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Evaluation of pathogenicity of certain mitosporic ascomycete fungi to the house fly, Musca domestica L. (diptera: muscidae)
Journal of Saudi Chemical Society (2013) 17, 97–100
King Saud University
Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Evaluation of pathogenicity of certain mitosporic ascomycete fungi to the house fly, Musca domestica L. (diptera: muscidae) Ebtesam M. Al-Olayan Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Received 22 November 2011; accepted 29 November 2011 Available online 6 December 2011
KEYWORDS Fungal extracts; Pathogenicity; Mortality percentage; House fly; Musca domestica
Abstract The virulence of five fungal strains, Acremonium cephalosporium, Aspergillus niger, Penicillium chrysogenum, Trichoderma viride, and Verticillum albo-atrum by application of 108 conidia mL1 to larvae and adults of house flies, Musca domestica was determined. A. niger produced the highest percentage of mortality to larvae, 85.1 ± 1.60, and adults, 98.2 ± 3.10, of house flies. Topical application of the fungal strains to larvae showed concentration-dependent response and caused mortality ranged between 41% and 83% with LT50 values varying between 5.22 and 7.81 days. The highest percentage of mortality, 83.9 ± 3.52, was produced by 107 conidia mL1 of A. niger with LT50 5.22. The lowest mortality percentage, 41.2 ± 3.00 with LT50 7.81, was produced by 105 conidia mL1 of V. alboatrum. On the other hand, the highest percentage of mortality to adults of house flies, 97.3 ± 3.52, was produced by 107 conidia mL1 ofA. niger with LT50 3.49. While the lowest mortality percentage, 59.1 ± 2.38 with LT50 6.91, was produced by 105 conidia mL1 of V. albo-atrum. ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction The house fly; Musca domestica L., is one of the most important and cosmopolitan insect pests, from both a medical and social standpoint (Axtell and Arends, 1990). It can affect E-mail addresses: [email protected], [email protected] 1319-6103 ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2011.11.019
Production and hosting by Elsevier
livestock performance, cause irritation to human beings and possesses considerable potential for the mechanical transmission of 100 pathogens on human beings. The development of resistance to pesticides by the house fly has increased the willingness of those involved in their production to seek alternative methods for fly management (Kaufman et al., 2005). On the other hand, the use of entomopathogenic fungi in the area of fly management has generated attention. Numerous studies are available documenting the attempted use of entomopathogenic fungi against house fly under laboratory and field conditions including Entomophthoramusca (Cohn) Fresenius (Mullens, 1986; Geden et al., 1993), Beauveria bassiana (Bals.) Vuill (Barson et al., 1994; Geden et al., 1995; Watson et al., 1995, 1996; Lecouna et al., 2005; Kaufman et al., 2005). However, isolates of fungi vary in their virulence to a given insect host but what factors contribute to this variation often
98 remain unclear (Anderson et al., 2011). The differences in isolate virulence may be attributed to the position of the phenotype of a particular isolate occupies on a continuum between two main strategies; an isolate may produce a large amount of toxins or may focus their energy into vegetative growth. The objectives of the present study were firstly, to evaluate the efficacy of 5 fungal species against the adults of house fly and secondly, to compare between the efficiency of the pathogenicity of those fungi for control house fly. 2. Materials and methods 2.1. House flies The house flies were collected from a poultry house using sweeping net and were transferred to the laboratory where they were reared at 27 C and 50 ± 5% RH with a 12:12 light:dark photoperiod. Adults were maintained in cages (40 · 40 · 40 cm3) covered by gauze. Adult flies were provided access to food, consisting of a 1:1 ration of powdered milk and granulated sugar, and water with replenished every 24–48 h. Larval medium comprised of 55 g wheat bran, 3 g date extract and 2 g dried alfalfa suspended in 140 mL water. One cup (250 mL) of this medium was left in each cage for oviposition by adults and subsequent development of larvae. The food was replaced every 24–48 h. 2.2. Fungal isolates Five isolates of mitosporic Ascomycete fungi; namely, Acremonium cephalosporium, Aspergillus niger, Penicillium chrysogenum, Trichoderma viride, and Verticillum albo-atrum were used in this study. The fungi were obtained from the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. The fungi were cultured on Sabourad dextrose agar (SDA) (Oxoid, UK) in 9 cm diameter Petri dishes and incubated at 27 C for 10 days. The sporulating cultures were harvested by scraping the dry conidia from the surface of the culture plate with a scalpel and transferred to sterile distilled water containing 0.01% Tween-80. The concentration of the suspension was determined using a hemocytometer (Butt and Goettel, 2007).
