Abstract: Virulence of three indigenous Iranian isolates of the fungus, Metarhizium anisopliae (Metsch.) Sorokin (Deuteromycotina: Hyphomycetes), named DEMI001, IRAN 715C and IRAN 1018C, were evaluated as well as their virulence and their ability to suppress populations of the granary weevil, Sitophilus granarius L. (Coleoptera: Curculionidae). Five aqueous suspensions were prepared from each isolates, in a logarithmic series in Tween 80 (0.05% v/v). LT50 values ranged from 5.54 to 7.9 days following immersion in aqueous suspensions. Lowest LC50 on day 10 was 1/4x105 conidia mL-1 for DEMI001. Cumulative mortality 10 days after treatment varied from 9.4 to 88.88% for IRAN 1018C at low and high concentration, respectively.
INTRODUCTION
The granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae) is a very serious primary pest of stored grain products, which is able to cause considerable economic losses (Hill, 1990). Application of insecticides is one means of preventing some losses during storage. However, the choice of insecticides for storage pest control is very limited because of the strict requirements imposed for the safe use of synthetic insecticides on or near food (Padin et al., 2002). The continuous use of chemical insecticides for control of storage grain pests has also resulted in serious problems such as resistance to the insecticides, pest resurgence, elimination of economically beneficial insects and toxicity to humans and wildlife (Adane et al., 1996; Padin et al., 2002).
Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes) is a mitosporic haploid fungus with a global distribution. It represents a pathogen for many insect species including a wider range of important agricultural pests and therefore, it holds great potential for use as biological control agent (Butt et al., 2001).
Until present, limited number of published articles on biocontrol of stored grain insects using entomopathogenic fungi are available. Beauveria bassiana, for example, has proven highly effective against the major stored grain insects: Sitophillus oryzae, Rhyzopertha dominica, Oryzaephilus surinamensis, Prostephanus truncatus and Tribolium castaneum (Smith et al., 1999; Tanya and Doberski, 1984). In contrast, M. anisopliae has been less frequently reported for control of stored grain insects although it has been used effectively to control other insects especially termites, black field crickets, grasshoppers and locusts, tobacco whitefly and red spidermites (Batta and Abu Safieh, 2005).
Investigations since mid 1980s by Tanya and Doberski (1984) followed by Adane et al. (1996), Hidalgo et al. (1998), Bello et al. (2000), Ekesi et al. (2001) and Padin et al. (2002) suggested that isolates of B. bassiana and M. anisopliae are potential microbial control agents against some stored product pests.
In this research, the susceptibility of the granary weevil, S. granarius to three Iranian isolates of entomopathogenic fungus M. anisopliae was appraised. All experiments were carried out in room conditions to evaluate efficacy of different isolates. These isolates showed virulence on other stored product insect (Personal observations).
MATERIALS AND METHODS
S. granarius culture: Adults of S. granarius were collected from a laboratory culture, was kept on whole wheat; at 27±1°C, 65±5% RH and continuous darkness that kept in laboratory cultures for > 3 years without exposing insecticides in the Department of Entomology in Urmia University, Iran. Adults used in the experiments were < 7 days old.
Source of Metarhizium anisopliae isolates: All fungal isolates; DEMI001, IRAN 715C and IRAN 1018C were obtained from the collection maintained by the Plant Pest and Diseases Research Institute, Tehran, Iran. The isolates were cultured and stored at 4°C on Sabouraud Dextrose Agar (SDA). The three isolates of M. anisopliae were used for the virulence test (Table 1).
