Naturally occurring entomopathogenic fungi infecting stored grain insect species in Punjab, Pakistan.

The occurrence of entomopathogenic fungi isolated from stored grain insect pests sampled from various geographical regions of Punjab, Pakistan, was investigated. In total, 25,720 insects from six different species were evaluated, and 195 isolates from 24 different fungal species were recovered. These included the Ascomycetes Beauveria bassiana sensu lato (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae), Metarhizium anisopliae sensu lato (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae), Purpureocillium lilacinum (Thorn) Samson (Hypocreales: Ophiocordycipitaceae), and Lecanicillium attenuatum (Zare and W. Gams) (Hypocreales: Clavicipitaceae). The cadavers of red flour beetle Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae) were significantly infected with the fungi followed by rice weevil Sitophilus oryzae (L.) (Coleoptera: Curculionidae), lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), rusty grain beetle Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae), and cowpea weevil Callosobruchus maculatus (F.) (Coleoptera: Bruchidae); however, the least were recovered from khapra beetle Trogoderma granarium (Everts) (Coleoptera: Dermestidae). The geographical attributes (altitude, longitude, and latitude) greatly influenced the occurrence of entomopathogenic fungi with highest number of isolates found from >400 (m) altitude, 33°-34' N latitude, and 73°-74' E longitude. The findings of the current surveys clearly indicated that the entomopathogenic fungi are widely distributed in the insect cadavers, which may later be used in successful Integrated Pest Management programs.

Grain commodities are commonly infested by insect pests during storage ( Haq et al. 2005 ). It is generally considered that 5–15% grain losses in different stored grain commodities occur as a result in insect pest infestations ( Padin et al. 2002 ), which reduces the commercial value, quality of stored grain and seed viability. The most economically important stored grain and pest species include the red flour beetle Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae), the lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), and the rice weevil Sitophilus oryzae (L.) (Coleoptera: Curculionidae) ( Bello et al. 2001 ; Chanbang et al. 2007 ; Michalaki et al. 2007 ). These pests are often managed using synthetic chemical insecticides applied to the grain store; however, excessive insecticide use can result in control failure following the evolution of insecticide resistance and has also raised concerns about human and environmental safety ( Cox 2004 ). As an alternative to these insecticides, entomopathogenic fungi have received some attention for use in stored product Integrated Pest Management (IPM) ( Gillespie 1988 ). The potential benefits of using entomopathogenic fungi include human safety, the development of an epizootic ( Hidalgo et al. 1997 ), and repeated infection cycles of propagules in the microenvironment ( Thomas et al. 1995 ; Wood and Thomas 1996 ), which increases the longevity and persistence of the biological control agent ( Bateman et al. 1993 ). Successful control of stored grain pests with entomopathogenic fungi has been reported by a number of researchers ( Latge and Moletta 1988 ; McCoy et al. 1988 ; McCoy 1990 ; Ferron et al. 1991 ; Roberts and Hajek 1992 ; Tanada and Kaya 1993 ; Hajek and St. Leger 1994 ; Wakil and Ghazanfar 2010 ). The entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin ( Akbar et al. 2004 ; Lord 2007 ) and Metarhizium anisopliae (Metschnikoff) Sorokin ( Batta 2004 , 2005 ; Kavallieratos et al. 2006 ; Michalaki et al. 2006 ), in particular, have both proven to be efficacious against many insect pests of stored products. These fungi do not decrease the quality of the grains and thus enhances the marketability of stored products ( Steenberg 2005 ). For instance, Adane et al. (1996) and Kavallieratos et al. (2006) tested B. bassiana isolates for controlling the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae) and the cowpea Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and noted their high virulence. Likewise, Cherry et al. (2005) and Athanassiou et al. (2008) found M. anisopliae effective against S. oryzae and R. dominica on rice, wheat, and maize grains. High mortality rates of R. dominica were attributed to using a local isolate of B. bassiana on stored wheat under laboratory conditions ( Wakil et al. 2011 , 2012 ). However, despite the clear potential of entomopathogenic fungi for use in IPM, little is known about the natural occurrence of fungal infections in populations of stored product pests, and this could be an impediment to the identification of the most effective candidate fungal strains. In the current study, we quantified the entomopathogenic fungal flora occurring on populations of grain insects in Pakistan in order to better understand the relationship, if any between fungal occurrence and geographical location.

