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

Spore persistence in soil. Six-eight soil cores (9 cm diameter, 10 cm high) were extracted
at random in each plot. They were pooled on a plastic bag to get ca. 1-1.5 k of soil and
cooled (5-10° C) prior to analysis. Spores number was estimated through dilution plating
method described in detail by Chase et al., 1986. We used dodine fomulated as Syllit 65
WP (ANASAC, Santiago, Chile). The procedure was as follows:
In the laboratory, the soil was sieved and litter and plant parts 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. 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 in soil 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 foliage were cut with scissors and
measured to complete 32 cm 2 . 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. Just ryegrass leaves were included
in this analysis. Sampling was performed at 1, 4 and 7 days after spraying.
The dilution/transference process was repeated 4 times, therefore dilutions from 10 -1 to 10 -3
were available, both soil and foliage samples. Estimates from dilution 10 -2 were used to
draw Figure 1.
Arthropod identification.
Specimens were stored on 70% ethanol. Samples were sorted in the laboratory with the aid
of a microscope and identification was performed with reference to Artigas (1994), CSIRO
(1991) and with the help of a reference collection identified by Dr. Roberto Carrillo
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