Bioassay procedures
2.4.1. Effect of application period and soil cover on fungal anti-tick efficacy
Seasonal effects on fungal efficacy were determine din 5-l buckets (Ø 20 cm), with drainage holes in the bot-tom, filled to two-thirds their volume with sandy loam and placed in an open field. A 50-ml suspension of 1 × 108M. brunneum-7 conidia/ml (about 1.6 × 107 conidia/cm2)was sprayed on the soil surface of each bucket. Twenty engorged female ticks were scattered in each bucket 2 hafter spraying and covered with a 2-cm layer of dry euca-lyptus foliage. The buckets were covered with a light metalnet to prevent tick predication by birds or rodents. The buck-ets were irrigated daily at midday with 50 ml water/bucket.The tests were performed in December–January (winterperiod in Israel) and June (summer period in Israel).To determine the effect of soil coverage on fungal effi-cacy, three different covers were tested in the 5-l buckets:dry eucalyptus foliage (2–3 cm thick), gravel (2–3 cm thick)and growing grass (6–10 cm thick). The latter consisted of pre-grown grass placed on the soil surface several days before the start of the experiment and irrigated daily. The experiments with different ground covers were performed in the spring (March–April).The effects of the EPF on ticks were evaluated in all experiments by recording female tick mortality, egg-mass size, and percentage of egg hatch. For approximate rela-tive estimation of egg-mass size, the length and width of each egg mass were recorded, and the averages of the two measurements were used as the “egg-mass size” (Ø mm).Egg contamination with fungus was assessed visually after transferring the egg masses that were laid on the soil to Petri dishes and incubating for 10 days at 25◦C and ca 90% RH. Percent egg hatch was measured by placing 50 eggs from each collected egg mass on moistened filter paper in a Petri dish and incubating for 3 weeks at 25◦C. The number of hatched eggs was then recorded.