E.M. Al-Olayan distilled water. For testing the virulence of the fungi on the larval stage of M. domestica, groups of 25 larvae were immersed in conidial suspension for 10 s and were then placed on damp filter paper kept in a white disposable plastic container (100 mL volume). Containers were sealed with lids. The control larvae were dipped in 0.01% Tween-80 solution. Each treatment was replicated three times. Each group of larvae was transferred after 24 h to the container with 70 g of larval medium. The tops of the dishes were covered with gauze and incubated at 27 C with photoperiod 12:12 (light:dark). Emerged adults were recorded as the rate of survival and subsequently the rate of mortality was calculated. For testing the effect of fungal isolates on adults, 3–4 day adults were anesthetized with CO2 and batches of 25 were dipped in a suspension containing 108 conidia mL1 for 10 s and then placed on a piece of filter paper in small cages (20 · 20 · 20 cm). They were provided with sugar and powdered milk as solid medium and water. Cages were kept at 27 C with photoperiod 12:12 (light:dark). The control flies were treated with 0.01% solution of Tween-80. All treatments were replicated three times. Mortality was recorded for 7 days at 24 h intervals. 2.5. Larval treatment Suspensions of the most virulent fungal strains were prepared and serially diluted to give 107, 106, and 105 conidia mL1. Groups of 25 larvae were immersed simultaneously for 10 s in the conidial suspension and each group placed on damp filter paper in a white disposable plastic container. Control larvae were dipped in 0.01% Tween-80 solution. Larvae were transferred to the larval medium after 24 h. there were three replicates per treatment. All containers were incubated at 27 C with photoperiod 12:12 (light:dark). Mortality was recorded after treatment at intervals of 24 h until pupation occurred. Dead larva and pupa were held to monitor fungiinduced mortality. Externally sporulated cadavers were recorded as dead due to fungal infection. 2.6. Adult treatment
For each experiment, conidia viability was determined by enumerating the percentage of germinated conidia, 24 h after spreading on SDA medium. A 0.01 mL of conidial suspension (107 conidia mL1) was spread on 9 cm Petri dish containing 15 mL of SDA medium and incubated at 27 C for 24 h for germination (formation of distinct germ tube). Percentage germination was determined by first placing three 15 mm square cover slips directly on to the surface of the medium and counting the number of germinated conidia and total number of conidia per field of view at 200· magnification. Three to four fields of view were observed per cover-slip (Quesada-Moraga et al., 2006).
Four doses of conidia from the fungal strains were tested on cohorts of twenty five 2–3 days old, feeding male and female flies housed in small cages (20 · 20 · 20 cm). Each cage contained a 9 cm diameter Petri dish lined with Whatman filter paper and 10 g bait (6 g sugar, 2 g powder milk and 2 mL distilled water). For better dispersion of fungus, 1 mL of the stock suspension of the fungal strains under test was dispersed on the surface of bait to give doses 1 · 107, 1 · 106, and 1 · 105 conidia g 1 . The treated baits were left in the cages for 48 h before being removed and replaced with dry bait, water was provided ad lib. The experiments were run in a completely randomized design with three replicates per treatment. Cages were checked daily over a period of 7 days following presentation of fungus. Cadavers were removed daily and surface sterilized and transferred to a sterile Petri dish with a damp filter paper. Sporulating cadavers were recorded as mortality due to fungal infections.
2.4. Screening of fungal isolates virulence
3. Data analysis
An aqueous suspension of each fungal strain was prepared and the concentration was adjusted to 108 conidia mL1, in sterile
Three mortality counts of between 10% and 90% were obtained for each experiment. However, experimental tests
2.3. Fungal viability
Evaluation of pathogenicity of certain mitosporic ascomycete fungi to the house fly, that demonstrated more than 20% control mortality were discarded and repeated. When the control mortality ranged from 5% to 20%, the observed percentage of mortality (% M) was corrected by Abbott’s formula (1925): %M¼
% test mortality % control mortality X100 100 % control mortality
The mean lethal time (LT50) required killing one-half of the larvae and adults was estimated using probit analysis of SAS software (SAS, 1998). 4. Results and discussion Preliminary screening of five fungal strains by the application of 108 conidia mL1 to both larvae and adults of house flies showed that the A. niger was the most virulent strain for larvae and adults and produced mortality rates 85.1 ± 1.60 and 98.2 ± 3.10, respectively (Table 1). However, T. viride produced mortality rates 82.8 ± 4.10 and 83.5 ± 1.97 and V.
