EFFECTS OF LOCAL ENTOMOPATHOGENIC BEAUVERIA BASSIANA ISOLATES AGAINST SITOPHILUS GRANARIUS

ml respectively. Mycosis was observed in all the treatments except the control. Our study indicates that all the isolates could be used as potential biological control agents. Further studies are ongoing for determination of the efficacy of this isolate under storage conditions.


INTRODUCTION
Stored grains and their by-products are infested by insect pests causing approximately 10-25% losses worldwide.Losses caused by insects include not only the direct feeding damage resulting in loss of weight but they also severely reduce nutrients, lowering percentage of seeds germination, reducing grade and lowering their marketing value due to the accumulation of waste, webbing and insect cadavers (Hill, 1990).Most of these are coleopterans (Vinuela et al. 1993).Among them, the granary weevil Sitophilus granarius L. (Coleoptera: Curculionidae) and is known for its economic importance.The damages consist of the reduction in weight, quality, commercial value and seed viability (Hill, 1990).Residual insecticides have been employed to control insect pests of stored grains, but alternative control strategies are desirable because of the loss of insecticides due to pest resistance and consumer desire for pesticide-free grain (Arthur, 1996).
The most significant impetus for the growth of biopesticides comes from the growing awareness by farmers of the value of integrated pest management as a more environmentally sound, economical, safer and a selective approach to crop protection (Menn, 1996).Therefore, it is necessary to find out safer alternative control strategies such as the use of microbial control agents against stored-product insect pests.Using fungi and selected insecticides can potentially reduce the use of chemical insecticides and subsequently their residues and side effects in agriculture.Beauveria bassiana and Metarhizium anisopliae are naturally occurring entomopathogenic fungi with a broad host range (Sheeba et al. 2001, Wakil and Ghazanfar, 2010, Sewify et al. 2014).
The entomopathogenic fungus B. bassiana bears a considerable potential for the control of the different stored product pests.Cherry et al. (2005) suggested that B. bassiana is a potential microbial control agent against some stored product pests.Nowadays, several B. bassiana formulations (Boverosil ®, Mycotrol® ES, Mycotrol® 22WP, Naturalis® SC) are commercially available and are registered for use in storage facilities.Entomopathogenic fungi within stored food products can be employed to treat empty stores to control residual pests before the new harvest is brought in or may be applied as the direct admixture of conidia to grain, either as preventative or curative treatments of bulk grain.The present work aimed to evaluate the efficacy of the local entomopathogenic B. bassiana isolates against the granary weevil S. granarius.

MATERIAL AND METHODS
Test Insect Rearing: Tested stored product insect species Sitophilus granarius L. were obtained from laboratory cultures reared for several generations at the Department of Plant Protection, Agricultural Faculty, Gaziosmanpasa University, Tokat/Turkey.The insects were reared on whole wheat grains.Insect cultures were maintained in glass jars (2 litres) covered with a muslin cloth.All insects were reared under laboratory conditions of 24±2 °C and 75±2% R.H.

Fungal Isolates:
The entomopathogenic fungi, B. bassiana, isolates F-52, F-53, and F-56 were initially isolated from the meadow soil at Kelkit Valley.The fungi were grown on Potato dextrose agar (PDA) medium.The media was autoclaved at 121 °C for 15 minutes and poured into Petri dishes (9 cm diameter x 1.5 cm).The fungal isolates kept at 25 ±2 °C and 85± 5 % RH.The fungal isolate was sub-cultured every 14 -30 days and kept at 4 °C.
Bioassay: The isolates F-52, F-53, and F-56 were grown on Potato Dextrose Agar (PDA) medium at 27 o C for four weeks to get sporulation.The conidia were harvested under sterile conditions by flooding the plate with 10 ml of sterile distilled water containing 0,02% tween 80 then scraping the colony with a sterile glass hockey stick.The spore suspension was filtered through four layer sterile cheesecloths to remove mycelia.The concentrations of conidial suspensions were determined, using a Neubauer hemocytometer.The conidial suspensions were stored at 4 o C for up to 1 week until used in the assays.The viability of conidia was determined by spread-plating 0.1 ml of the suspension on the PDA plates.A sterile microscope coverslip was placed on each plate.Plates were incubated at 27 o C and examined after 24 h.The percentage of germination was determined by counting 100 spores for each plate, and over 95% of the spores germinated.For the dose-mortality test, Sitophilus granarius healthy adults were randomly selected and used for bioassays.Conidial suspensions of fungal isolates were prepared as described above, and a series of dilutions was prepared as 1×10 3 , 1×10 5 , 1×10 7 , 1×10 8 , and 1×10 9 conidia/ml.Ten adults were dipped in conidia suspension for 5 seconds than transferred into the glass wails containing ten wheat grains as food.The experiment was laid out in a completely randomised block design with five replications and replicated two times.The control insects treated with sterile distilled water containing 0,02% tween 80. Mortalities were recorded on the 1 st , 3 rd , 5 th , and seventh days of incubation.Dead insects were transferred in humid sterile 90 mm glass petri dishes for 14 days to determine the mycosis rates.The percentage mortality and LC50 were estimated.

Statistical Analysis
The data were analysed by analysis of variance (ANOVA) and the means compared by Tukey's multiple comparison test.All statistical analyses were carried out using the SPSS Release 16 packet program.The lethal concentration (LC50) was calculated using probit analysis.(Finney 1978).

RESULTS AND DISCUSSION
Several studies documented the high potential of entomopathogenic fungi for the control of insect pests in stored grains and their byproducts (Moore et al., 2000, Cox et al. 2003and 2004, Cherry et al. 2005).The insecticidal efficacy of B. bassiana is highly influenced by several factors such as characteristics of host insects, and physiology of pathogen fungi (enzymes and toxins) (Fargues et al., 1996, Cox et al., 2004).So, the testing of different isolates of B. bassiana against different storage pests is needed.In the present study it was found that three B. bassiana isolates tested (F-52, F-53, and F-56) were pathogenic in S. granarius adults.
The percentages of mortality varied from 76% to 84% at 1x10 8 conidia/ml concentration 7 day after inoculation (Table 1, 2, and 3).The control group had 8% mortality.In all the experiments, mortality increased with increase in conidia concentrations and incubation periods while the highest mortality was observed after seven days of incubation at 1x10 9 conidia/ml concentrations.The results indicated that isolate F-53 was effective against S. granarius and resulted in a high mortality 98% at the end of the 7 th day incubation period at 1×10 9 conidia/ml.
Followed by isolates F-52 and F-56 with 94% mortality (table 1,2 and 3).Mycosis was observed in all the treatments except the control.The present study agrees with the results of previous studies where higher doses produce the highest percentage of mortality (Athanassiou andSteenberg 2007, Khashaveh et al., 2011;Magda and Mohamed, 2015).Probit analysis was carried out to determine LC50.The parameters of the probit analysis and LC50 are given in Table 4. LC50 values confirmed that S. granarius was more susceptible to the isolate F-52 than the other two isolates F-53 and F-56 where the LC50's were 1x10 5 , 2x10 5 , and 5x10 5 conidia/ml respectively (Table 4).
* SEM: Standard error of the mean; ** DAT: Days after treatment; *** Means in a column followed by the same letter are not statistical significantly different (P < 0.05).* SEM: Standard error of the mean; ** DAT: Days after treatment; *** Means in a column followed by the same letter are not statistical significantly different (P < 0.05).