Materials and Methods

Collection of Stored Grain Insects

Systematic surveys of grain storage facilities were carried out in different districts of Punjab (Pakistan). The relevant geographical attributes ( Table 1 ) and the positions of the sites are given in Figure 1 . In total, five to six grain storage facilities were sampled from 15 sites ( Table 1 ). Each sample consisted of ∼500 g grain collected from each of five different points selected at random within the grain storage facility (i.e., a total of 2.5 kg grain was collected from each store). At the time of sample collection, any insects with abnormal movement or showing lethargic activity in the immediate vicinity of the subsample were also collected and put into sterile plastic vials. The grain samples were put into zip lock bags and were brought back to the laboratory; however, the vials containing insects were kept in a cooler maintained at 10°C before reaching the laboratory. The grain samples were sieved to separate any insects present from the grain. Insect cadavers were stored at 4°C in the refrigerator for further processing.

Table 1.

Sampling sites with geographical characteristics for the isolation of fungi from insect cadavers

Sites	Altitude (m)	Latitude	Longitude
Bahawalpur	109	29° 24′ N	71° 40′ E
Lodhran	111	29° 53′ N	71° 63′ E
Basti Maluk	119	29° 51′ N	71° 32′ E
Multan	124	30° 20′ N	71° 48′ E
Khanewal	128	30° 17′ N	71° 55′ E
Shorkot	126	30° 95′ N	72° 08′ E
Jhang	158	31° 27′ N	72° 31′ E
Faisalabad	184	31° 30′ N	73° 05′ E
Sargodha	193	32° 10′ N	72° 40′ E
Sheikhupura	213	31° 71′ N	73° 97′ E
Lahore	210	31° 35′ N	74° 20′ E
Gujranwala	223	32° 10′ N	74° 12′ E
Ghakhar	224	32° 30′ N	74° 15′ E
Jhelum	220	32° 55′ N	73° 43′ E
Rawalpindi	497	33° 58′ N	73° 08′ E

Fig. 1.

Mapping different sampling sites from Punjab, Pakistan for the isolation of fungi from insect cadavers: 1. Bahawalpur; 2. Lodhran; 3. Basti Maluk; 4. Multan; 5. Khanewal; 6. Shorkot; 7. Jhang; 8. Faisalabad; 9. Sheikhupura; 10. Lahore; 11. Sargodha; 12. Gujranwala; 13. Ghakhar; 14. Jhelum; 15. Rawalpindi.

Isolation and Identification of Fungi

The collected insects (live and dead) were identified to species level according to taxonomic keys ( Rees 2004 ). All dead insects were washed in 75% ethanol solution (aq.) for 1 min, then transferred to 3% NaClO for 3 min, followed by immersion in 75% ethanol for 1 min. Cadavers were then dried on filter paper and then placed in petri dishes containing Sabouraud Dextrose Agar (SDA) supplemented with 0.1 g/liter of streptomycin sulfate and 0.05 g/liter of chloramphenicol. Plates were sealed with parafilm and incubated at 25°C for up to 5–7 d. Insects were examined under a binocular microscope every 24 h for the appearance of any fungal growth on the medium. Where more than one fungal colony was present, the colonies were separated by subculturing on SDA. The fungi were identified on the basis of morphological traits using taxonomic keys ( Barnett and Hunter 1999 ; Domsch et al. 2007 ) and their pathogenicity (Koch’s postulates) was confirmed on their respective hosts. The slides and the culture of identified entomopathogenic fungal isolates were deposited in the IPM ( Insect Pathology ) Laboratory, Department of Entomology, University of Agriculture, Faisalabad, Pakistan.

