Abstract: In the present study, the occurrence and diversity of fungi associated with beetles and white grub, Brahmina coriacea (Hope) (Coleoptera: Scarabaeidae) in potato fields of North Western Indian hills was explored for the first time. Sixteen species belonging to 9 genera and two non-sporulating fungi were isolated and identified from white grub cadavers, adult beetles and soil samples in question. Total 1404 out of 14164 (9.91%) beetles were found infected with fungi during year 2007-09 at studied areas. Most abundantly occurring fungi associated with beetles were Metarhizium anisopliae, 423 out of 1404 (30.12%); followed by Aspergillus flavus, 322 (23.0%); Fusarium oxysporum, 262 (18.66%); Beauveria bassiana, 143 (10.2%); Aspergillus clavatus, 130 (9.25%) and Fusarium solani, 120 (8.6%). Whereas, 21 out of 1351 (1.6%) white grubs were found infected with fungi. A. clavatus showed high frequency of infection to grubs (33.8%), followed by A. flavus, (23.8%) and Rhizopus oryzae, (19.04%). Each Aspergillus niger and Alternaria alternata showed an infection frequency of 9.53% while 4.8% was noticed for Penicillium griseofulvum. Total ten fungi were identified from soil samples with high frequency of R. oryzae (27.60%) and Cunninghamella elegans (24.13%). Preliminary pathogenicity tests were performed on late first instar grubs for all isolated fungi except non-sporulating. B. bassiana was found highly pathogenic with 63.70% mortalities followed by 60.37% by A. flavus and 50.00% by M. anisopliae after 15 days of treatment. F. oxysporum and F. solani showed mortalities up to 49.63 and 42.59%, respectively.
INTRODUCTION
Entomopathogenic fungi is well distributed and its occurrence in various agricultural soils had been investigated by several workers (Chandler et al., 1997; Bidochka et al., 1998; Ali-Shtayeh et al., 2002; Klingen et al., 2002; Keller et al., 2003; Meyling and Eilenberg, 2006). Entomopathogenic fungi, Beauveria, Conidiobolus, Metarhizium and Paecilomyces are frequently found in soil (Domsch et al., 1980). Various other fungal species have also been reported from soil. Mucor species have been isolated from the larval cadavers of Troglophilus neglectus Krauss (Gunde-Cimerman et al., 1998). F. solani had been reported from Tetanops myopaeformis Roder and Hypothenemus hampei (Ferrari) (Rojas et al., 1999; Majumdar et al., 2008). Aspergillus species were found to be most common among 28 species representing 15 fungal genera, associated with different stages of Spodoptera littoralis (Boisduval) (Ismail and Abdel-Sater, 1993). Eleven species of fungi belonging to 5 genera including Penicillium have been isolated from mosquito larvae (Da Costa and de Oliveira, 1998; Sur et al., 1999). Aspergillus flavus and Fusarium species were identified from Chilo partellus Swinhoe (Atwal et al., 1973) and many other fungal species have been reported from diseased soil-inhabiting insects. According to Ali-Shtayeh et al. (2002) weak pathogenic fungi can become causal agent of epizootics in predisposed insects. These workers have reported pathogenicity of isolates of the genera Absidia, Aspergillus, Fusarium and Mucor against the larvae of Galleria mellonella (L.) (Ali-Shtayeh et al., 2002). Insects in the soil are always prone to infection by various pathogens. Even avirulent pathogens have been reported to cause various degree of pathogenecity in insects (Thomas et al., 2003). Fungal epizootics in soil insect populations are documented by many workers (Samson et al., 1988; Keller and Zimmerman, 1989; Klingen and Haukeland, 2006). Lot of work had been done on pathogenicity of Metarhizium anisopliae and Beauveria bassiana against several insect species (Demirel and Cranshaw, 2006; Khashaveh et al., 2008; Abood et al., 2010; Cheong et al., 2010; Sharififard et al., 2011). B. bassiana, M. anisopliae, Fusarium species near Fusarium solani, Cordyceps species, Penicillium species and Aspergillus species were isolated from beetles of Phyllophaga species, in Southern Quebec, Canada (Poprawski and Yule, 1991). Aspergillus species were reported from all stages of white grub in India (Yadav and Mathur, 1987). Fusarium species have also been reported from coleopteran insects with weak pathogenicity (Teetor-Barsch and Roberts, 1983). The knowledge of occurrence and diversity of naturally associated fungi can help to explore the potential fungal pathogens other than well known insect pathogenic fungi. It is very useful to get more information on mycoflora associated with different insect pests so that possibilities of bio-control can be explored extensively. Perusals of the literature revealed that no studies have been carried out on the fungal species associated with white grub Brahmina coriacea (Hope) (Coleoptera: Scarabaeidae). Therefore, in present study, in order to explore bio-control agents against white grub, a survey was undertaken. Further, to distinguish the relationship between fungi and white grubs, all fungi except non-sporulating species were tested for their pathogenicity.
