An Improved Bioassay Method for Entomopathogenic Fungi of Sugarcane Pests and Its Evaluation in Studies of Virulence in Subcultures

An improved bioassay method was developed for evaluating entomopathogenic fungi (EPF) using pests and fungi from sugarcane crop system. Third instar larvae of white grub Holotrichia serrata F. (Coleoptera: Scarabaeidae) and shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) surface-contaminated with suspensions (10 8 spores/ml) of Beauveria brongniartii (Sacc.) Petch and Beauveria bassiana (Balsamo-Criv.) Vuill., respectively were subjected to starvation and feeding regimes. Holotrichia serrata grubs maintained in starvation regime showed lower overall mortality and higher LT 50 values than those kept in feeding regime in one test; the trend was, however, opposite in the second test. On the other hand, C. infuscatellus larvae in starvation regime produced higher mortality and lower LT 50 values than those in feeding regime in both tests. Further, spore output per C. infuscatellus larval cadaver was equal in both regimes. In degeneration studies, spore output of Metarhizium anisopliae Metch. maintained on nutrient broth for 15 continuous subcultures showed overlapping differences and a decreasing trend after the first six subcultures. In virulence studies with M. anisopliae from eight subcultures against diet-reared C. infuscatellus , third instar larvae treated with the fungus and maintained under starvation regime showed two peaks of LC 50 and LT 50 values. In comparative studies with field-collected C. infuscatellus , third instar larvae treated with 10 8 spores/ml of the fungus from 15 subcultures and fed with sugarcane shoots showed indistinct peaks in per cent mortality which reached significantly lowest value in the 15th subculture. The advantages of the modified bioassay method in virulence tests and the importance of degeneration studies for candidate EPF such as B. brongniartii are discussed.