Characterization of Growth and Virulence of Five Nigerian Isolates of Entomopathogenic Fungi Using Galleria mellonella Larvae for Pathogenicity Testing

Abstract

Entomopathogenic fungi (EF) are naturally occurring insect population regulators, with several species that are exploited as biocontrol agents against insect pests. Five EF consisting of two strains of Isaria farinosa, (IF-I and IF-II) and one strain each of Metarhizium anisopliae, Beauveria bassiana and Entomophaga sp. (Sensu lato) were isolated from soil using Tribolium castaneum (Coleoptera: Tenebrionidae) (Herbst) larvae as bait. The isolates were cultured on standard Potato Dextrose Agar (PDA) (Sigma-Aldrich, UK) and identified based on phenotypic appearance and micro-morphology. Growth rates (mm day-1), number of conidia per cm2 colony area after incubation for 14 days at ambient temperature (25±2°C), viability of conidia (% germination), based on 24-hours incubation period and virulence of the infective conidia against Galleria mellonella were evaluated. The data on growth was subjected to analysis of Variance (ANOVA) procedure and means were separated using Tukeys Honestly Significant Difference (P=0.05). The number of conidia produced by Entomophaga sp was (7.0×105 conidia cm 2 per colony area), while the isolate of M. anisopliae produced (5.2×104 conidia cm2 per colony area). The number of conidia produced by the two isolates of I. farinosa, IF-I and IF-II and B. bassiana were 9.4×104, 7.2×104 and 2.1×105 conidia cm2 per colony area respectively. Eighty percent of Entomophaga sp conidia germinated after incubating for 24 hours at 25°C while 100% germination occurred in the other fungal isolates. There were statistically significant variabilities in the rates of growth of the EF isolates F(4,2.064) =12.97, P=0.001. The B. bassiana isolate had the fastest growth rate, with statistically significant value of 3.3 mm day-1. The rates of growth of the two I. farinosa isolates: IF-I, IF-II and M. anisopliae were comparable, being 1.53, 1.4 and 1.28 mm day-1 respectively, without statistically significant difference. The growth rate of Entomophaga sp was 2.0 mm day-1, which was significantly higher than the growth rates of I. farinosa and M. anisopliae. The mean percentage mortality values of, G. mellonella larvae treated with 1×108 conidia ml-1 of the infective conidia of I. farinosa, IF-I, IF-II, M. anisopliae, after five days were 70, 60, 60% respectively while Entomophaga sp and B. bassiana caused 50% mortality. The results suggest that the five isolates examined can potentially be developed into experimental formulations and tested against important horticultural pests in future studies.