MATERIALS AND METHODS
Collection of white grubs, adult beetles and soil samples: A survey was undertaken during three years (2007-09) at different locations of North Western Indian hills. All naturally occurring fungi associated with grubs in North Western Indian hills was included. White grubs were collected from potato farms at three different locations, Shimla (31°N -77°E; 2,202 m amsl), Shillaroo (31°N-77°E; 1,820 m amsl) and Kheradhar (30°N-77°E; 1,048 m amsl) of North Western hills of India, during year 2007-08. Sampling was done from root zone of randomly selected potato plants in 30 cm2 area and minimum of 10 samples from each quadrate of selected location were taken. To collect adults (beetles) of white grubs, two light traps at potato farms (Shimla and Fagu, 32°N-77°E; 2,502 m amsl) were installed during year 2007-09 and beetles were caught weekly. Grubs and beetles were reared and observed for any natural fungal infection. A total of 35 soil samples originating from potato fields of above regions were collected. Only those soil samples were taken in which dead grubs were found. The samples were taken by cylindrical soil core borer to a depth of 20 cm. The samples were placed into plastic bags and stored at 4°C.
Isolation of fungi: Visible conidia from grubs and adult beetles were harvested directly or scraped off on to media plates. Alternatively grub cadavers without external fungal growth were surface sterilized for 2 min each with 70% ethanol, 1% sodium hypochlorite and sterile water (2-3 washes) and were homogenized. Small quantity of homogenate was plated onto the SDAY media (4% Sabouraud dextrose agar media + 2% yeast extract + 0.1% streptomycin) (Papierok and Hajek, 1997). Soil samples were passed through sieve (pore size 2 mm) to remove plant tissues and gravels and mixed thoroughly. Isolation of fungi from soil samples was carried out through soil dilution plate method. One gram soil was mixed in 9 mL (w/v) sterile water. The sample was homogenized on mechanical shaker for 60 min. Aliquots of 100 μL were spread using L-shaped glass rod on above mentioned media. Cultures were incubated at 25°C for one week.
Identification of fungi: All isolated fungi were identified morpho-taxonomically from Maharashtra Association for the Cultivation of Science, Agharkar Research Institute Pune, India.
Preliminary pathogenicity test (Koch’s postulates): All fungal isolates with no knowledge about their pathogenicity to the white grubs except non sporulating fungi were bio-assayed. The fungi were grown on SDAY media plate for 10-12 days. Late first instar grubs of B. coriacea permitted to walk on the sporulated fungus culture for 5 min (Goettel and Inglis, 1997) and transferred to pots individually filled with sterile soil: Farm Yard Manure (FYM) (1:1) (w/w). Pots were incubated at the temperature 25°C in darkness. Maize plants were grown in the pots so that grubs could feed on roots of maize plants. Thirty laboratory reared grubs were taken for each fungus with three replications of each. Observations on mortality of grubs were recorded at periodic intervals (7, 15, 21 and 30 days after treatment). The cadavers were surface disinfected and fungus was re-isolated.
Data analysis: Mortality in control group was corrected using method suggested by Abbott (1925):
where, n is number of grubs, T is treatment and CO is control.
Corrected mortality data were statistically analyzed by using one-way analysis of variance after Arc sine transformation. Mean difference was tested using Duncan’s multiple range test. The difference of two means between treatments exceeding Critical Differences value was taken significant. MSTAT-C software was used for analysis.
