MATERIALS AND METHODS

Insect rearing: The initial stock of R. dominica was obtained from the Department of Plant Protection, Faculty of Agriculture, Urmia University. Adults of R. dominica were reared on dried, healthy and mature wheat grains, Zarin variety. Grains of Zarin variety were stored at -5°C for 1 week to eliminate natural unwanted infestations. Two hundred adults of R. dominica of mixed sexes and different ages were introduced in each jar (15 cm diameter and 20 cm height) containing 400 g of wheat grain and were maintained under the laboratory conditions (28±2°C, 65±5% R.H.) with a natural photoperiod. The jars were covered with cheese cloth fastened by rubber bands. The original adults were removed from the jars after two weeks of infestation. Newly emerged adults (males and females) were used in the experiments.

Table 1:	Host, origin and germination of Beauveria bassiana and Metarhizium anisopliae isolates used in the present study

Fungal isolates: Three Iranian isolates of B. bassiana and two isolates of M. anisopliae were obtained from the fungal culture collection maintained by the Plant Pest and Diseases Research Institute, Tehran, Iran. Detail information’s about the isolates are given in Table 1.

Preparation of conidial suspensions: Fungal isolates were cultured on Potato Dextrose Agar (PDA) in 8 cm diameter Petri dishes and were incubated under dark conditions at 25°C for 14 days for complete sporulation. A mixture of conidia and hyphae was harvested by flooding the Petri dishes with sterile distilled water containing 0.05% (v/v) Tween 80 and agitating with a glass rod. All samples were vortexed for 3 min to break up the conidial chains or clumps. Conidia were separated from hyphae and substrate materials by filtration of the suspension through five layers of cheese-cloth. The concentrations of fungal conidia in suspension were determined using a haemocytometer (Improved Neubauer, 0.1 mm depth). Viability of conidia was determined by spreading a drop of conidial suspensions onto the surface of glass slides held in Petri dishes lined with moistened sterile filter paper. Three glass slides per isolate representing three replicates were used and scored for germination after 24 h at 25±2°C. Conidia with germ tubes equal or greater than the width were considered to have germinated.

Bioassay: After preliminary assays, 5 different conidial concentrations were prepared in sterile distilled water containing Tween 80 (0.05% v/v) based on the logarithmic series. For each replicate, thirty (7-14 day old) R. dominica adults were inoculated by immersing them for 5 sec in 5 mL of conidial suspension. Control insects were treated using sterile distilled water containing Tween 80 (0.05% v/v).

The treated insects were transferred into petri dishes containing sterile filter paper (9 cm diameter) and sealed with parafilm to prevent them from escaping. The filter paper helped to absorb excess moisture and increased conidial load in each insect by allowing secondary conidia growth (Adane et al., 1996). The treated insects were starved for 24 h then transferred into glass pots (7 cm diameter and 8.5 cm height) with perforated lids containing 30 g wheat grains and kept at 27±1°C and 70±5% R.H. for 14 days. The experiment was arranged in a completely randomized design with four replications. Mortality was counted for 14 days. Dead insects from each treatment were washed three times in sterile distilled water and kept separately in Petri dishes. The dishes were then incubated in a plastic box with high R.H. (approximately 100%) to observe the outgrowth of fungus.

Statistical analysis: Cumulative mortality counts obtained from experiments were corrected for natural mortality using Abbott’s formula (Abbott, 1925) and were normalized using arcsine transformation prior to analysis. Data were statistically analyzed using ANOVA (SAS, 2000) and means were separated using the Duncan’s Multiple range test at p = 0.05. Probit analysis was used to estimate both LC₅₀ and LC₉₅ of the isolates with 95% Confidence Limits (CL) as well as LT₅₀ values (SPSS, 1999).

RESULTS

Germination of all B. bassiana and M. anisopliae isolates tested varied from 80 to 94% (Table 1). All the isolates tested were pathogenic to adults of R. dominica in immersion bioassays, but they had different virulence. Mortality rates increased with increasing conidial concentration (B. bassiana Iran 187C: F = 66.54, p<0.0001), (B. bassiana Iran 429C: F = 64.95, p<0.0001), (B. bassiana Iran 441C: F = 80.67, p<0.0001), (M. anisopliae DEMI001: F = 79.40, p<0.0001) (M. anisopliae Iran 715C: F = 63.66, p<0.0001). The B. bassiana 441C and M. anisopliae DEMI001 isolates were the most virulent isolates (Table 2). The maximum and minimum percentage of mortality observed in M. anisopliae DEMI001 and B. bassiana Iran 441C isolates, respectively. The lowest LC₅₀ value was observed in B. bassiana Iran 187C isolate. The LT₅₀ values for B. bassiana isolates varied from 6.77 to 9.28 days, with an average of 7.77 days, while those for M. anisopliae isolates varied from 7.48 to 8.25 days with an average of 7.86 days (Table 3). Among B. bassiana isolates, Iran 441C had the shortest LT₅₀ of 6.77 days and among M. anisopliae isolates, DEMI001 had the shortest LT₅₀ of 7.48 days (Table 4).