Virulence of Isolated Fungi

The isolated entomopathogenic fungi were subcultured at 25°C and 75% r.h. with 16-h illumination per day on SDAY (32.5 g SDA; 7.5 g of Bacto Agar; 5 g yeast in 1 liter distilled water) plates for the production of conidia. After 14 d of incubation, the plates were air-dried for one week. Fungal conidia were harvested by scraping the conidial layers formed on the surface of plates using a sterilized scalpel. The fungal conidia were suspended in 0.05% Tween-80 solution and filtered through muslin cloth to remove mycelial debris. A suspension of 1 × 10 6 conidia ml −1 (dose was standardized in preliminary bioassays) of each isolate was prepared and used to inoculate third larval instars of T. castaneum . This was done by immersing larvae in conidial suspension for five seconds before transferring larvae to petri plates (9 cm) lined with moist filter paper and sealed with parafilm. These plates were incubated at 25°C, and four replicates each having 10 larvae were used. The larvae treated with Tween-80 served as the control, and the same procedure for each isolate was repeated three times with four replications with new materials each time independently. The larval mortality was examined 7-d post application of fungi.

Statistical Analysis

The occurrence of fungi in different insect species ( T. castaneum, R. dominica, Trogoderma granarium, S. oryzae, the rusty grain beetle Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae), and C. maculatus ) collected from the different grain storage sites was compared using a χ2 test and one way analysis of variance The mortality of control in virulence assay was very low (0.07%), so that it was not included in the analysis. The statistical software Minitab 13.2 was used for the analysis of data (Minitab 2002 Software Inc., Northampton, MA, USA).

Results

Occurrence of Fungi

In total, 195 insect cadavers were separated out of 25,720 insects collected during the survey of different grain storage facilities ( Table 3 ). The major fungal genera isolated from the insect cadavers were Aspergillus sp. (22.6% occurrence), followed by Fusarium sp. (15.9%), Alternaria sp. (10.8%), Rhizopus sp. (8.2%), Penicillum sp. (6.7%); however, the least was Mucor sp. (1%) ( Table 2 ). These fungal species were isolated from different insects as 68 from T. castaneum , 41 from S. oryzae , 26 from R. dominica , 21 each from C. ferrugineus and C. maculatus , and 18 from the khapra beetle T. granarium (Everts) (Coleoptera: Dermestidae). The fungus were distributed in all the collection sites surveyed ( Table 3 ). The highest frequency (23.1%) of fungi was recorded from the various storage facilities in Rawalpindi district, followed by 12.8% from Ghakhar, 12.3% from Jhelum, and 8.7% both from Sargodha and Faisalabad; however, the lowest fungi was occurred (0.5%) in Lodhran, Punjab province, Pakistan.

Table 3.

Total number of insects collected (and those from which entomopathogenic fungi were isolated) and frequency distribution (%) from the insect cadavers