RESULTS
Occurrence of insect associated fungi: Total 1404 out of 14164 (9.91%) adult beetles were found infected with fungi during year 2007-09 at Shimla and Fagu. Most frequently occurring fungus was M. anisopliae. Out of 1404 infected beetles, 423 (30.12%) were infected by M. anisopliae followed by A. flavus, 322 (23%) and F. oxysporum, 262 (18.66%). Occurrence of infection was comparatively less by B. bassiana, 143 (10.2%), A. clavatus, 130 (9.25%) and F. solani 120 (8.6%). M. anisopliae was found with high frequency during three years 2007-09. An infection frequency of 2.96 and 3.29% of M. anisopliae out of 9.92 and 9.80% total fungal infection was observed at Shimla and Fagu, respectively. Similar trend was observed with A. flavus (2.28 and 2.12%) and F. oxysporum (1.86 and 1.65%), respectively. Well known insect pathogenic fungus B. bassiana (1.01 and 1.02%) was significantly less prevalent at both the above mentioned places during all the three years. A. clavatus and F. solani were also least prevalent with infection frequencies of 0.92 and 0.86% at Shimla and 0.86 and 0.72% at Fagu, respectively (Table 1). Figure 1 shows naturally infected beetles with A. flavus and B. bassiana.
| Table 1: | Location wise fungal species found associated with adult beetles of B. coriacea during year 2007-09 |
| Fig. 1(a-b): | (a) Natural infection to Brahmina coriacea beetle by Aspergillus flavus (b) Natural infection by Beauveria bassiana |
During the years 2007-08 fungal infection of grubs was very less as compared to adult beetles, at all studied places. Only 21 out of 1351 (1.55%) grubs were found infected. A. clavatus was found to cause high frequency of infection among grubs, 7 out of 21 (33.8%), A. flavus, 5 out of 21 (23.8%) followed by Rhizopus oryzae, 4 out of 21 (19.04%), both A. niger and A. alternata 2 (9.53%) and Penicillium griseofulvum, 1 (4.8%) out of 21 were found infected. In Shimla, 3.19% grub population was found infected with fungi of which A. clavatus (1.45%) was dominating followed by A. flavus, 0.87 and 0.53% of each of A. niger, R. oryzae and P. griseofulvum during 2007-08. Fungal infection in white grubs was less at Shillaroo (0.60%) with 0.3% each of two species, A. flavus and A. clavatus.
| Table 2: | Fungal species found associated with white grubs of B. coriacea during year 2007-08 |
At Kheradhar, 1.73% grubs were found infected with fungi and dominating fungal species were R. oryzae (0.86%), A. alternata (0.58%) and A. niger (0.29%) (Table 2). Total fungal infection to white grubs and adult beetles was high during year 2008 than in 2007 and least in 2009 (beetles only) probably due to low rainfall or drought in year 2009, as for optimum growth of fungi high humidity with warm-wet environment are more congenial.
Among 35 soil samples, 26 (74.28%) soil samples were found positive for the presence of fungi, out of which 10 species were identified. 14 out of 29 isolates were found at Shimla, showed more diversity of fungal species as compared to Fagu (7) and Shillaroo (5). Only 3 fungal species were found in soil samples collected from Kheradhar.
| Table 3: | Fungi isolated from soil samples in potato fields |
| Table 4: | Mortality among late first instar grubs of B. coriacea |
| Corrected % mortality was calculated by Abbots formula, Dat: Day after treatment, Values in brackets are transformed value, Results are based on isolated fungal species recorded in their preliminary tests, CD: Critical difference | |
Out of 29 isolates, R. oryzae were 8 (27.6%), Cunninghamella elegans 7 (24.13%), Curvularia prasadii 3 (10.34%), each C. pallescens, A. raphani and A. niger were 2 (6.89%) and 1 (3.44%) of each F. equiseti and A. terreus and 1 (3.44%) each of non-sporulating hyaline form and non-sporulating form (Table 3).