Table 1 Mortality of Acremonium cephalosporium, Aspergillus niger, Penicillium chrysogenum, Trichoderma viride, and Verticillum albo-atrum to larvae and adults of Musca domestica after exposure to a suspension of 108 conidia mL1. Fungal species
Acremonium cephalosporium Aspergillus niger Penicillium chrysogenum Trichoderma viride Verticillum albo-atrum
Table 2
Mortality (%) ± SE Larvae
Adults
28.0 ± 2.00 85.1 ± 1.60 24.3 ± 2.20 82.8 ± 4.10 73.6 ± 1.48
29.7 ± 2.76 98.2 ± 3.10 27.6 ± 2.65 83.5 ± 1.97 77.9 ± 2.08
99
albo-atrum 73.6 ± 1.48 and 77.9 ± 2.08 to larvae and adults, respectively. On the other hand, P. chrysogenum and A. cephalosporium produced the lowest mortality to larvae and adults; 24.3 ± 2.20 and 27.6 ± 2.65 and 28.0 ± 2.00 and 29.7 ± 2.76, respectively. The most virulent fungal strains; A. niger, T. viride, and V. albo-atrum were selected for further investigations. Three doses; 105, 106, and 107 of conidia mL1 for each fungal strain were prepared. The virulence of all fungal doses to larvae and adults of M. domestica were determined (Tables 2 and 3). The highest mortality to the larvae of M. domestica was produced by A. niger (Table 2). However, V. albo-atrum produced mortality to larvae higher than T. viride. Topical application of the fungal strains to larvae showed concentration-dependent response and caused mortality ranged between 41% and 83% with LT50 values varying between 5.22 (4.03–6.67) and 7.81 (5.90–8.14) days (Table 2). The highest percentage of mortality, 83.9 ± 3.52, was produced by 107 conidia mL1 of A. niger with LT50 5.22 (4.03–6.67). The lowest mortality percentage, 41.2 ± 3.00 with LT50 7.81 (5.90–8.14), was produced by 105 conidia mL1 of V. albo-atrum. The results revealed that the percentage of mortality to larvae and LT50 of the investigated fungal strains were dependent on the concentration of conidia mL1. The increase in conidia concentration increased the percentage of mortality and decreased the LT50 values (Table 2). Steinkraus et al. (1990) mentioned that 35–52% of mortality in third instars larvae of the house fly was produced by B. bassiana and Watson et al. (1995) obtained mortality rates between 48–56% in the second instar with high doses (1010 conidia mL1) while rates were negligible with lower doses. On the other hand, the highest mortality to adults of M. domestica was produced by A. niger (Table 3). However, unlike larvae, T. viride produced mortality to larvae higher than V.
Effect of Aspergillus niger, Trichoderma viride, and Verticillum albo-atrum on mortality of larvae of Musca domestica.
Fungal species
Dose (conidia mL1)
Mortality (%)
LT50 (CI)
Aspergillus niger
105 106 107 105 106 107 105 106 107
53.2 ± 3.42 67.5 ± 3.80 83.9 ± 3.52 49.2 ± 2.56 60.4 ± 2.83 69.0 ± 4.10 41.2 ± 3.00 55.1 ± 3.42 78.2 ± 4.43
6.58 6.23 5.22 7.33 6.47 5.46 7.81 6.28 5.91
Trichoderma viride
Verticillum albo-atrum
Table 3
(6.10–7.41) (4.96–7.03) (4.03–6.67) (5.67–8.23) (5.76–7.11) (4.53–6.65) (5.90–8.14) (5.65–7.13) (4.56–6.12)
Effect of Aspergillus niger, Trichoderma viride, and Verticillum albo-atrum on mortality of adults of Musca domestica.