Sites	Number of insects collected (percentage of insects in sample group)						Infected insects ( N )	Total ( N )	Distribution frequency (%)
	T. castaneum	C. ferrugineus	R. dominica	T. granarium	S. oryzae	C. maculatus
Bahawalpur	123 (30.22)	31 (7.62)	85 (20.88)	120 (29.48)	42 (10.32)	6 (1.47)	3	407	1.54
Lodhran	266 (43.61)	43 (7.05)	93 (15.25)	145 (23.77)	54 (8.85)	9 (1.48)	1	610	0.51
Basti Maluk	304 (42.40)	56 (7.81)	114 (15.90)	160 (22.32)	69 (9.62)	14 (1.95)	2	717	1.03
Multan	321 (39.58)	75 (9.25)	139 (17.14)	171 (21.09)	82 (10.11)	23 (2.84)	5	811	2.56
Khanewal	383 (41.01)	84 (8.99)	152 (16.27)	189 (20.24)	90 (9.64)	36 (3.85)	6	934	3.08
Shorkot	412 (39.96)	97 (9.41)	161 (15.62)	214 (20.76)	103 (9.99)	44 (4.27)	4	1031	2.05
Jhang	454 (39.10)	134 (11.54)	178 (15.33)	225 (19.38)	113 (9.73)	57 (4.91)	7	1161	3.59
Faisalabad	543 (38.27)	168 (11.84)	215 (15.15)	278 (19.59)	147 (10.36)	68 (4.79)	17	1419	8.72
Sheikhupura	674 (39.14)	193 (11.21)	267 (15.51)	331 (19.22)	183 (10.63)	74 (4.30)	12	1722	6.15
Lahore	688 (34.37)	242 (12.09)	351 (17.53)	366 (18.28)	223 (11.14)	132 (6.59)	14	2002	7.18
Gujranwala	734 (32.56)	278 (12.33)	387 (17.17)	403 (17.88)	275 (12.20)	177 (7.85)	13	2254	6.66
Ghakhar	897 (34.41)	347 (13.31)	426 (16.34)	434 (16.65)	314 (12.04)	189 (7.25)	25	2607	12.82
Sargodha	983 (33.65)	392 (13.42)	469 (16.06)	497 (17.01)	367 (12.56)	213 (7.29)	17	2921	8.72
Jhelum	1,023 (31.67)	434 (13.44)	507 (15.70)	559 (17.31)	386 (11.95)	321 (9.94)	24	3,230	12.31
Rawalpindi	1,066 (27.38)	548 (14.07)	757 (19.44)	868 (22.29)	421 (10.81)	234 (6.01)	45	3,894	23.08
Total	8,871	3,122	4,301	4,960	2,869	1,597	195	25,720	100

Table 2.

Distribution and frequency (positive %) of fungal species from stored grain insect cadavers collected from storage facilities of Punjab, Pakistan

Fungal species	Number of insects collected ( N )						χ 2	P	Distribution frequency (%)	Total isolates ( N )
	T. castaneum	C. ferrugineus	R. dominica	T. granarium	S. oryzae	C. maculatus
Alternaria alternata	3	1	3	2	2	1	2.68	0.74	6.15	12
Alternaria solani	2	1	1	0	3	2	3.16	–	4.62	9
Aspergillus flavus	5	1	2	1	1	1	1.43	0.92	5.64	11
Aspergillus fumigatus	4	3	2	1	1	1	3.58	0.61	6.15	12
A. parasiticus	2	1	1	3	1	1	6.84	–	4.62	9
Asprgillus niger	4	2	1	1	1	3	4.05	0.54	6.15	12
B. bassiana	5	0	0	0	1	1	5.44	–	3.59	7
Bipolaris maydis	1	0	0	0	0	0	1.87	–	0.51	1
Bipolaris oryzae	1	1	1	2	5	1	6.76	0.23	5.64	11
Curvularia lunata	2	1	1	2	0	0	6.12	–	3.08	6
Fusarium oxysporium	7	2	1	1	2	3	2.9	0.71	8.21	16
Fusarium solani	7	0	3	2	1	2	4.92	0.42	7.69	15
Helminthosporium oryzae	4	2	2	0	5	0	5.18	0.39	6.67	13
L. attenuatum	1	0	0	0	1	0	1.83	–	1.03	2
M. anisopliae	2	0	1	0	1	0	1.97	–	2.05	4
Mucor sp.	1	0	0	0	1	0	1.83	–	1.03	2
P. lilacinum	1	0	1	0	0	1	3.61	–	1.54	3
Penicillium capsulatum	3	1	1	1	2	1	0.08	–	4.62	9
Penicillium chrysogenum	1	2	1	0	0	0	8.04	–	2.05	4
Phomopsis sp.	3	2	3	1	1	0	4.85	–	5.13	10
Pyricularia oryzae	1	0	0	0	7	0	22.4	–	4.10	8
Pythium sp.	1	0	0	0	1	1	2.67	–	1.54	3
Rhizopus oryzae	5	0	0	1	4	1	4.51	0.47	5.64	11
Rhizopus stolonifers	2	1	1	0	0	1	2.57	–	2.56	5
Total	68	21	26	18	41	21	–	–	–	12
Occurrence (%)	0.26	0.08	0.10	0.07	0.16	0.08	–	–	–	195

