Naturally Occurring Entomopathogenic Fungi Infecting

Journal of Insect Science RESEARCH

Naturally Occurring Entomopathogenic Fungi Infecting Stored Grain Insect Species in Punjab, Pakistan Waqas Wakil,1,3 Muhammad Usman Ghazanfar,2 and Muhammad Yasin1 1Department of Entomology, University of Agriculture, Faisalabad, Pakistan 2Department of Plant Pathology, University College of Agriculture, University of Sargodha, Pakistan 3Corresponding author, e-mail: [email protected]

Subject Editor: Stefan Jaronski

J. Insect Sci. 14(182): 2014; DOI: 10.1093/jisesa/ieu044

ABSTRACT. 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, 33–340 N latitude, and 73–740 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.

Key Words: occurrence, entomopathogenic fungi, stored grain insects, localities, virulence

Grain commodities are commonly infested by insect pests during stor- 2005). For instance, Adane et al. (1996) and Kavallieratos et al. (2006) age (Haq et al. 2005). It is generally considered that 5–15% grain losses tested B. bassiana isolates for controlling the maize weevil Sitophilus in different stored grain commodities occur as a result in insect pest zeamais (Coleoptera: Curculionidae) and the cowpea Callosobruchus infestations (Padin et al. 2002), which reduces the commercial value, maculatus (F.) (Coleoptera: Bruchidae) and noted their high virulence. quality of stored grain and seed viability. The most economically im- Likewise, Cherry et al. (2005) and Athanassiou et al. (2008) found M. portant stored grain and pest species include the red flour beetle anisopliae effective against S. oryzae and R. dominica on rice, wheat, Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae), the lesser and maize grains. High mortality rates of R. dominica were attributed to grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), and using a local isolate of B. bassiana on stored wheat under laboratory the rice weevil Sitophilus oryzae (L.) (Coleoptera: Curculionidae) conditions (Wakil et al. 2011, 2012). However, despite the clear poten- (Bello et al. 2001; Chanbang et al. 2007; Michalaki et al. 2007). These tial of entomopathogenic fungi for use in IPM, little is known about the pests are often managed using synthetic chemical insecticides applied natural occurrence of fungal infections in populations of stored product to the grain store; however, excessive insecticide use can result in con- pests, and this could be an impediment to the identification of the most trol failure following the evolution of insecticide resistance and has also effective candidate fungal strains. In the current study, we quantified raised concerns about human and environmental safety (Cox 2004). As the entomopathogenic fungal flora occurring on populations of grain an alternative to these insecticides, entomopathogenic fungi have re- insects in Pakistan in order to better understand the relationship, if any ceived some attention for use in stored product Integrated Pest between fungal occurrence and geographical location. Management (IPM) (Gillespie 1988). The potential benefits of using entomopathogenic fungi include human safety, the development of an Materials and Methods epizootic (Hidalgo et al. 1997), and repeated infection cycles of propa- Collection of Stored Grain Insects. Systematic surveys of grain stor- gules in the microenvironment (Thomas et al. 1995; Wood and Thomas age facilities were carried out in different districts of Punjab (Pakistan). 1996), which increases the longevity and persistence of the biological The relevant geographical attributes (Table 1) and the positions of the control agent (Bateman et al. 1993). Successful control of stored grain sites are given in Figure 1. In total, five to six grain storage facilities pests with entomopathogenic fungi has been reported by a number of were sampled from 15 sites (Table 1). Each sample consisted of 500 g researchers (Latge and Moletta 1988; McCoy et al. 1988; McCoy 1990; grain collected from each of five different points selected at random Ferron et al. 1991; Roberts and Hajek 1992; Tanada and Kaya 1993; within the grain storage facility (i.e., a total of 2.5 kg grain was col- Hajek and St. Leger 1994; Wakil and Ghazanfar 2010). The entomopa- lected from each store). At the time of sample collection, any insects thogenic fungi Beauveria bassiana (Balsamo) Vuillemin (Akbar et al. with abnormal movement or showing lethargic activity in the immedi- 2004; Lord 2007) and Metarhizium anisopliae (Metschnikoff) Sorokin ate vicinity of the subsample were also collected and put into sterile (Batta 2004, 2005; Kavallieratos et al. 2006; Michalaki et al. 2006), in plastic vials. The grain samples were put into zip lock bags and were particular, have both proven to be efficacious against many insect pests brought back to the laboratory; however, the vials containing insects of stored products. These fungi do not decrease the quality of the grains were kept in a cooler maintained at 10C before reaching the laboratory. and thus enhances the marketability of stored products (Steenberg The grain samples were sieved to separate any insects present from the