Fungal species
Dose (conidia mL1)
Mortality (%)
LT50 (CI)
Aspergillus niger
105 106 107 105 106 107 105 106 107
71.1 ± 1.00 84.6 ± 1.90 97.3 ± 1.15 65.4 ± 1.35 73.7 ± 2.10 89.0 ± 3.57 59.1 ± 2.38 70.8 ± 2.65 81.1 ± 1.60
4.15 (3.89–5.02) 3.87(3.55–4.37) 3.49 (3.40–3.58) 6.83 (5.52–7.37) 6.36 (5.75–7.89) 5.52 (4.71–6.96) 6.91 (6.26–8.23) 6.41 (5.12–7.12) 5.67 (4.75–7.23)
Trichoderma viride
Verticillum albo-atrum
100 albo-atrum. Baiting application of fungus to adults showed concentration-dependent response and caused mortality ranged between 59% and 97% with LT50 values varying between 3.49 (3.40–3.58) and 6.91 (6.26–8.23) days (Table 3). The highest percentage of mortality, 97.3 ± 3.52, was produced by 107 conidia mL1 of A. niger with LT50 3.49 (3.40–3.58). The lowest mortality percentage, 59.1 ± 2.38 with LT50 6.91 (6.26–8.23), was produced by 105 conidia mL1 of V. albo-atrum (Table 3). The percentage of mortality produced to adults and LT50 of the investigated fungal strains were dependent on the concentration of conidia mL1. The increase in conidia concentration increased the percentage of mortality and decreased the LT50 values (Table 3). Similar results were obtained by Lecouna et al. (2005) which reported that only 5 out of 19 fungal species produced mortality rates exceeded 85% to adult house fly at 3 · 108 conidia/10 g sugar. Geden et al. (1995) found that baits containing 108 conidia/100 mg of two B. bassiana strains killed 78–88% of the adult house fly 5 days after exposure and 100% after 6 days. Despite that a 5–6 days interval between treatment and death is long compared with the rapid control that can be achieved with chemical insecticides, this long period may be acceptable to producers where flies have become resistant to such chemicals that they can no longer be used as control agents. The results of the current study revealed that the fungi such as A. niger, T. viride, and V. albo-atrum are effective for the control of the house fly, M. domestica and could be used as biological control agents. Similar results were previously reported by many researchers (Mullens, 1986; Geden et al., 1993, 1995; Barson et al., 1994; Watson et al., 1995, 1996; Renn et al., 1999; Kaufman et al., 2005; Lecouna et al., 2005; Mwamburi et al., 2010; Sharififard et al., 2011a,b).
Acknowledgments The author extends her appreciation to the deanship of scientific research at King Saud University for funding the work through the research group project no. RGP-VPP-074. The author also wish to thank Prof. Mohamed E. Zain, Professor of Microbiology, Botany and Microbiology Dept., College of Science, King Saud University for his useful advice for preparing the fungal cultivation, maintenance, conidial doses during this study. References Abbot, W.S., 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–266. Anderson, R.D., Bell, A.S., Blanford, S., Paaijmans, K.P., Thomas, M.B., 2011. Comparative growth kinetics and virulence of four different isolates of entomopathogenic fungi in the house fly (Musca domestica L.). J. Invert. Pathol. 107, 179–184. Axtell, R.C., Arends, J.J., 1990. Ecology and management of arthropod pests of poultry. Ann. Rev. Entomol. 35, 101–126.
E.M. Al-Olayan Barson, G., Renn, N., Bywater, A.F., 1994. Laboratory evaluation of six species of entomopathogenic fungi for control of house fly Musca domestica L., a pest of intensive animal units. J. Invert. Pathol. 64, 107–113. Butt, T.M., Goettel, M.S., 2007. Bioassay of entomogenous fungi. In: Navon, A., Ascher, K.R.S. (Eds.), Bioassay of microbes and nematodes. CABI Publishing, Wallingford, pp. 141–195. Geden, C.J., Steinerkraus, D.C., Rutz, D.A., 1993. Evaluation of two methods for release of Entomophthora musca (Entomophthorales: Entomophthoraceae) to infect house flies (Diptera: Muscidae) on dairy farms. J. Environ. Entomol. 20, 1201–1208. Geden, C.J., Rutz, D.A., Steinerkraus, D.C., 1995. Virulence of different isolates and formlation of Beauveria bassiana for house flies and the parasitoid muscidofurax raptor. J. Bio. Conf. 5, 615– 621. Kaufman, P.E., Reasor, C., Rutz, D.A., Ketzis, J.K., Arends, J., 2005. Evaluation of Beauveria bassiana applications against adult house fly, Musca domestica, in commercial caged-layer poultry facilities in New York State. J. Bio. Conf. 33, 360–367. Lecouna, R.E., Turica, M., Tarocco, F., Crespo, D.C., 2005. Microbial control of Musca domestica (Diptera: Musciadae) with selected strains of Beauveria bassiana. J. Med. Entomol. 42, 332–336. Mullens, B.A., 1986. A method for infecting large numbers of Musca domestica (Diptera: Musciadae). J. Econ. Entomol. 