Serial in vitro passage of Nomuraea rileyi (Farlow) Samson (UFL 78-6 pathotype) altered both its growth and development on in vitro and in vivo substrates. After only six conidial transfers on Sabouraud maltose and yeast extract agar (SMAY) plates, a reduction in the ability of these cultures to produce the yeastlike, hyphal body stage was observed. Conidia derived from transfers 6-10, although virulent to neonatal Anticarsia gemmatalis Hubner larvae, failed to produce progeny conidia on larvae killed by N. rileyi. Continued serial conidial transfer (< 10) of this pathotype on SMAY resulted in conidia with reduced virulence against neonatal larvae. By the 16th conidial transfer, progeny conidia were avirulent to larvae. The attenuation of N. rileyi was associated with the sporulation process. After multiple vegetative transfers (<80) of the hyphal body stage on SMAY plates, no decrease in virulence to A. gemmatalis was observed.

Nomuraea rileyi (UF178-6) conidia isolated from larval cadavers of Anticarsia gemmatalis and from an attenuated culture (UF178-6a) were used to establish separate hyphal body and mycelial cultures in liquid media, respectively. Studies on the growth of each strain were performed under submerged liquid fermentation conditions. Analysis of the respective utilization of carbohydrates and amino acids from the media, production of extracellular polysaccharide, and alterations in pH of the media demonstrated differences between the mycelial and hyphal body cultures. Using an enzyme mixture of β-glucuronidase and chitinase, protoplasts were produced from hyphal bodies and mycelia throughout each fermentation. Maximum yields of protoplasts were isolated from each strain midway through their vegetative growth phases.

A heterologous rDNA probe was used to detect restriction fragment length polymorphisms in Entomophaga rDNA sequences. Six Canadian strains of Entomophaga aulicae, isolated from the spruce bud worm or hemlock looper in Ontario or Newfoundland, showed no detectable rDNA variation at 10 different restriction enzyme loci: BamHI, DraI, EcoRI, EcoRV, HindIII, HinfI, MspI, PstI, RsaI, or TaqI. A total of 14 isolates of E. aulicae representative of several different geographic and host origins were compared at the DraI and HindIII rDNA loci and two different banding patterns were detected. Of these, 12 showed the same patterns and were designated E. aulicae type 1. The two members which differed were designated E. aulicae type 2. The variations in rDNA restriction sites did not appear to be geographically dependent. Entomophaga maimaiga, a recently reclassified species from the E. aulicae complex, displayed an rDNA banding pattern clearly distinguishable from the E. aulicae patterns with DraI, EcoRI, EcoRV, or HindIII. Members of the E. grylli species complex exhibited patterns which clearly differed from the patterns seen with either E. aulicae or E. maimaiga isolates. However, members of the E. grylli species complex appeared to be more heterogeneous than those in the E. aulicae complex. Among four E. grylli members, three different rDNA banding patterns were detected with either HindIII or DraI. These were designated as E. grylli type 1, type 2, and type 3. An undesignated Entomophaga isolate from a dipteran host displayed rDNA polymorphisms not previously noted in either the lepidopteran or orthopteran isolates. Our results suggest that RFLPs in rDNA are useful in the delineation of genera and species within the Entomophthorales, but may not be as useful at lower taxonomic levels. These and other RFLPs can however provide useful information regarding the epidemiology of Entomophaga epizootics.