Production of conidial suspension: All fungal isolates were cultured on Potato Dextrose Agar (PDA, Merck and Co., Inc., Germany) in 9 cm diameter Petri dishes and then placed in dark at 24±2°C and 45±5% RH (in room conditions) for 15 days for complete sporulation. After this period, a mixture of conidia and hyphae was harvested by flooding the Petri dishes with sterile distilled water containing 0.05% (v/v) Tween 80 (Sigma Chemical, St. Louis, MO, USA) and agitating with glass rod. All sample 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 a five layers of cheese-cloth. The conidia concentration was counted with Haemocytometer (Improved Neubauer, 0.1 mm depth). Germination was assessed by counting 300 conidia after fixing with lacto phenol cotton blue (Kassa et al., 2002).
Dose-response bioassay by immersion method: Five aqueous suspension were prepared from 1x108 down to 1x104 conidia mL-1 in Tween 80 (0.05%v/v) for primary experiments. On the basis of preliminary tests, for each isolate and insect five concentrations of conidia were prepared for main experiments (Robertson and Preisler, 1992). Each concentration was replicated four times. For each replicate, thirty adults ( >7 days old ) were treated by immersion for 5 sec in 5 mL suspension. The control insect were immersed in sterile distilled water with Tween 80 (0.05%v/v). The treated insects and the suspension (1 mL) were subsequently poured into a plate containing filter paper (9 cm diameter) and sealed with parafilm to prevent insects from escaping. The filter paper helped to absorb the excess moisture and increased conidial load in each insect by allowing secondary spore pick up. (Adane et al., 1996). The treated insects were kept without food for 24 h at 24±2°C and 45±5% r.h. After 24 h, the treated insects in each replicate were transferred into glass pots (7 cm diameter and 8.5 cm height) with perforated lid containing 30 g wheat grains (variety Zarrin) and then kept at 24±2°C and 45±5% RH (at room conditions) for 10 days. The experiment was arranged in a completely randomized design and mortality was recorded at 48 h interval. Dead insects from each treatment were washed in 70% ethanol, rinsed in sterile distilled water three times and kept separately in Petri dishes. These plates were then incubated in a plastic box with high RH (approximately 100%) to observe the outgrowth of fungus. The same procedure were carried out for the control insects.
Data analysis: Control mortality was corrected by using Abbott`s (1925) formula. For dose-mortality bioassay, cumulative mortality percentage was normalized using arcsine transformation and subjected to analysis of variance (ANOVA) using SAS (1999). Means were separated by using the Tukey-Kramer honestly significant difference test at p≤0.05. Probit analysis was used to estimate LC50, LC95 and LT50 of the isolates with 95% Confidence Limits (CL) after 10 days (SPSS, 2002).
RESULTS AND DISCUSSION
Results of this study sowed that the mean viability of conidia of all M. anisopliae isolates ranged between 89 to 94% (Table 1). The mortality within the control group was very low (2.49±1.59%) and no fungal growth was observed on the control insects. LT50 values for M. anisopliae isolates varied from 5.54 to 7.9 days, with an average of 6.63 days (Table 2). Among the M. anisopliae isolates, the isolate DEMI001 demonstrated the shortest LT50. The parameters of the probit analysis and LC50 and LC95 are given in Table 3. At all isolates, M. anisopliae were pathogenic. The lowest and highest LC50 and LC95 values were observed in the isolates DEMI001 (1.4x105 and 1.5x107) and IRAN 715C (1x107 and 5.1x1011), respectively. Mortality percentages for adults increased with increasing conidial concentration in all isolates (DEMI001: F = 38.973, p≤0.0001), (IRAN 1018C: F = 80.408, p<0.0001), (IRAN 715C: F = 61.085, p≤0.0001). Maximum and minimum mortality rates observed in IRAN 1018C (88.88 and 9.4%, respectively). In general isolate DEMI001 had better effect on S. granarius because the range of concentration was very low and the high concentration in this isolate was lower rather than the two other isolates and had same mortality rate with IRAN 1018C (88.03%) (Table 4).