Entomopathogenic Fungi

The entomopathogenic fungi were widely distributed in the cadavers collected from the grain-storage facilities located in various districts. There were four entomopathogenic fungal genera found from a total of 25,720 insects. The fungus B. bassiana was the most dominantly occurring species (3.6%) isolated from the cadavers compared with other fungi. On the other hand, the fungus M. anisopliae was the next important distributed with 2.1% occurrence. The distribution of fungus Purpureocillium lilacinum was minimal with 1.5% followed by Lecanicillium attenuatum (1.03%). In the present study, the geography of collection sites influenced the distribution of entomopathogenic fungi.

Among the four genera of entomopathogenic fungi, B. bassiana (43.8%) ranked at the top followed by M. anisopliae (25%), P. lilacinum (18.8), and L. attenuatum (12.5%) ( Table 4 ). T. castaneum was the most contaminated (0.3%) insect species with all types of mycoflora followed by S. oryzae (0.2%), R. dominica (0.1%), C. ferrugineus, and C. maculatus (0.1%) occurrence ( Table 2 ). Among the entomopathogenic fungi, B. bassiana was the most frequent (5 isolates out of 195) isolated from T. castaenum ; however, the least occurred were P. lilacinum and L. attenuatum (1 isolate of each out of 195). The geographical parameters were also studied, which influenced the occurrence of entomopathogenic fungi, in insects. The geographical coordinates (altitude, latitude, and longitude) of all the localities have influenced the distribution of entomopathogenic fungi as highest number of isolates were found from >400 altitude, 33°–34′ N latitude, and 73°–74′ E longitude ( Fig. 2 ).

Table 4.

Distribution, frequency and virulence (% ± SE) of different isolates of entomopathogenic fungi against T. castaneum ( F3,15  = 9.28, P  ≤ 0.01). HSD test P  = 5% level

Fungal species	Distribution frequency (%)	Total isolates ( N )	Mortality	Range of mortality (%)
B. bassiana	43.75	7	85.81 ± 3.54a	76.44–92.75
L. attenuatum	12.50	2	59.79 ± 4.84c	48.28–71.93
M. anisopliae	25.00	4	76.86 ± 2.88ab	71.19–84.75
P. lilacinum	18.75	3	67.83 ± 3.20bc	62.50–77.01
Total	100.00	16	–	–

a,b,c: Mortality means sharing common letters do not differ significantly.

Fig. 2.

Effect of geographical attributes (a, altitude; b, latitude; c, longitude) on the occurrence of entomopathogenic fungi from the insect cadavers from storage facilities of Punjab, Pakistan. The variables are categorized in groups indicated in the respective legend of the plots.

Virulence Test

The virulence (percentage mortality at 7-d post inoculation) of the recovered isolates from insect cadavers was quantified against larvae of T. castaneum , and the mortality was significantly ( P  < 0.05) different among the isolates ( Table 4 ). The mortality range from the lowest to the highest of L. attenatnum was (48.2–71.9%) with 59.8% mortality, followed by P. lilacinum (62.5–77.0%) with 67.8% mortality, and M. anisopliae (71.2–84.7%) with 76.9% mortality, and B. basssiana proved to be more virulent (76.4–92.7%) which gave 85.8% mortality ( Table 4 ).

Discussion

Entomopathogenic fungi consist of a diverse group of species ( Chandler et al. 1997 ) and have been recorded from all major taxa of arthropods ( Roberts and Humber 1981 ). The key to successfully exploiting their potential as biological control agents lies in comprehensive knowledge of their ecology and life history ( Fisher et al. 2011 ). There is a wide range of literature available about the natural occurrence of entomopathogenic fungi in the soil ( Klingen et al. 2002 ; Shapiro-Ilan et al. 2003 ; Meyling et al. 2009 ; Batalla-Carrera et al. 2013 ; Wakil et al. 2013 ). However, to our knowledge, there have been few previous reports on the natural distribution of entomopathogenic fungi from stored grains insect pests from geographically distinct storage facilities.