99, 1943–1947. Mwamburi, L.A., Laing, M.D., Miller, R.M., 2010. Laboratory screening of insecticidal activities of Beauveria bassiana and Paecilomyces lilacinus against larval and adult house fly (Musca domestica L.). Afr. Entomol. 18, 38–46. Quesada-Moraga, E., Ruiz-Garacia, A., Saniago-Alvarez, C., 2006. Laboratory evaluation of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae against puparia and adults of Ceratitis capitat (Diptera: Tephritidae). J. Eocn. Entomol. 99, 1955–1966. Renn, N., Bywater, A.F., Barson, G., 1999. A bait formulation with Metarhizium anisopliae for the control of Musca domestica (Diptera: Musciadae) assessed in large scale laboratory enclosures. J. Applied Entomol. 123, 309–314. SAS (1998). SAS/STAT User’s Guide. 9th Edn., SAS Institute, Cary, North Caroina. Sharififard, M., Mossadegh, M.S., Vazirian-zadhe, B., Mahmoudabadi, A.Z., 2011a. laboratory pathogenicity of entomopathogenic fungi, Beauveria bassiana (Bals) Vuill. and Metarhizium anisopliae (Metch.) Sorok. To larvae and adult of house fly, Musca domestica L. (Diptera: Muscidae). Asian J. Bio. Sci. 4 (2), 128–137. Sharififard, M., Mossadegh, M.S., Vazirian-zadhe, B., Mahmoudabadi, A.Z., 2011b. Interactions between entomopathogenic fungus, Metarhizium anisopliae and sublethal doses of spinosad for control of house fly, Musca domestica. Iran J. Arthropod-Borne Dis. 5 (1), 28–36. Steinkraus, D.C., Geden, C.J., Rutz, D.A., Kramer, J.P., 1990. First report of the natural occurrence of Beauveria bassiana (Moniliales: Moniliaceae) in Musca domestica (Diptera: Muscidae). J. Med. Entomol. 27, 309–312. Watson, D.W., Geden, C.G., Long, S.J., Rutz, D.A., 1995. Efficacy of Beauveria bassiana for controlling the house fly and stable fly (Diptera: Muscidae). J. Bio. Control 5, 405–411. Watson, D.W., Rutz, D.A., Long, S.J., 1996. Beauveria bassiana and sawdust bedding for the management of the house fly, Musca domestica (Diptera: Muscidae) in calf hutches. J. Bio. Control 7, 221–227.
King Saud University
Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Evaluation of pathogenicity of certain mitosporic ascomycete fungi to the house fly, Musca domestica L. (diptera: muscidae) Ebtesam M. Al-Olayan Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Received 22 November 2011; accepted 29 November 2011 Available online 6 December 2011
KEYWORDS Fungal extracts; Pathogenicity; Mortality percentage; House fly; Musca domestica
Abstract The virulence of five fungal strains, Acremonium cephalosporium, Aspergillus niger, Penicillium chrysogenum, Trichoderma viride, and Verticillum albo-atrum by application of 108 conidia mL1 to larvae and adults of house flies, Musca domestica was determined. A. niger produced the highest percentage of mortality to larvae, 85.1 ± 1.60, and adults, 98.2 ± 3.10, of house flies. Topical application of the fungal strains to larvae showed concentration-dependent response and caused mortality ranged between 41% and 83% with LT50 values varying between 5.22 and 7.81 days. The highest percentage of mortality, 83.9 ± 3.52, was produced by 107 conidia mL1 of A. niger with LT50 5.22. The lowest mortality percentage, 41.2 ± 3.00 with LT50 7.81, was produced by 105 conidia mL1 of V. alboatrum. On the other hand, the highest percentage of mortality to adults of house flies, 97.3 ± 3.52, was produced by 107 conidia mL1 ofA. niger with LT50 3.49. While the lowest mortality percentage, 59.1 ± 2.38 with LT50 6.91, was produced by 105 conidia mL1 of V. albo-atrum. ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction The house fly; Musca domestica L., is one of the most important and cosmopolitan insect pests, from both a medical and social standpoint (Axtell and Arends, 1990). It can affect E-mail addresses: [email protected], [email protected] 1319-6103 ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2011.11.019
Production and hosting by Elsevier
livestock performance, cause irritation to human beings and possesses considerable potential for the mechanical transmission of 100 pathogens on human beings. The development of resistance to pesticides by the house fly has increased the willingness of those involved in their production to seek alternative methods for fly management (Kaufman et al., 2005). On the other hand, the use of entomopathogenic fungi in the area of fly management has generated attention. Numerous studies are available documenting the attempted use of entomopathogenic fungi against house fly under laboratory and field conditions including Entomophthoramusca (Cohn) Fresenius (Mullens, 1986; Geden et al., 1993), Beauveria bassiana (Bals.) Vuill (Barson et al., 1994; Geden et al., 1995; Watson et al., 1995, 1996; Lecouna et al., 2005; Kaufman et al., 2005). However, isolates of fungi vary in their virulence to a given insect host but what factors contribute to this variation often
