IDENTIFICATION AND DIFFERENTIATION OF THE ENTOMOPATHOGENIC FUNGUS BEAUVERIA-BASSIANA USING POLYMERASE CHAIN-REACTION AND SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS
Dd. Hegedus et Gg. Khachatourians, IDENTIFICATION AND DIFFERENTIATION OF THE ENTOMOPATHOGENIC FUNGUS BEAUVERIA-BASSIANA USING POLYMERASE CHAIN-REACTION AND SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS, Journal of invertebrate pathology, 67(3), 1996, pp. 289-299
A series of genomic DNA probes which exhibit specificity for Beauveria
bassiana demonstrated a level of sensitivity to approximately 152 ng
of fungal DNA (Hegedus and Khachatourians, 1993a). To improve the sens
itivity of a DNA-based monitoring system for detection of this entomop
athogenic fungus we have developed a PCR-based method. Using sequence
information from a region of the B. bassiana-specific probe pBb22, pri
mers P1 (5'AAGCTTCGACATGGTCTG) and P3 (5'GGAGGTGGTGAGGTTCTGTT) were ge
nerated. This primer set amplified similar-sized products from several
B. bassiana isolates, Beauveria brongniartii, Beauveria caledonica, a
nd B. densa, but not Tolypocladium nivea, Tolypocladium cylindrospora,
Metarhizium anisopliae, Verticillium lecanii, and Paecilomyces farino
sus or migratory grasshoppers and locusts. Hybridization with the prob
e indicated the presence of DNA homology between the products. Restric
tion enzyme analysis of the PCR products, however, showed that sequenc
e heterogeneity existed which was confirmed by partial sequencing of t
he products. Another primer, P5 (5'AGGAGAGAGCTCGACGGTCA), was develope
d to exploit the sequence variations. When the P1-P5 primer set was us
ed, a product was amplified from most B. bassiana isolates, including
two which failed to amplify with the P1-P3 set. This amplification was
not observed with the other Beauveria species tested. The nature of t
he sequence variations within the P1-P3 PCR-amplified region suggests
the placement of B. densa and B. caledonica outside the species B. bas
siana, confirming previous evidence with mitochondrial DNA and DNA pro
bes. PCR with the P1-P3 and P1-P5 primer sets was used to discriminate
isolates of B. bassiana found to be infecting a population of the mig
ratory grasshopper, Melanoplus sanguinipes, collected in Saskatchewan.
The PCR products derived from the P1-P3 primer set with the various B
eauveria spp. could be differentiated by single-strand conformation po
lymorphisms (SSCP). Thus, analysis of PCR products using restriction e
nzymes, sequencing, or SSCPs allows positive differentiation of a part
icular B. bassiana isolate from others. The great sensitivity of these
techniques should help the release and monitoring of entomopathogenic
fungi in the environment. (C) 1996 Academic Press, Inc.