VC The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 JOURNAL OF INSECT SCIENCE VOLUME 14

sterilized scalpel. The fungal conidia were suspended in 0.05% Tween- Table 1. Sampling sites with geographical characteristics for the 80 solution and filtered through muslin cloth to remove mycelial debris. isolation of fungi from insect cadavers A suspension of 1 106 conidia ml1 (dose was standardized in pre- Sites Altitude (m) Latitude Longitude liminary bioassays) of each isolate was prepared and used to inoculate Bahawalpur 109 29 240 N71 400 E third larval instars of T. castaneum. This was done by immersing larvae Lodhran 111 29 530 N71 630 E in conidial suspension for five seconds before transferring larvae to Basti Maluk 119 29 510 N71 320 E petri plates (9 cm) lined with moist filter paper and sealed with parafilm. Multan 124 30 200 N71 480 E These plates were incubated at 25C, and four replicates each having 0 0 Khanewal 128 30 17 N7155 E 10 larvae were used. The larvae treated with Tween-80 served as Shorkot 126 30 950 N72 080 E Jhang 158 31 270 N72 310 E the control, and the same procedure for each isolate was repeated Faisalabad 184 31 300 N73 050 E three times with four replications with new materials each time inde- Sargodha 193 32 100 N72 400 E pendently. The larval mortality was examined 7-d post application of 0 0 Sheikhupura 213 31 71 N7397 E fungi. Lahore 210 31 350 N74 200 E Gujranwala 223 32 100 N74 120 E Statistical Analysis. The occurrence of fungi in different insect spe- Ghakhar 224 32 300 N74 150 E cies (T. castaneum, R. dominica, Trogoderma granarium, S. oryzae, the Jhelum 220 32 550 N73 430 E rusty grain beetle Cryptolestes ferrugineus (Stephens) (Coleoptera: Rawalpindi 497 33 580 N73 080 E Cucujidae), and C. maculatus) collected from the different grain storage sites was compared using a v2 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 dis- Fig. 1. Mapping different sampling sites from Punjab, Pakistan for tributed in all the collection sites surveyed (Table 3). The highest fre- the isolation of fungi from insect cadavers: 1. Bahawalpur; 2. quency (23.1%) of fungi was recorded from the various storage Lodhran; 3. Basti Maluk; 4. Multan; 5. Khanewal; 6. Shorkot; 7. facilities in Rawalpindi district, followed by 12.8% from Ghakhar, Jhang; 8. Faisalabad; 9. Sheikhupura; 10. Lahore; 11. Sargodha; 12. 12.3% from Jhelum, and 8.7% both from Sargodha and Faisalabad; Gujranwala; 13. Ghakhar; 14. Jhelum; 15. Rawalpindi. however, the lowest fungi was occurred (0.5%) in Lodhran, Punjab province, Pakistan. grain. Insect cadavers were stored at 4C in the refrigerator for further Entomopathogenic Fungi. The entomopathogenic fungi were processing. widely distributed in the cadavers collected from the grain-storage Isolation and Identification of Fungi. The collected insects (live facilities located in various districts. There were four entomopathogenic and dead) were identified to species level according to taxonomic keys fungal genera found from a total of 25,720 insects. The fungus B. bassi- (Rees 2004). All dead insects were washed in 75% ethanol solution ana was the most dominantly occurring species (3.6%) isolated from (aq.) for 1 min, then transferred to 3% NaClO for 3 min, followed by the cadavers compared with other fungi. On the other hand, the fungus immersion in 75% ethanol for 1 min. Cadavers were then dried on filter M. anisopliae was the next important distributed with 2.1% occurrence. paper and then placed in petri dishes containing Sabouraud Dextrose The distribution of fungus Purpureocillium lilacinum was minimal Agar (SDA) supplemented with 0.1 g/liter of streptomycin sulfate and with 1.5% followed by Lecanicillium attenuatum (1.03%). In the 0.05 g/liter of chloramphenicol. Plates were sealed with parafilm and present study, the geography of collection sites influenced the distribu- incubated at 25C for up to 5–7 d. Insects were examined under a binoc- tion of entomopathogenic fungi. ular microscope every 24 h for the appearance of any fungal growth on Among the four genera of entomopathogenic fungi, B. bassiana the medium. Where more than one fungal colony was present, the colo- (43.8%) ranked at the top followed by M. anisopliae (25%), P. lilaci- nies were separated by subculturing on SDA. The fungi were identified num (18.8), and L. attenuatum (12.5%) (Table 4). T. castaneum was the on the basis of morphological traits using taxonomic keys (Barnett and most contaminated (0.3%) insect species with all types of mycoflora Hunter 1999; Domsch et al. 2007) and their pathogenicity (Koch’s pos- followed by S. oryzae (0.2%), R. dominica (0.1%), C. ferrugineus, and tulates) was confirmed on their respective hosts. The slides and the cul- C. maculatus (0.1%) occurrence (Table 2). Among the entomopatho- ture of identified entomopathogenic fungal isolates were deposited in genic fungi, B. bassiana was the most frequent (5 isolates out of 195) the IPM (Insect Pathology) Laboratory, Department of Entomology, isolated from T. castaenum; however, the least occurred were P. lilaci- University of Agriculture, Faisalabad, Pakistan. num and L. attenuatum (1 isolate of each out of 195). The geographical Virulence of Isolated Fungi. The isolated entomopathogenic fungi parameters were also studied, which influenced the occurrence of ento- were subcultured at 25C and 75% r.h. with 16-h illumination per day mopathogenic fungi, in insects. The geographical coordinates (altitude, on SDAY (32.5 g SDA; 7.5 g of Bacto Agar; 5 g yeast in 1 liter distilled latitude, and longitude) of all the localities have influenced the distribu- water) plates for the production of conidia. After 14 d of incubation, the tion of entomopathogenic fungi as highest number of isolates were plates were air-dried for one week. Fungal conidia were harvested by found from >400 altitude, 33–340 N latitude, and 73–740 E longitude scraping the conidial layers formed on the surface of plates using a (Fig. 2). 2014 WAKIL ET AL.: ENTOMOPATHOGENIC FUNGI INFECTING STORED GRAIN INSECTS 3