| Table 1: | The host, location and germination percentage of the isolates of Metarhizium anisopliae used |
| Table 2: | LT50 values (day) with 95% confidence limits following immersion of S. granarius adults in aqueous suspensions of M. anisopliae isolates in high concentration |
| *Non calculated | |
| Table 3: | LC50 and LC95 values (with 95% confidence limit) and probit analysis parameters for adults of S. granarius 10th day after immersion in aqueous conidial suspensions of M. anisopliae (three isolates) |
| Table 4: | Cumulative mortality percentage (corrected) ±SE of S. granarius adults 10th day after immersion in aqueous conidial suspensions of M. anisopliae (three isolates)* |
| *Mean within a row followed by the same letter do not differ significantly by Tukey-Kramer test at p≤0.05 | |
Insect cuticle, the first barrier against fungal pathogens, consists of a thin outer epicuticle, containing lipid and proteins and a thick procuticle, consisting of chitin and proteins. Entomopathogenic fungi produce proteases, chitinases and lipases which can degrade insect cuticle (Weiguo et al., 2005). Laboratory assessment of entomopathogenic fungi is an essential step in identifying virulent strain prior to field or large scale use. Entomopathogenic fungi are being developed worldwide for the control of insect pests and some products are already available commercially (Ekesi et al., 2001). Results of the current study indicated that all fungal isolates were virulent to granary weevil. The isolate DEMI001 can provide better control of S. granarius because it had lower LT50, LC50 and LC95 . Within these taxa, individual isolates can exhibit substantially restricted host range and isolates recovered from a target host and closely related species are generally more virulent than isolates from non-related species (Inglis et al., 2001). Because the isolate DEMI001 was originally from a curculionid pest, its potential for the control of S. granarius was great. This observation highlights the need for screening for more virulent isolates against storage pests for use in the management of these pests. Other investigators have reported that treatment of stored grain pests with entomopathogenic fungi, especially M. anisopliae and B. bassiana can be effective (Hidalgo et al., 1998; Kassa et al., 2002; Batta, 2005). Obtained results are in accordance with their results. Wakefield et al. (2005) demonstrated that some B. bassiana isolates can provide 100% mortality in Oryzaephilus surinamensis (saw-toothed grain beetle- organophosphate resistant strain), Ephestia kuehniella (Mediterranean flour moth), Epinotus patruelis (black domestic psocid) and Acarus siro (flour mite) 10 days after treatment in 1x108 conidia mL-1. Adane et al. (1996) demonstrated that several isolates of B. bassiana tested against Sitophilus zeamais (Motsch.) adults showed pathogenicity to insects, but there were highly significant differences among the isolates with respect to virulence. These isolates caused 37 to 100% mortality in S. zeamaiz. They also had been recorded median lethal time for different isolates. The lowest time (2.74 days) was recorded for isolate 189-481. Rodrigues and Pratissoli (1990) reported 6 months protection of maize grains and bean grains from damage by S. Zeamais and Acanthoscelides obtectus (Say) following treatment with Beauveria brongniartii (Sacc.) Petch. and M. anisopliae at a dose of 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 could cause 77.5, 88.75 and 90% of S. oryzae adults mortality, respectively (Hendrawan and Ibrahim, 2006). Bourassa et al. (2001) found that B. bassiana IMI330194 led to 100% mortality of P. truncatus larvae. Cherry et al. (2005), also have demonstrated that different isolates from M. anisopliae and B. bassiana could provide good control of C. maculatus by immersion bioassay. They represented that the LT50 values for B. bassiana and M. anisopliae isolates was varied from 3.11 to 6.13 days (with an average of 4.61 days) and 3.27 to 5.62 days (with an average of 4.60 days).
In conclusion, study research showed a high susceptibility of adult S. granarius to M. anisopliae. Based on this finding, we may suggest M. anisopliae as a useful candidate for the management of the storage pests, S. granarius. However, further investigations are strongly recommended to be carried out on the possibility of field application as well as finding other isolates of entomopathogenic fungi that have potential as biopesticides against such storage pests.