Our findings clearly indicated the diversity in the occurrence of microbiota in storage insect pests. Along with entomopathogenic fungi, there were some other fungal genera recorded comprising of opportunistic pathogen and secondary colonizers. The present findings can be compared with a few available reports about the distribution of entomopathogenic fungi in storage structures: Odour et al conducted the survey of 124 farms of maize in 12 districts of Kenya and found association of B. bassiana with S. zeamais , Tribolium sp., and Carpophilus sp. Thakur and Sandhu (2010) isolated 246 entomopathogenic fungal isolates, which belong to seven species of the fungi from Lepidopteran, Coleopteran, and Dipteran host insects. Hatting et al. (1999) collected eight species of entomopathogenic fungi during the survey of South Africa, which includes six Entomophthorales and two Hyphomycetes that infected and killed the aphid hosts. Similar to our observations, Mar et al. (2012) , in search of indigenous isolates of entomopathogenic fungi, isolated B. bassiana (2 isolates), M. flavoviride (1 isolate), M. anisopliae (1 isolate), P. lilacinus (1 isolate), and I. tenuipes (1 isolate) from dead insect cadavers at four locations in Chiang Mai province Thailand. The occurrence of entomopathogenic fungi in soil samples has been reported by Barra et al. (2013) when they isolated P. lilacinus by baiting Tribolium confusum in maize ecosystem.

The insect cadavers collected from the fields and incubated in the laboratory may contribute to epizootics in nature because these entomopathogenic fungi occur naturally in the environment. The occurrence of insect pathogenic fungi may be lower in grain stores compared with other environments. For example, Meyling et al. (2012) isolated 32 Beauveria sp. from the pollen beetles Meligethes aeneus (Coleoptera: Nitidulidae) from oilseed rape fields at different sites. Similarly, Kim et al. (2010) collected a total of 542 entomopathogenic fungal isolates from dead insects from mountains and islands in several locations in Korea from 2003 to 2007. Morphological and cultural characteristics of the fungal fruiting bodies produced on the insect samples were examined and three species of Beauveria , eight species of Cordyceps, and four species of Isaria were mainly isolated from the insect samples. The other entomopathogenic fungi isolated from the insect samples were M. anisopliae , Nomuraea rileyi (Farlow) (Samson), Paecilomyces sp., and Verticillium sp. Various entomopathogenic fungi viz. Beauveria brongniartii , Conidiobolus obscurus, C. thromboides, Entomophthora muscae, E. aphidis, Entomophthora sp., B. bassiana , M. anisopliae , and Verticillium lecanii were recorded from diseased insects from central and western parts of Latvia ( Jankevica 2004 ). Approximately 3,400 insects were collected in three different surveys ( Barragán et al. 2004 ) from the tropical forest and an agricultural area at El Eden Ecological Reserve, Quintana Roo, Mexico: one isolate of Aspergillus sp., two of Penicillium sp., three of Paecilomyces marquandii , and three of Verticillium sp. from three insect orders, Hymenoptera, Diptera, and Isoptera in the tropical forest. On the other hand, a higher number of fungal isolates were recovered from the agricultural areas: 3 isolates of Aspergillus parasiticus , 100 of Fusarium moniliforme , 1 of Aschersonia sp., and 246 of Fusarium oxysporum out of 3,100 insects (11.3%) from three insect orders, Homoptera, Coleoptera, and Lepidoptera. In support of the findings of this study, Padanad and Krishnaraj (2009) isolated entomopathogenic fungus N. rileyi from Spodoptera litura (F.) (Lepidoptera: Noctuidae) and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) insect cadavers collected from India. The results of Pilz et al. (2008) are also in accordance with the present study as they collected M . anisopliae and Beauveria sp. from field-collected larvae (1.4%), pupae (0.2%), and adults (0.05%) of western corn rootworm. The presence of entomopathogenic fungi B . bassiana was highest ( Oliveira et al. 2012 ) in the phyllophagous larvae and pupae of olive moth. During the survey of cereal crops in Argentina Manfrino et al. (2013) recovered three species of entomophthoroid fungi from the cadavers of six aphid species. For the biological control of Aphis gossypii (Glover) (Hemiptera: Aphididae) vector of curly virus disease aphid nymphs and adults, larvae of Lepidoptera, Hemiptera, and other insect were collected. The results showed the explorations of 25 isolates of enthomopathogenic fungi consisting 10 isolates of B. bassiana and 15 isolates of M. anisopliae ( Herlinda et al. 2010 ). Entomopathogenic fungi B. bassiana MaW1 were directly isolated from a cadaver of adult Monochamus alternatus ( Shin et al. 2009 ), the vector of pine wilt pathogen. The lower recovery of entomopathogenic fungi from the dead insect is due to their difficulty to isolate as the infected insects often were contaminated by air fungi ( Herlinda et al. 2006 ).

