were collected at random in each plot
(10-15 points) and pooled. In the laboratory, pieces of leaves were cut with scissors,
measured with a rule and added still to complete 32 cm 2 per plot. Leaf pieces were added to
a tube with 25 ml of sterilized distilled water and drops of Tween-20. Then, the same
procedure of soil samples was adopted. Spore numbers are expressed by fresh leaf area.
Only ryegrass leaves were included in this analysis. Sampling was performed at 1, 4 and 7
days after spraying.
The dilution/transference process was repeated 3 times, therefore dilutions from 10 -1 to 10 -3
were available, both soil and foliage samples. Only estimation from dilution 10 -2 were used
to draw the soil persistence curve and the foliage persistence curve was drawn from 10 -1
dilution data (Figure 1).
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