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) v2 P Distribution Total frequency (%) 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

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 Total (N) Distribution insects (N) 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

Virulence Test. The virulence (percentage mortality at 7-d post inoc- ulation) of the recovered isolates from insect cadavers was quantified Table 4. Distribution, frequency and virulence (% 6 SE) of different against larvae of T. castaneum, and the mortality was significantly isolates of entomopathogenic fungi against T. castaneum (F3,15 ¼ (P < 0.05) different among the isolates (Table 4). The mortality range 9.28, P 0.01). HSD test P ¼ 5% level from the lowest to the highest of L. attenatnum was (48.2–71.9%) with Fungal species Distribution Total Mortality Range of 59.8% mortality, followed by P. lilacinum (62.5–77.0%) with 67.8% frequency (%) isolates (N) mortality (%) 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 B. bassiana 43.75 7 85.81 6 3.54a 76.44–92.75 L. attenuatum 12.50 2 59.79 6 4.84c 48.28–71.93 85.8% mortality (Table 4). M. anisopliae 25.00 4 76.86 6 2.88ab 71.19–84.75 P. lilacinum 18.75 3 67.83 6 3.20bc 62.50–77.01 Discussion Total 100.00 16 – – Entomopathogenic fungi consist of a diverse group of species a,b,c: Mortality means sharing common letters do not differ signiﬁcantly. (Chandler et al. 1997) and have been recorded from all major taxa of 4 JOURNAL OF INSECT SCIENCE VOLUME 14