Virulence (speed to kill) of individual entomopathogenic fungi showed great variation, and the factors that contribute this variation are still unclear ( Anderson et al. 2011 ). In the present study, although all the entomopathogenic fungal strains were pathogenic, significant variations were recorded among isolates of different genera and even with in same genera. In support of this statement, Alston et al. (2005) and Gindin et al. (2009) reported the differences in the larval mortality of Plum curculio, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), and Lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), by the application of entomopathogenic fungi. Different strains of Beauveria and Metarhizium are known to vary in virulence and other pathogenicity-related characteristics ( Zimmermann 2007a , b ; Anderson et al. 2011 ) are inconformity with our results. The present findings are in line with those of Aemprapa (2007) as he screened 7 isolates of Metarhizium sp., 12 isolates of Beauveria sp., and 1 isolate of Hirsutella citriformis against oriental fruit fly ( Bactrocera dorsalis Hendel Diptera: Tephritidae). The mortality rate of the fruit fly was from 2 to 68%; however, Beauveria sp. (isolate 6,241) killed 50% of the fruit flies. Similarly, Er et al. (2008) tested the pathogenicity of eight entomopathogenic fungi ( P. farinosus , P. fumosoroseus , B. bassiana , Lecanicillium lecanii , and M. anisopliae ) against Coccinella septempunctata L. (Coleoptera: Coccinellidae). The mortality rate after 8 d varied between 27 and 51%, and there were statistically significant differences among the effects of the tested fungi. The pathogenicity of B. bassiana , I. fumosorosea , and M. anisopliae was confirmed against Leptoglossus occidentalis (Heidemann) (Heteroptera: Coreidae) and in the laboratory LC 50 values were highest for I. fumosorosea and lowest for M. anisopliae ( Barta 2010 ). In the previous study conducted by Italian authors ( Rumine and Barzanti 2008 ), the B. bassiana virulence was proved against western conifer seed bug, L. occidentalis , adults under laboratory conditions. The five entomopathogenic fungal isolates ( C. obscurus 79, C. obscurus 79-3, C. obscurus E 68, and C. thromboides and Basidiobolus ranarum ) were used against aphids Aphis fabae (Scopoli) and Metopeurum fuscoviride (Stroyan) (Hemiptera: Aphididae) and all fungal isolates tested were virulent ( Halimona and Jankevica 2011 ). In another study, Sookar et al. (2008) tested 14 isolates of M. anisopliae , P. fumosoroseus, and B. bassiana against Bactrocera zonata (Saunders) (Diptera: Tephritidae) and B. cucurbitae and reported all were pathogenic. In our study, B. bassiana isolates caused highest mortality followed by M. anisopliae ; this could be due to the use of native isolates as these isolates might be better adapted or prepared to infect a particular host that cohabit in the same location ( Batalla-Carrera et al. 2013 ).

The entomopathogenic fungi are distributed in the insect species from the grain storage facilities; however, their presence was confined to the localities with relatively cooler climatic conditions. These fungi have great virulence against the stored grain insect species because of their pathogenicity against T. castaneum larvae. Further research is required for the exploration of indigenous isolates of these fungi so that they may play key role for the environment friendly and safer management of stored grain insect pests.