Table 2:	Cumulative percentage mortality (corrected ± SE) of Rhyzopertha dominica adults 14 days after immersion in aqueous conidial suspensions of Beauveria bassiana and Metarhizium anisopliae isolates
Means followed by the same letter in the row are not significantly different (Duncan’s Multiple range test at p = 0.05). Cumulative mortality data were corrected for natural mortality using Abbott’s formula and normalized using arcsine transformation before analysis

Table 3:	LC50 and LC95 (conidia mL-1) values with 95 fiducial limits and probit analysis parameters for adults of Rhyzopertha dominica

Table 4:	LT50 values in days with 95 fiducial limits following immersion of Rhyzopertha dominica adults in aqueous suspensions of Beauveria bassiana and Metarhizium anisopliae isolates

DISCUSSION

On the other hand, one of the key elements of Insect Pest Management (IPM) in stored-products is the combination of several, reduced-risk, control methods, because storage pests are not always effectively controlled by the application of only one measure (Kavallieratos et al., 2006). However, Several studies documented that entomopathogenic fungi B. bassiana and M. anisopliae can be used with success against stored product insect pests and some products are already available commercially (Ekesi et al., 2001; Ferron et al., 1991).

The results of the present study indicated that although all tested isolates were pathogenic and caused mortality in R. dominica, they had different virulence. B. bassiana Iran 441C caused significantly higher mortality (89.35%) than the other isolates while M. anisopliae, DEMI001 isolate caused high mortality (79.99%) at the highest conidia concentration (4.6x10⁹ conidia mL^-1).

Our results for LT₅₀ indicated that the present isolates are not effective as much as other isolates cited in literatures. For instance, Batta (2005) reported that high mortalities of adult R. dominica were obtained 7 days after treatment of newly emerged adults with M. anisopliae conidia. Wakefield et al. (2005) reported that some B. bassiana isolates provided 100% mortality in Oryzaephilus surinamensis L., Ephestia kuehniella (Zeller) and Acarus siro L. 10 days after treatment in 1x10⁸ conidia mL^-1. Unformulated conidia of M. anisopliae isolate MaPs and B. bassiana isolates BbPs and BbGc at the rate of 0.15 g a.i. to 50 g a.i. rice grain caused mortality ranging from 77.5- 90% in adults of Sitophilus oryzae (Hendrawan and Ibrahim, 2006). Rodrigues and Pratissoli (1990) reported 6 months protection of maize and bean grains from damage by Sitophilus zeamais (Motsch) and Acanthoscelides obtectus (Say) following treatment with B. brongniartii (Sacc.) Petch. and M. anisopliae at a dose of 1x10⁸ conidia mL^-1. Cherry et al. (2005) also have demonstrated that different isolates from M. anisopliae and B. bassiana can provide good control of Callosobruchus maculatus F. by immersion bioassay. On the contrary, other investigators have reported that treatment of S. oryzae on wheat grains with M. anisopliae alone was not effective, but that 50% adult mortality of the pest was achieved 30 days after treatment with a conidial suspension mixture of M. anisopliae and B. bassiana (Dal-Bello et al., 2001). The difference in M. anisopliae effectiveness obtained in the above studies may be attributed to differences in aggression of the fungal strains used, host susceptibility, experimental conditions and food substrate used (Batta, 2005). Among the isolates tested, M. anisopliae Iran 715C and B. bassiana Iran 187C caused lower mortality that may be attributed to their low percentage of conidia germination (Table 1).

In conclusion, our research showed high susceptibility of adult of R. dominica to B. bassiana and M. anisopliae. However, further experiments are needed to be carried out for screening for more virulent isolates for biocontrol of storage pest.

ACKNOWLEDGMENTS

We are grateful to Dr. Rasol Zare for providing the Fungal isolates for experimentation. The resources made available by Urmia University are also gratefully acknowledged.