Development of formulations and delivery systems to control economically important ticks with entomopathogenic fungi

Abstract

Due to the rapid development of tick resistance to synthetic chemical acaricides and the potential risk posed by these chemicals to non-target species, efforts are intensifying towards the development of entomopathogenic fungi as an environmentally benign alternative tick control strategy. The main objective of this study is the development of formulations and delivery systems for control of ticks with entomopathogenic fungi. Screening of entomopathogenic fungal isolates is the first step towards the development of mycoacaricides. Twelve isolates of Metarhizium anisopliae (Metschnik.) Sorok. and three isolates of Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) were evaluated for pathogenicity against unfed Rhipicephalus pulchellus Gerstäcker (Acari: Ixodidae) adults under laboratory conditions. Out of the 15 isolates screened, 4 isolates were virulent against R. pulchellus, inducing significantly (F value = 11.86; DF = 14, 75; P < 0.0001) higher tick mortalities; i.e. ICIPE 78 (76.1 ± 5.9%), ICIPE 69 (62.6 ± 5%), ICIPE 62 (49.8 ± 5.8%) and ICIPE 60 (49.6 ± 7.7%), four weeks post-treatment compared to the others. The results of this study demonstrate that R. pulchellus ticks were susceptible to some M. anisopliae isolates. Since the process of strain selection is an important step in the development of insect pathogens for biological control, an appropriate bioassay technique for infecting different life stages of a host is of paramount importance. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using R. pulchellus as a model. In a preliminary experiment, an oil-based formulation of M. anisopliae titred at 109 conidia ml-1 was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon’s spray tower (52.3% tick mortality) , although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 x 104 ± 1.1 x 103 conidia ml-1) after spraying by Burgerjon’s spray tower compared to 1 x 106 conidia ml-1 obtained with the microapplicator. Thus, inoculation by Burgerjon’s spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC50 values following contamination of nymphs (LC50 = 1.4 x 107 conidia ml-1) and adults (LC50 = 6.7 x 107 conidia ml-1) in SS were significantly lower compared to SP; nymphs (LC50 = 5.7 x 108 conidia ml-1) and adults (LC50 = 5.3 x 109 conidia ml-1) and SW; nymphs (LC50 = 5 x 108 conidia ml-1) and adults (LC50 > 1010 conidia ml-1). Although the LC50 value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at two weeks post-treatment) and (23.3% at three weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8% respectively) and SW (0.0 and 0.0). Results show that among the modes of contamination tested, SP was the most appropriate for contaminating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil. The integrated use of semiochemicals and entomopathogenic fungus (hypocreales) may optimise the performance of a fungus as a biocontrol agent against ticks. Initially, experiments were conducted to evaluate the attraction of the hunter tick Amblyomma variegatum Fabriscius (Acari: Ixodidae) to semiochemicals. In one of the experiments, the simultaneous release of 1-octen-3-ol and AAAP together with CO2 from a trap in simulated field plots attracted up to 94 ± 6% of adult ticks from a distance of 6 m, and up to 24.0 ± 5.1% from 8 m. Formulations of M. anisopliae (dry powder, oil, and emulsifiable) applied within the trap baited with AAAP and 1-octen-3-ol resulted in high levels of contamination of the ticks attracted to the traps. However, 48 hr after autoinoculation, 89.1 and 33.3% of conidia were lost in dry powder and oil formulations, respectively. Emulsifiable formulation lost the lowest number of propagules (17.1%). Samples of ticks attracted to the baited traps were transferred to plastic basins containing grass and maintained for 5 weeks. The experiment was conducted in the rainy and dry seasons. The emulsifiable formulation gave the highest relative reduction in tick numbers in both seasons: 54.7 and 46.5% in the rainy and dry seasons, respectively, followed by the oil formulation (32 and 23.8%) and the powder formulation (38.0 and 24.4%). Following the high attraction and contamination of A. variegatum under semi-field conditions, experiments were done to evaluate the efficacy of M. anisopliae-treated semiochemical-baited traps for control of A. variegatum under field conditions. Unfed A. variegatum adults (118) were seeded in each 100-m plot. An emulsifiable formulation of M. anisopliae (consisting of 49.5% sterile distilled water, fungal conidia, 49.5% corn oil [CHEF cooking oil, Premier Oil Mills LTD] and 1% Tween 80) titrated at 109 conidia ml-1- was applied in a semiochemical-baited traps (900 cm2) which were placed at 5 spots within the plot. The control and fungal treatments were repeated after 14 and 28 days soon after rotating the traps clockwise (45o) in order to cover different sections of the plot. In the control plots, traps baited with semiochemicals only were used. Six weeks after the initiation of the experiments, five semiochemical-baited traps (untreated) were deployed in each plot for 3 successive days to trap ticks in the treated and control plots. The percentage of ticks recovered in the fungus-treated plots were significantly lower (31.1 ± 5.2%) than in the control plots (85.6% ± 3%) (P < 0.001), which represented a relative tick reduction of 63.7%. Mortality of 93.8 ± 2.3% was observed among the ticks that were recovered from the field and maintained in the laboratory for two weeks; while only 3.3 ± 0.9% died from the control plots. The results of this study open up the possibility of developing an environmentally friendly, low cost product to control these economically important ticks.