98 remain unclear (Anderson et al., 2011). The differences in isolate virulence may be attributed to the position of the phenotype of a particular isolate occupies on a continuum between two main strategies; an isolate may produce a large amount of toxins or may focus their energy into vegetative growth. The objectives of the present study were firstly, to evaluate the efficacy of 5 fungal species against the adults of house fly and secondly, to compare between the efficiency of the pathogenicity of those fungi for control house fly. 2. Materials and methods 2.1. House flies The house flies were collected from a poultry house using sweeping net and were transferred to the laboratory where they were reared at 27 C and 50 ± 5% RH with a 12:12 light:dark photoperiod. Adults were maintained in cages (40 · 40 · 40 cm3) covered by gauze. Adult flies were provided access to food, consisting of a 1:1 ration of powdered milk and granulated sugar, and water with replenished every 24–48 h. Larval medium comprised of 55 g wheat bran, 3 g date extract and 2 g dried alfalfa suspended in 140 mL water. One cup (250 mL) of this medium was left in each cage for oviposition by adults and subsequent development of larvae. The food was replaced every 24–48 h. 2.2. Fungal isolates Five isolates of mitosporic Ascomycete fungi; namely, Acremonium cephalosporium, Aspergillus niger, Penicillium chrysogenum, Trichoderma viride, and Verticillum albo-atrum were used in this study. The fungi were obtained from the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. The fungi were cultured on Sabourad dextrose agar (SDA) (Oxoid, UK) in 9 cm diameter Petri dishes and incubated at 27 C for 10 days. The sporulating cultures were harvested by scraping the dry conidia from the surface of the culture plate with a scalpel and transferred to sterile distilled water containing 0.01% Tween-80. The concentration of the suspension was determined using a hemocytometer (Butt and Goettel, 2007).
E.M. Al-Olayan distilled water. For testing the virulence of the fungi on the larval stage of M. domestica, groups of 25 larvae were immersed in conidial suspension for 10 s and were then placed on damp filter paper kept in a white disposable plastic container (100 mL volume). Containers were sealed with lids. The control larvae were dipped in 0.01% Tween-80 solution. Each treatment was replicated three times. Each group of larvae was transferred after 24 h to the container with 70 g of larval medium. The tops of the dishes were covered with gauze and incubated at 27 C with photoperiod 12:12 (light:dark). Emerged adults were recorded as the rate of survival and subsequently the rate of mortality was calculated. For testing the effect of fungal isolates on adults, 3–4 day adults were anesthetized with CO2 and batches of 25 were dipped in a suspension containing 108 conidia mL1 for 10 s and then placed on a piece of filter paper in small cages (20 · 20 · 20 cm). They were provided with sugar and powdered milk as solid medium and water. Cages were kept at 27 C with photoperiod 12:12 (light:dark). The control flies were treated with 0.01% solution of Tween-80. All treatments were replicated three times. Mortality was recorded for 7 days at 24 h intervals. 2.5. Larval treatment Suspensions of the most virulent fungal strains were prepared and serially diluted to give 107, 106, and 105 conidia mL1. Groups of 25 larvae were immersed simultaneously for 10 s in the conidial suspension and each group placed on damp filter paper in a white disposable plastic container. Control larvae were dipped in 0.01% Tween-80 solution. Larvae were transferred to the larval medium after 24 h. there were three replicates per treatment. All containers were incubated at 27 C with photoperiod 12:12 (light:dark). Mortality was recorded after treatment at intervals of 24 h until pupation occurred. Dead larva and pupa were held to monitor fungiinduced mortality. Externally sporulated cadavers were recorded as dead due to fungal infection. 2.6. Adult treatment
For each experiment, conidia viability was determined by enumerating the percentage of germinated conidia, 24 h after spreading on SDA medium. A 0.01 mL of conidial suspension (107 conidia mL1) was spread on 9 cm Petri dish containing 15 mL of SDA medium and incubated at 27 C for 24 h for germination (formation of distinct germ tube). Percentage germination was determined by first placing three 15 mm square cover slips directly on to the surface of the medium and counting the number of germinated conidia and total number of conidia per field of view at 200· magnification. Three to four fields of view were observed per cover-slip (Quesada-Moraga et al., 2006).
