SUSCEPTIBILITY OF SCHIZONYCHA AFFINIS BEETLES TO NATIVE STRAINS OF BEAUVERIA BRONGNIARTII IN SOUTH AFRICA

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1 SHORT, NON-REFEREED PAPER SUSCEPTIBILITY OF SCHIZONYCHA AFFINIS BEETLES TO NATIVE STRAINS OF BEAUVERIA BRONGNIARTII IN SOUTH AFRICA GOBLE TA 1,3, CONLONG DE 1,2 AND HILL MP 3 1 South African Sugarcane Research Institute, P/Bag X02, Mount Edgecombe, 4300, South Africa 2 School of Biological and Conservation Sciences, University of KwaZulu-Natal, Pietermaritzburg Campus, Scottsville, 3209, South Africa 3 Department of Zoology and Entomology, Rhodes University, PO Box 94, Grahamstown, 6140, South Africa tarryn.goble@sugar.org.za des.conlong@sugar.org.za m.p.hill@ru.ac.za Abstract Beauveria brongniartii (Saccardo) Petch is a fungal species which attacks insects, particularly beetles in the family Scarabaeidae. Different fungal isolates were recovered at two sites in the Dalton area of the Midlands in South Africa on two wattle chaffer beetle species, Hypopholis sommeri Burmeister and Schizonycha affinis Boheman (Coleoptera: Scarabaeidae). The pathogenicity of four, genetically distinct B. brongniartii isolates and one isolate of B. bassiana s.s. (Balsamo) Vuillemin were tested under laboratory conditions against the adult stage of a known sugarcane pest, S. affinis. Preliminary results indicated a dose-dependent response by the beetles to six concentrations (1x10 8-1x10 3 ) of fungal conidia (F (5,90) =280.53; P=0.001). Two B. brongniartii isolates in particular, C13 and HHE37 showed a greater virulence, as measured by insect mortality, towards S. affinis beetles than the rest of the tested isolates (F (4,90) =37.873; P=0.001). Over 85% of test beetles died at the highest concentration when B. brongniartii isolate C13 was considered, compared with 53% when B. bassiana (4222) was used at the same concentration. Beauveria brongniartii isolates (C13 and HHE37) produce a powerful red, mycotoxin called oosporein in large amounts when grown in submerged culture. It is therefore hypothesised, based on the preliminary bioassay results, that isolates producing large amounts of oosporein have greater pathogenicity. This study also served to confirm that B. brongniartii isolates are superior potential biological control agents than B. bassiana 4222, as a greater amount of insect mortality was observed in S. affinis adults. Keywords: biological control, wattle chaffer beetles, bioassays, sugarcane, fungal pathogens Introduction In 2010, two farms, namely Harden Heights and Canema, were found to harbour Beauveria brongniartii fungal epizootics. During a collection study, 60 B. brongniartii and 11 B. bassiana isolates were collected from the two farms from infected white grub larvae and pupae of the species S. affinis and H. sommeri. Fungal isolates were also collected from soils baited with greater wax moth, Galleria mellonella (Zimmermann, 1986), and various plant surfaces (Meyling and Eilenberg, 2006). There appears to be a global trend in the association 129

2 of B. brongniartii and white grubs of the sub-family Melolonthinae. The most commonly reported B. brongniartii epizootics have occurred on European cockchafer, Melolontha melolontha Fabricius in Switzerland, Austria, Italy and France (Hurpin and Robert, 1972; Keller, 1986; Neuveglise et al., 1997; Piatti et al., 1998). In India, there are reports of B. brongniartii on the melolonthid species, Holotrichia serrata Hope; a serious soil pest in tropical upland rice (Rombach et al., 1994). New Zealand has reported success in the use of a local B. brongniartii isolate in controlling the melolonthid pests, Pyronota festiva Fabricius and P. setose Given (Townsend et al., 2010). B. brongniartii isolates produce a major secondary metabolite called oosporein in submerged culture; the compound is a C2 symmetrical red 2,5-dihydroxybenzoquinone derived from the biosynthesis of a broad variety of soil borne fungi, including B. bassiana (Strasser et al., 2000). Research has shown that oosporein is not phytotoxic and is usually not taken up by plants (Strasser et al., 2000). Further, there are no published or reported data on the side effects of oosporein on human and mammals but because the B. brongniartii cannot grow above 33 C, pathogenicity to warmblooded animals can be excluded (Strasser et al., 2000). Previous research has shown that a high level of insecticidal activity (92% mortality) can be achieved when oosporein extract is combined with fungal conidia and used to treat whitefly, Bemisia tabaci (Amin et al., 2010). It is hypothesised that B. brongniartii isolates which produce oosporein are more virulent (as measured by insect mortality) than non-producing isolates. The aim of the study was thus to compare insect mortality when S. affinis beetles were exposed to different concentrations of fungal isolates. A second aim was to determine the virulence of B. bassiana isolates compared to B. brongniartii against the white grub species S. affinis, and to understand which fungal species is the superior biological control agent. Methods and Materials Collection of insects Three wall-mounted light traps, consisting of a 3 m PVC gutter leading into a funnel placed inside the neck of a 25 L plastic drum, were erected just below security lights at workshops at three sites in the Dalton area of the KZN Midlands North. Annual mass emergences of H. sommeri and S. affinis occur from late October to the end of November, and thousands of beetles can be caught in a single light trap in one night. Traps were collected daily in the mornings and beetles were sorted into species according to morphological descriptions by Sweeny (1967). Fungal cultures Four genetically distinct B. brongniartii isolates and one isolate of B. bassiana s.s. were selected for topical bioassays. Fungal isolates were grown on Sabouraud Dextrose Agar (SDA) supplemented with 50 mg/l Dodine, 50 mg/l Chloramphenicol, 50 mg/l Ampicillin, or 50 mg/l Rifampicin and maintained at 22 C. Preparation of spore suspensions and insect inoculation Fungal conidia were harvested from 3-week-old cultures by scraping with a glass rod. Conidia were suspended in 10 ml of sterile distilled water supplemented with 0.05% Triton X-100 in sterile glass bottles containing 3 mm glass beads, and mixed. Conidial concentrations were determined using a Neubauer haemocytometer following serial dilution in sterile distilled water, and conidial suspensions were used within 3 h of enumeration. Six concentrations were tested (1x10 3 to 1x10 8 conidia/ml -1 ). Viability of conidia (germination potential) was determined according to Ekesi et al. (2002). Thirty adult beetles were used per 130