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. arthropods (Roberts and Humber 1981). The key to successfully there were some other fungal genera recorded comprising of opportun- exploiting their potential as biological control agents lies in comprehen- istic pathogen and secondary colonizers. The present findings can be sive knowledge of their ecology and life history (Fisher et al. 2011). compared with a few available reports about the distribution of entomo- There is a wide range of literature available about the natural occur- pathogenic fungi in storage structures: Odour et al. (2000) conducted rence of entomopathogenic fungi in the soil (Klingen et al. 2002; the survey of 124 farms of maize in 12 districts of Kenya and Shapiro-Ilan et al. 2003; Meyling et al. 2009; Batalla-Carrera et al. found association of B. bassiana with S. zeamais, Tribolium sp., and 2013; Wakil et al. 2013). However, to our knowledge, there have been Carpophilus sp. Thakur and Sandhu (2010) isolated 246 entomopatho- few previous reports on the natural distribution of entomopathogenic genic fungal isolates, which belong to seven species of the fungi from fungi from stored grains insect pests from geographically distinct stor- Lepidopteran, Coleopteran, and Dipteran host insects. Hatting et al. age facilities. (1999) collected eight species of entomopathogenic fungi during the Our findings clearly indicated the diversity in the occurrence of survey of South Africa, which includes six Entomophthorales and two microbiota in storage insect pests. Along with entomopathogenic fungi, Hyphomycetes that infected and killed the aphid hosts. Similar to our 2014 WAKIL ET AL.: ENTOMOPATHOGENIC FUNGI INFECTING STORED GRAIN INSECTS 5 observations, Mar et al. (2012), in search of indigenous isolates of ento- Gindin et al. (2009) reported the differences in the larval mortality of mopathogenic fungi, isolated B. bassiana (2 isolates), M. flavoviride (1 Plum curculio, Conotrachelus nenuphar (Herbst) (Coleoptera: isolate), M. anisopliae (1 isolate), P. lilacinus (1 isolate), and I. tenuipes Curculionidae), and Lesser mealworm, Alphitobius diaperinus (1 isolate) from dead insect cadavers at four locations in Chiang Mai (Panzer) (Coleoptera: Tenebrionidae), by the application of entomopa- province Thailand. The occurrence of entomopathogenic fungi in soil thogenic fungi. Different strains of Beauveria and Metarhizium are samples has been reported by Barra et al. (2013) when they isolated P. known to vary in virulence and other pathogenicity-related characteris- lilacinus by baiting Tribolium confusum in maize ecosystem. tics (Zimmermann 2007a,b; Anderson et al. 2011) are inconformity The insect cadavers collected from the fields and incubated in the with our results. The present findings are in line with those of laboratory may contribute to epizootics in nature because these entomo- Aemprapa (2007) as he screened 7 isolates of Metarhizium sp., 12 iso- pathogenic fungi occur naturally in the environment. The occurrence of lates of Beauveria sp., and 1 isolate of Hirsutella citriformis against ori- insect pathogenic fungi may be lower in grain stores compared with ental fruit fly (Bactrocera dorsalis Hendel Diptera: Tephritidae). The other environments. For example, Meyling et al. (2012) isolated 32 mortality rate of the fruit fly was from 2 to 68%; however, Beauveria Beauveria sp. from the pollen beetles Meligethes aeneus (Coleoptera: sp. (isolate 6,241) killed 50% of the fruit flies. Similarly, Er et al. Nitidulidae) from oilseed rape fields at different sites. Similarly, Kim (2008) tested the pathogenicity of eight entomopathogenic fungi (P. far- et al. (2010) collected a total of 542 entomopathogenic fungal isolates inosus, P. fumosoroseus, B. bassiana, Lecanicillium lecanii, and M. from dead insects from mountains and islands in several locations in anisopliae) against Coccinella septempunctata L. (Coleoptera: