Characterization of Some Beauveria bassiana Isolates and Their Virulence toward the Coffee Berry Borer Hypothenemus hampei AMANDA VARELA* AND ESPERANZA MORALES† ,1 *Departamento de Microbiología, Universidad Javeriana, Carrera 7 No. 43 - 82, Santafé de Bogotá, Colombia; and †Laboratorio de Investigaciones sobre la Química del Café, Colombia Received May 6, 1995; accepted September 28, 1995 Some morphological and physiological characteris- tics and electrophoretic esterase profiles of six iso- lates of Beauveria bassiana were evaluated, and labo- ratory bioassays were performed to assess their viru- lence against the coffee berry borer Hypothenemus hampei. The relationship among these characteristics and virulence against H. hampei and their utility in selecting isolates for further studies are discussed. © 1996 Academic Press, Inc. KEY WORDS: Beauveria bassiana; Hypothenemus hampei; biological control; entomopathogenic fungus; coffee berry borer; characterization; pathogenicity; li- pase; esterases. INTRODUCTION The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin has been extensively used for the control of many important pests of various crops around the world and it has been tested on different target insects (Trefi, 1984; Campbell et al., 1985; Rom- bach et al., 1986a,b; Aguda et al., 1987; Hajek et al., 1987; Andersoni et al., 1988; Ming-Guang and Johnson, 1990; Leathers and Gupta, 1993). The coffee berry borer Hypothenemus hampei is now considered the most important coffee pest (Bustillo et al., 1993). It lives the greatest part of its life cycle in- side the coffee berry. The female insect bores through the coffee berry and can fly outside it, while the un- winged male cannot (Cárdenas, 1993). The time when the female leaves the berry is strategic for the dissemi- nation of a pest control agent. Due to the lifestyle of H. hampei, contagious biocontrol agents could in theory be much more effective than chemical control. Fungi are potentially the most versatile entomopathogens be- cause many have wide host ranges, infect different ages and stages of their hosts, and often cause natural epizootics (Ferron, 1978; Fuxa, 1987). Some previous studies look for particular traits of this fungus that can be related to virulence, such as physiological characteristics (Paris and Ferron, 1979; Paris et al., 1985; Ming-Guang and Johnson, 1990) and enzyme production (Bidochka and Kachatourians, 1990; Sosa-Gómez et al., 1994). However, for its use as a bioinsecticide, it is important to identify a range of characteristics favorable to survival and virulence against H. hampei. In this study we report the variations among six iso- lates of B. bassiana based on microscopic and macro- scopic morphology and physiological, and biochemical characteristics and we assess their significance and correlation to virulence. MATERIALS AND METHODS Isolates. Six isolates, which had been obtained from different hosts and geographical locations (Table 1), were selected from the culture collection at Laboratorio de Investigaciones sobre la Química del Café (LIQC). Stock cultures were freeze-dried and, when required, they were reconstituted and plated onto fresh SDAY (Sabouraud dextrose agar + 0.2% yeast extract) and incubated for 7 days. The inoculum for all experiments was obtained from these cultures. Fungal growth was harvested and suspended in sterile 0.05% Tween 20. This suspension was filtered with a No. 4 Millipore filter, which has a pore size of 5–15 m (Millipore Corp.). The conidial suspension obtained was adjusted to a concentration of 1 × 10 6 conidia/ml. All experi- ments were done in triplicate, and all incubations were performed at 27°C in the dark unless otherwise stated. Evaluation of conidial viability. Conidial viability was determined by a variation of the method of Samuels et al. (1989). The layer was divided into three equal sections and each was inoculated with a 0.6-ml aliquot of a conidial suspension of 1 × 10 6 conidia/ml. Slides were incubated in a humid chamber (>90% RH), for 24 hr, to determine the germination percentage. 1 Present address: Hoechst, Carrera 77A No. 45–61, SantaFé de Bogotá, Columbia. Fax: 57–1–2956051. JOURNAL OF INVERTEBRATE PATHOLOGY 67, 147–152 (1996) ARTICLE NO. 0022 147 0022-2011/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.