Conidial Germination of Two Biotypes of Nomuraea rileyi

Abstract

The entomopathogenic fungus Nomuraea rileyi has a worldwide distribution and has been reported from many different insects (Ignoffo 1981). Although geographical isolates of N. rileyi from different insects have very similar isozyme patterns (Boucias and Ignoffo unpub.), they exhibit widely different host spectra (Ignoffo 1981, Boucias et al. 1984, Ignoffo & Boucias 1991). As a specific example, a Mississippian biotype (Ms) will topically infect larvae of Heliocoverpa (Heliothis) zea and Heliothis virescens, while an Ecuadoran biotype (Ec), at equivalent rates, will infect only larvae of H. zea (Ignoffo & Garcia 1985). Infectivity and/or growth of N. rileyi may also be influenced by environmental conditions, nutritional factors, and/or intrinsic genetic factors (Gardner et al. 1977, Roberts & Campbell 1977, Boucias & Pendland 1984, Ignoffo & Garcia 1985). The objective of our research was to determine whether differences in germination might be used to further distinguish between these two morphologically identical biotypes. Differences in germination also might help explain why larvae of H. virescens are more resistant than H. zea to the Ec biotype. The Ms and Ec isolates of N. rileyi were selected because of their differential pathogenicity for these two species of closely related larvae and because earlier observations revealed differences in germination patterns. The source of the Ec and Ms biotypes of N. rileyi was previously described (Ignoffo & Garcia 1985). Conidia were produced from cultures grown on Sabouraud maltose agar fortified with 1% yeast extract (SMAY) after one passage through a susceptible host (Trichoplusia ni). The harvested conidia were then stored at -70?C prior to experiments described herein. The following procedure was used to determine the extent and rate of germination of conidia of both biotypes of N. rileyi. A series of disposable petri dishes (50 x 12 mm) containing SMAY were individually seeded with 107 conidia of either biotype using 50 ul per plate. Conidia were dispensed in sterile distilled water containing equal parts (0.01% ml/ml) streptomycin, + penicillin, and gentamycin. Inoculated petri plates were then incubated at 25 + 1?C. Three replicated plates of each biotype were removed at 24, 48, 72, 96, and 120 h of incubation. Each plate was washed with 3 successive rinses of 3 ml of sterile distilled water (a sterilized, angled-glass rod was used to aid in the removal of conidia), and the rinses from the 3 plates were pooled. A subsample of each pooled rinse was placed on a glass slide, and germinated and nongerminated conidia were counted under phase microscopy. A range of 250 to 750 conidia per biotype per incubation period was counted for each of three replicates. Heliothis larvae that had been dusted with conidia of either biotype were also examined with a scanning electronic microscope to determine whether germination had occurred on the cuticular surface. The average percent germination of conidia for each of the incubation periods for the Ec biotype ranged from ca. 18 to 35% (Table 1). Germination of conidia for the Ms biotype ranged from ca. 3 to 92% (Table 1). Initial germination was most rapid in the Ec biotype; however, total germination was almost 3 X less than that of the Ms biotype. The rapid rate but low total germination seems to be an inherent trait of the Ec biotype. Other culture media (e.g. potato-dextrose-agar, Sabouraud-dextrose-agar, oatmealagar, cuticle + SMAY) did not increase total germination of the Ec biotype. The highest germination of conidia for the Ec biotype (34.9 ? 6.9%) occurred after only 24 h of