Korea from 2003 to 2007. Morphological and cultural characteristics of Coccinellidae). The mortality rate after 8 d varied between 27 and 51%, the fungal fruiting bodies produced on the insect samples were exam- and there were statistically significant differences among the effects of ined and three species of Beauveria, eight species of Cordyceps, and the tested fungi. The pathogenicity of B. bassiana, I. fumosorosea, and four species of Isaria were mainly isolated from the insect samples. M. anisopliae was confirmed against Leptoglossus occidentalis The other entomopathogenic fungi isolated from the insect samples (Heidemann) (Heteroptera: Coreidae) and in the laboratory LC50 values were M. anisopliae, Nomuraea rileyi (Farlow) (Samson), Paecilomyces were highest for I. fumosorosea and lowest for M. anisopliae (Barta sp., and Verticillium sp. Various entomopathogenic fungi viz. 2010). In the previous study conducted by Italian authors (Rumine and Beauveria brongniartii, Conidiobolus obscurus, C. thromboides, Barzanti 2008), the B. bassiana virulence was proved against western Entomophthora muscae, E. aphidis, Entomophthora sp., B. bassiana, conifer seed bug, L. occidentalis, adults under laboratory conditions. M. anisopliae, and Verticillium lecanii were recorded from diseased The five entomopathogenic fungal isolates (C. obscurus 79, C. obscu- insects from central and western parts of Latvia (Jankevica 2004). rus 79-3, C. obscurus E 68, and C. thromboides and Basidiobolus rana- Approximately 3,400 insects were collected in three different surveys rum) were used against aphids Aphis fabae (Scopoli) and Metopeurum (Barragań et al. 2004) from the tropical forest and an agricultural area at fuscoviride (Stroyan) (Hemiptera: Aphididae) and all fungal isolates El Eden Ecological Reserve, Quintana Roo, Mexico: one isolate of tested were virulent (Halimona and Jankevica 2011). In another study, Aspergillus sp., two of Penicillium sp., three of Paecilomyces marquan- Sookar et al. (2008) tested 14 isolates of M. anisopliae, P. fumosoro- dii, and three of Verticillium sp. from three insect orders, Hymenoptera, seus, and B. bassiana against Bactrocera zonata (Saunders) (Diptera: Diptera, and Isoptera in the tropical forest. On the other hand, a higher Tephritidae) and B. cucurbitae and reported all were pathogenic. In our number of fungal isolates were recovered from the agricultural areas: 3 study, B. bassiana isolates caused highest mortality followed by M. ani- isolates of Aspergillus parasiticus, 100 of Fusarium moniliforme,1of sopliae; this could be due to the use of native isolates as these isolates Aschersonia sp., and 246 of Fusarium oxysporum out of 3,100 insects might be better adapted or prepared to infect a particular host that (11.3%) from three insect orders, Homoptera, Coleoptera, and cohabit in the same location (Batalla-Carrera et al. 2013). Lepidoptera. In support of the findings of this study, Padanad and The entomopathogenic fungi are distributed in the insect species Krishnaraj (2009) isolated entomopathogenic fungus N. rileyi from from the grain storage facilities; however, their presence was confined Spodoptera litura (F.) (Lepidoptera: Noctuidae) and Helicoverpa armi- to the localities with relatively cooler climatic conditions. These fungi gera (Hu¨bner) (Lepidoptera: Noctuidae) insect cadavers collected from have great virulence against the stored grain insect species because of India. The results of Pilz et al. (2008) are also in accordance with the their pathogenicity against T. castaneum larvae. Further research is present study as they collected M. anisopliae and Beauveria sp. from required for the exploration of indigenous isolates of these fungi so that field-collected larvae (1.4%), pupae (0.2%), and adults (0.05%) of they may play key role for the environment friendly and safer manage- western corn rootworm. 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