Pathogenicity tests: All fungal species, except non-sporulating were separately tested for their pathogenicity on late first instar white grubs. Corrected percent mortality data of fungi against late first instar grubs up to 30 days of treatment is presented in Table 4. B. bassiana was found highly pathogenic with 51.48% mortalities followed by 44.81% due to A. flavus and 41.48% by M. anisopliae after 7 days of treatments, whereas F . oxysporum, F. solani and A. clavatus had shown mortalities in range 17.04-37.78%. After 15 days of treatment, B. bassiana showed significantly higher mortalities of 63.70% followed by 60.37% by A. flavus and 50.00% by M. anisopliae. F. oxysporum and F. solani showed mortalities up to 49.63 and 42.59%, respectively. Significantly low mortalities were caused by A. clavatus (28.15%), P. griseofulvum (24.81%) and A. alternata (21.11%). The rest of tested fungal species viz., A. terreus, A. niger, F. equiseti, R. oryzae, C. prasadii, C. pallescens and A. raphani showed between 3.57-10.74% mortalities. B. bassiana caused highest mortalities 92.59% after 30 days of treatment followed by 89.26% by A. flavus, 85.93% by both, M. anisopliae and F. oxysporum, respectively and 53.70% by A. clavatus. Rest all isolates caused mortalities in the range 10.37-49.63%. A. flavus, F. solani and F. oxysporum were found significantly pathogenic in preliminary studies besides well known pathogenic fungi.
DISCUSSION
The present study is the first detailed investigation of fungi associated with white grub and adult beetles of B. coriacea. Aspergillus, Fusarium, Penicillium, Alternaria and Rhizopus species besides well known entomopathogenic species like Metarhizium and Beauveria were found associated with white grubs. Insect associated mycoflora have been reported earlier on several insects, including bees (Shoreit and Bagy, 1995), termites (Zoberi and Grace, 1990), lepidopterous (Ismail and Abdel-Sater, 1993), mosquitoes (Da Costa and de Oliveira, 1998), cockroaches (Moraes et al., 2001a), triatomines (Moraes et al., 1998) and beetles (Poprawski and Yule, 1991). In all of the above studies, Aspergillus, Fusarium and Penicillium have been found the predominating genera. B. bassiana, M. anisopliae, F. solani, Penicillium and Aspergillus species have been reported from white grubs Phyllophaga species earlier (Poprawski and Yule, 1991). A. flavus had been isolated from Opisina arenosella, Stephanitis typica and Proutista moesta (St Leger et al., 2000; Gupta and Gopal, 2002). In a survey of the fungal microbiota associated with adults of Triatoma infestans Klug, Penicillium, Aspergillus and Alternaria were the predominating genera (Marti et al., 2007). A. flavus, one Aspergillus and Fusarium species associated with one white grub species and Leucopholis burmeisteri Brenske have also been reported earlier (Yadav and Mathur, 1987; Padmanaban et al., 2003). The wide distributions and high occurrences of M. anisopliae and B. bassiana in coleopteran and other insects have been reported by many workers (Milner, 1992; Bidochka et al., 1998; Khashaveh et al., 2008; Abood et al., 2010). In present study, M. anisopliae was also found most frequently occurring in adult beetles. Earlier, M. anisopliae had been reported from Phytoscaphus species (Verma et al., 1988). The present study revealed that A. flavus and F. oxysporum were predominant species than B. bassiana. A. flavus and A. clavatus were found associated with both white grub and imago beetle in all locations. A. flavus had been found associated with all stages of S. littoralis, C. partellus and T. neglectus earlier (Atwal et al., 1973; Ismail and Abdel-Sater, 1993; Gunde-Cimerman et al., 1998). Recently, A. clavatus has been isolated from Oedaleus senegalensis Krauss (Seye et al., 2009). F. oxysporum, F. solani and A. flavus have also been reported from larvae and adults of Hypolixus haerens Boheman, Dendroctonus frontalis Zimmermann, T. myopaeformis and H. hampei (Moore, 1971, 1973; Rojas et al., 1999; Blodgett et al., 2004; Carrion and Bonet, 2004; Majumdar et al., 2008). Although in the present study fungal infection in grubs was less, which may be due to their aggressive behavior or conidia detachment from the host cuticle (Yaginuma et al., 2006). The most frequently occurring fungi in grubs were A. clavatus followed by A. flavus. Many of the fungi isolated in