3 replicate and four replicates were used per concentration (720 beetles per fungal isolate). Each beetle was inoculated on the ventral side of the abdomen with 10 µl of conidial suspension. Beetles were left in Petri dishes with fresh black wattle leaves, Acacia mearnsii, for 24 hours. Then replicate groups were released into small net cages (20 cm x 20 cm) and kept together for a week at 25 C. Results and Discussion Schizonycha affinis beetles exhibited a dose-dependent response when they were exposed to six different concentrations of fungal conidia (Figure 1). There effect of conidial concentration on the mortality of beetles was significantly greater at the higher concentrations (F (5,90) =280.53; P=0.001) with more beetle mortality observed. Two B. brongniartii isolates in particular, C13 and HHE37, showed a greater virulence, as measured by insect mortality, towards S. affinis beetles than the rest of the tested isolates (F (4,90) =37.873; P=0.001). The combined effect of conidial concentration and fungal isolate was also significantly different (F (20,90) =2.515; P=0.001). Over 85% of test beetles died at the highest concentration when B. brongniartii isolate C13 was considered compared to 53% when B. bassiana (4222) was used at the same concentration. A general trend in the intensity of oosporein production was observed which translated into greater virulence of that particular fungal isolate. Beauveria brongniartii isolates C13 and HHE37, which were significantly more potent than HH56, CHFE3 and 4222, produce large amounts of oosporein when grown in submerged culture. HH56 does produce oosporein, but at much lower rates in culture. However, CHFE3 and 4222 produce no observed oosporein. Oosporein is known to have antibiotic properties and it is proposed that when the fungus B. brongniartii penetrates the host insect, the oosporein it secretes may suppress the insect s gut flora, which in turn aids in rapid spore germination and proliferation of the fungus (Amin et al., 2010). The more oosporein secreted, the quicker the gut flora may be overcome, which increases the pathogenicity of the fungal isolate. However, it may also have an ecological role in that it protects the pathogen against antagonistic organisms during the early stages of insect infection in the soil environment (Strasser et al., 2000). The importance of having a biological control agent such as B. brongniartii which produces oosporein, means that when white grubs infected with the fungus die in the soil, degradation is much slower because saprophytic organisms are excluded from the insect s body. This allows the fungus to proliferate on the cadaver and act as a source of infection for other white grubs moving in the soil. 131

4 Figure 1. Virulence of native fungal isolates of B. brongniartii and B. bassiana (4222) towards Schizonycha affinis. Corrected mycosis of adult beetles after treatment with six concentrations of fungal conidia (conidia/ml -1 ). Points with the same letter(s) do not differ significantly by Tukey HSD test (P=0.001). REFERENCES Amin GA, Youssef NA, Bazaid S, and Saleh W (2010). Assessment of the insecticidal activity of red pigment produced by the fungus Beauveria bassiana. World Journal of Microbiology and Biotechnology 26(12): Ekesi S, Maniania N and Lux S (2002). Mortality in three tephritid fruit fly puparia and adults caused by the entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana. Biocontrol Science and Technology 12: Hurpin B and Robert PH (1972). Comparison of the activity of certain pathogens of the cockchafer Melolontha melolontha in plots of natural meadowland. Journal of Invertebrate Pathology 19: Keller S (1986). Quantitative ecological evaluation of the May beetle pathogen, Beauveria brongniartii, and its practical application. pp In: Samson RA, Vlak JM and Peters D (Eds), Fundamental and Applied Aspects of Invertebrate Pathology. Wageningen. Meyling N and Eilenberg J (2006). Isolation and characterisation of Beauveria bassiana isolates from phylloplanes of hedgerow vegetation. Mycological Research 110: Neuveglise C, Brygoo Y and Riba G (1997). 28S rdna group-i introns: A powerful tool for identifying strains of Beauveria brongniartii. Molecular Ecology 6:

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