Four doses of conidia from the fungal strains were tested on cohorts of twenty five 2–3 days old, feeding male and female flies housed in small cages (20 · 20 · 20 cm). Each cage contained a 9 cm diameter Petri dish lined with Whatman filter paper and 10 g bait (6 g sugar, 2 g powder milk and 2 mL distilled water). For better dispersion of fungus, 1 mL of the stock suspension of the fungal strains under test was dispersed on the surface of bait to give doses 1 · 107, 1 · 106, and 1 · 105 conidia g 1 . The treated baits were left in the cages for 48 h before being removed and replaced with dry bait, water was provided ad lib. The experiments were run in a completely randomized design with three replicates per treatment. Cages were checked daily over a period of 7 days following presentation of fungus. Cadavers were removed daily and surface sterilized and transferred to a sterile Petri dish with a damp filter paper. Sporulating cadavers were recorded as mortality due to fungal infections.
2.4. Screening of fungal isolates virulence
3. Data analysis
An aqueous suspension of each fungal strain was prepared and the concentration was adjusted to 108 conidia mL1, in sterile
Three mortality counts of between 10% and 90% were obtained for each experiment. However, experimental tests
2.3. Fungal viability
Evaluation of pathogenicity of certain mitosporic ascomycete fungi to the house fly, that demonstrated more than 20% control mortality were discarded and repeated. When the control mortality ranged from 5% to 20%, the observed percentage of mortality (% M) was corrected by Abbott’s formula (1925): %M¼
% test mortality % control mortality X100 100 % control mortality
The mean lethal time (LT50) required killing one-half of the larvae and adults was estimated using probit analysis of SAS software (SAS, 1998). 4. Results and discussion Preliminary screening of five fungal strains by the application of 108 conidia mL1 to both larvae and adults of house flies showed that the A. niger was the most virulent strain for larvae and adults and produced mortality rates 85.1 ± 1.60 and 98.2 ± 3.10, respectively (Table 1). However, T. viride produced mortality rates 82.8 ± 4.10 and 83.5 ± 1.97 and V.
Table 1 Mortality of Acremonium cephalosporium, Aspergillus niger, Penicillium chrysogenum, Trichoderma viride, and Verticillum albo-atrum to larvae and adults of Musca domestica after exposure to a suspension of 108 conidia mL1. Fungal species
Acremonium cephalosporium Aspergillus niger Penicillium chrysogenum Trichoderma viride Verticillum albo-atrum
Table 2
Mortality (%) ± SE Larvae
Adults
28.0 ± 2.00 85.1 ± 1.60 24.3 ± 2.20 82.8 ± 4.10 73.6 ± 1.48
29.7 ± 2.76 98.2 ± 3.10 27.6 ± 2.65 83.5 ± 1.97 77.9 ± 2.08
99
albo-atrum 73.6 ± 1.48 and 77.9 ± 2.08 to larvae and adults, respectively. On the other hand, P. chrysogenum and A. cephalosporium produced the lowest mortality to larvae and adults; 24.3 ± 2.20 and 27.6 ± 2.65 and 28.0 ± 2.00 and 29.7 ± 2.76, respectively. The most virulent fungal strains; A. niger, T. viride, and V. albo-atrum were selected for further investigations. Three doses; 105, 106, and 107 of conidia mL1 for each fungal strain were prepared. The virulence of all fungal doses to larvae and adults of M. domestica were determined (Tables 2 and 3). The highest mortality to the larvae of M. domestica was produced by A. niger (Table 2). However, V. albo-atrum produced mortality to larvae higher than T. viride. Topical application of the fungal strains to larvae showed concentration-dependent response and caused mortality ranged between 41% and 83% with LT50 values varying between 5.22 (4.03–6.67) and 7.81 (5.90–8.14) days (Table 2). The highest percentage of mortality, 83.9 ± 3.52, was produced by 107 conidia mL1 of A. niger with LT50 5.22 (4.03–6.67). The lowest mortality percentage, 41.2 ± 3.00 with LT50 7.81 (5.90–8.14), was produced by 105 conidia mL1 of V. albo-atrum. The results revealed that the percentage of mortality to larvae and LT50 of the investigated fungal strains were dependent on the concentration of conidia mL1. The increase in conidia concentration increased the percentage of mortality and decreased the LT50 values (Table 2). Steinkraus et al. (1990) mentioned that 35–52% of mortality in third instars larvae of the house fly was produced by B. bassiana and Watson et al. (1995) obtained mortality rates between 48–56% in the second instar with high doses (1010 conidia mL1) while rates were negligible with lower doses. On the other hand, the highest mortality to adults of M. domestica was produced by A. niger (Table 3). However, unlike larvae, T. viride produced mortality to larvae higher than V.
