Evaluation of entomopathogenic fungi (ascomycota) for the control of cydia pomonella (lepidoptera: tortricidae)
Codling moth ([Cydia pomonella (Lepidoptera: Tortricidae]) infest pomes resulting in high production losses to fruit farmers in South Africa and globally. Many farmers are adopting biocontrol methods including the use of entomopathogens as alternatives to synthetic insecticides for sustainable management of Arthropod pests. Research activities on screening and application of entomopathogenic fungi (EPF) have intensified in recent years. This study was carried out to isolate and identify virulent indigenous entomopathogenic fungal strains from soils of selected locations in the Western Cape region of South Africa that are pathogenic against codling moth (Cydia pomonella (Linn) [Lepidoptera: Totricidae]) and to evaluate the possiblility of combining semiochemicals and fungal inoculums in a simulated attract-and-fungus contamination system for codling moth control. Soil samples were collected from 10 locations in the Western Cape, South Africa for the isolation of entomopathogenic fungi (EPF) by baiting the soil samples with 5th instar larvae of codling moths. Fungal strains were identified and characterized using light microscopy and DNA analysis (internal transcribed spacer region) and elongation factor 1-α (EF 1-α) genes of fungal cultures. Thirty-nine (39) isolates belonging to six species were obtained; Purpureocillium lilacinum (8 isolates), Fusarium oxysporum (five isolates), Fusarium polyphialidicum (two isolates), Beauveria pseudo-bassiana (one isolate), Aspergillus flavus (three isolates) and Metarhizium robertii (20 isolates). Generally, M. robertsii was the most frequently encountered species representing 51% of the total number of isolates collected from the soil samples. This is the first report of the isolation of M. robertsii in southern Africa. A screening test to identify the most virulent fungal strains against codling moth was carried out on 39 isolates by immersing 5th instar larvae of C. pomonella into aqueous spore suspension 1 x 108 conidia ml-1. Mortality data from the immersion bioassay indicated that the 39 fungal isolates were pathogenic against fifth instar larvae of codling moth inducing 47-85% insect mortality at an infective concentration of 1 x108 spores’ ml-1, 5 days post-treatment. Two fungal strains MTL151 and GW461 induced over 85% larval mortality and were selected for further evaluations. The effect of MTL151 and GW461 on egg hatchability of 0-day old eggs was evaluated by exposing freshly laid eggs on wax papers that were pre-treated with fungal spores ranging from 103 -108 spores/ml. Egg hatchability reduced significantly from 93-71% (GW461) and 95-66% (MTL151) as spore concentration increases from1 x 103 to 1 x 108 spores ml-1, respectively. The highest spore concentration significantly inhibited egg hatchability resulting in lower egg hatchability compared to that obtained with the cypermethrin containing commercial insecticide (Fruitfly [registered by Kombat (PTY) Ltd] ) tested at the recommended dose of 0.25 ml/250 ml of water. The potential of the two M. robertsii strains to protect apples from infestation by codling moth neonates was assessed in an apple fruit bioassay under laboratory and field bioassay. Codling moth neonates were exposed to apples that were sprayed topically with varied conidia concentrations (103 - 108 sporesml-1). The mean number of participating apple fruits having developing/developed larvae in the core/flesh significantly reduced from 5.3 to 1 and 7.6 to 1 for MTL151 and GW461, respectively as spore concentrations increased from 1 x103 to 1x 108 spores/ml-1. A concurrent decrease in apple fruit rot as conidia concentration increased was observed. Up to 90% of apples treated with 1 x 108 spores/ml-1 had no larva present in their cores and this result compared favourably with the commercial pesticide (Fruitfly) used at a recommended dose of 0.25g/250 ml of water. No significant difference was found between the EC50 values of 1.2 x 103 (CI=1.2 x 102 -1 x 105) (MTL151) and 1.1 x 105 (CI =7 x103 – 5.6x105) (GW461) spores ml-1. However, neither the two M. robertsii strains nor the insecticide deterred the neonates from feeding on the surface of the fruits. The attraction responses of male and female adult codling moths to butyl hexanoate and codlemone blend; butyl hexanoate (0.025 mg / ml of hexane) and codlemone (0.1 mg / ml of hexane) in a ratio of 1:1 (0.5 ml: 0.5 ml from both solutions) were assessed in a one-choice wind tunnel olfactory bioassay during scoto-phase in the absence of fungal spores. The blend attracted more females (69%) than males (66.7%), however these responses were not significantly different (P > 0.5). The prospect of integrated use of entomopathogenic fungal conidia and semiochemical blend for control of C. pomonella adults was assessed in a wind tunnel containing the combo lure and M. robertsii (MTL151) spores formulated as a powder (250 mg) or aqueous spore suspension (3.1 x 108 spores m-1 ± 7 x 102 sporesml-1). Insect attraction, mean number of spores picked per insect and insect mortality was assessed. No significant difference (P > 0.5) was observed in the number of inoculi picked by unsexed adult moths in the two conidial formulations tested; spore powder (3.1 x 103 ± 1 x 103 spores ml-1) and aqueous spore suspension (3.1 x 103 ± 7 x 102 spores ml-1). No mortality was recorded among fungus or control-treated moths. Both M. robertsii (MTL151) spore formulations did not significantly (P > 0.05) affect the attraction responses of the insects to the combo lure; powder (73.3 ± 3.3%) and aqueous (68.6 ± 2.9%) compared to the control treatment (without the fungal spores) (70 ± 0.5 %). These results suggest that the fungus did not inhibit the attraction of moths by semiochemicals. Despite the successful autoinoculation of moths with fungal spores, the level of contamination was too low to induce mortality among adult moths. This study opens up opportunities for research in attract-and-fungus contamination delivery systems. This is the first report of the occurrence of M. robertsii in the southern Africa. Further, results from this study indicates that C. pomonella is susceptible to indigenous South African entomopathogenic fungi and 2 indigenous M. anisopliae strains, MTL151 and GW461 provided adequate protections against codling moth larval infestations of apple fruits. Future research endeavours to improve adherence of conidia to adult moth cuticle is recommended. Key words: Entomopathogenic fungi (EPF), codling moth, B. pseudobassiana, M. robertsii, larvae, pathogenicity and biological control, coddlemone, butyl hexanoate, attraction, semiochemicals.