present study had never been reported from grubs elsewhere in earlier studies. Further, in present study not even a single grub out of 21 infected grubs was found infected with well known insect pathogenic fungi M. anisopliae and B. bassiana. However, adult beetles had shown higher infection of M. anisopliae. A few reports about A. alternata, P. griseofulvum, A. niger and R. oryzae associated with other insects have already been published (Christias et al., 2001; Moraes et al., 2001a-d; Greif and Currah, 2007; Sun and Liu, 2008) but there are no such reports on white grubs. In present study, soil sample containing dead grubs had shown predominance of R. oryzae and C. elegans. Distribution of these species within agricultural field soils had been investigated in earlier studies (Ali-Shtayeh et al., 2002; Meyling and Eilenberg, 2006). In preliminary pathogenecity test, A. flavus, F. oxysporum and F. solani resulted in the significant mortality of grubs besides well-known pathogenic species. M. anisopliae and B. bassiana had earlier been reported pathogenic to various insect species including white grubs (Shahid et al., 2003; Mohi-ud-din et al., 2007; Khan et al., 2008; Mahdneshin et al., 2009; Dal Pogetto et al., 2011). A. flavus and Fusarium species have also been reported pathogenic to white grub L. burmeister (Padmanaban et al., 2003). According to earlier studies, chitinase producers can be considered as potential pathogens (Sur et al., 1999; Kumar et al., 2004). High chitinolytic activities were observed with A. flavus on S. littoralis, Bombyx mori (L.) and termites (Domsch et al., 1980; Gunde-Cimerman et al., 1998). F. oxysporum and F. solani are proven entomo-pathogenic species for several insects groups including Coleoptera (Miller et al., 1985; Hajek et al., 1993; Ali-Shtayeh et al., 2002). A. flavus, F. oxysporum and F. solani had also been reported to cause mortalities of G. mellonella to the tune of 30.8, 80.0 and 66.7%, respectively in preliminary tests (Sun and Liu, 2008). F. oxysporum had caused 100% mortality of Nilaparvata lugens (Stal) (Kuruvilla and Jacob, 1979, 1980). F. solani had been found pathogenic to Scolytus scolytus F., T. myopaeformis and lobster (Barson, 1976; Claydon et al., 1977; Majumdar et al., 2008). Low levels of moralities by A. clavatus, P. griseofulvum and A. alternata to grubs were also observed in the present study. A. clavatus had already been reported highly pathogenic to the larvae of Aedes aegypti (L.), Anopheles gambiae Giles and Culex quinquefasciatus (Say) (Seye et al., 2009). 100% mortality by A. alternata had been reported to the aphids (Christias et al., 2001). P. griseofulvum had been reported pathogenic to termites earlier (Moraes et al., 2001a, 2001b).
CONCLUSION
Present work is a first report of listing 16 species belonging to 9 genera with two non-sporulating fungi associated with B. coriacea white grubs, adult beetles and soil containing dead grubs. This study showed the diversity and spectrum of the fungal species associated with white grubs and beetles. More over many fungal species tested for their pathogenicity against first instar grubs had shown pathogenicity with more or less virulence. Fungi which caused significant mortalities were A. flavus, F. oxysporum and F. solani besides well known pathogenic fungi. These species could be further developed as a potential biological control agent against white grubs in their initial life cycle stage. However, present studies conducted are able to provide framework for further investigation and generating hypothesis to conduct preliminary pathogenicity required to test in different developmental stages of white grubs. Further studies are needed for fungal strains optimization and development of better substrates for mass production and practical uses. Characterization and application of toxins on different stages of grubs including eggs and beetles are also needed to understand their killing effects. Side effects and safety tests with beneficial insects, mammals and human cells need to be conducted. Further, it reveals that opportunistic pathogens, largely been overlooked till date can be tested to prove their pathogenicity.
ACKNOWLEDGMENTS
Authors are grateful to Director CPRI, Dr. S.K. Pandey for providing necessary facilities to conduct this work and Indian Council of Agricultural Research for funding this project.