Effect of Aspergillus niger, Trichoderma viride, and Verticillum albo-atrum on mortality of larvae of Musca domestica.
Fungal species
Dose (conidia mL1)
Mortality (%)
LT50 (CI)
Aspergillus niger
105 106 107 105 106 107 105 106 107
53.2 ± 3.42 67.5 ± 3.80 83.9 ± 3.52 49.2 ± 2.56 60.4 ± 2.83 69.0 ± 4.10 41.2 ± 3.00 55.1 ± 3.42 78.2 ± 4.43
6.58 6.23 5.22 7.33 6.47 5.46 7.81 6.28 5.91
Trichoderma viride
Verticillum albo-atrum
Table 3
(6.10–7.41) (4.96–7.03) (4.03–6.67) (5.67–8.23) (5.76–7.11) (4.53–6.65) (5.90–8.14) (5.65–7.13) (4.56–6.12)
Effect of Aspergillus niger, Trichoderma viride, and Verticillum albo-atrum on mortality of adults of Musca domestica.
Fungal species
Dose (conidia mL1)
Mortality (%)
LT50 (CI)
Aspergillus niger
105 106 107 105 106 107 105 106 107
71.1 ± 1.00 84.6 ± 1.90 97.3 ± 1.15 65.4 ± 1.35 73.7 ± 2.10 89.0 ± 3.57 59.1 ± 2.38 70.8 ± 2.65 81.1 ± 1.60
4.15 (3.89–5.02) 3.87(3.55–4.37) 3.49 (3.40–3.58) 6.83 (5.52–7.37) 6.36 (5.75–7.89) 5.52 (4.71–6.96) 6.91 (6.26–8.23) 6.41 (5.12–7.12) 5.67 (4.75–7.23)
Trichoderma viride
Verticillum albo-atrum
100 albo-atrum. Baiting application of fungus to adults showed concentration-dependent response and caused mortality ranged between 59% and 97% with LT50 values varying between 3.49 (3.40–3.58) and 6.91 (6.26–8.23) days (Table 3). The highest percentage of mortality, 97.3 ± 3.52, was produced by 107 conidia mL1 of A. niger with LT50 3.49 (3.40–3.58). The lowest mortality percentage, 59.1 ± 2.38 with LT50 6.91 (6.26–8.23), was produced by 105 conidia mL1 of V. albo-atrum (Table 3). The percentage of mortality produced to adults and LT50 of the investigated fungal strains were dependent on the concentration of conidia mL1. The increase in conidia concentration increased the percentage of mortality and decreased the LT50 values (Table 3). Similar results were obtained by Lecouna et al. (2005) which reported that only 5 out of 19 fungal species produced mortality rates exceeded 85% to adult house fly at 3 · 108 conidia/10 g sugar. Geden et al. (1995) found that baits containing 108 conidia/100 mg of two B. bassiana strains killed 78–88% of the adult house fly 5 days after exposure and 100% after 6 days. Despite that a 5–6 days interval between treatment and death is long compared with the rapid control that can be achieved with chemical insecticides, this long period may be acceptable to producers where flies have become resistant to such chemicals that they can no longer be used as control agents. The results of the current study revealed that the fungi such as A. niger, T. viride, and V. albo-atrum are effective for the control of the house fly, M. domestica and could be used as biological control agents. Similar results were previously reported by many researchers (Mullens, 1986; Geden et al., 1993, 1995; Barson et al., 1994; Watson et al., 1995, 1996; Renn et al., 1999; Kaufman et al., 2005; Lecouna et al., 2005; Mwamburi et al., 2010; Sharififard et al., 2011a,b).
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