Molecular identification of species from the Penicillium roqueforti group associated with spoiled animal feed

Abstract

The Penicillium roqueforti group has recently been split into three species, P. roqueforti, Penicillium carneum, and Penicillium paneum, on the basis of differences in ribosomal DNA sequences and secondary metabolite profiles. We reevaluated the taxonomic identity of 52 livestock feed isolates from Sweden, previously identified by morphology as P. roqueforti, by comparing the sequences of the ribosomal internal transcribed spacer region. Identities were confirmed with random amplified polymorphic DNA analysis and secondary metabolite profiles. Of these isolates, 48 were P. roqueforti, 2 were P. paneum, and 2 were Penicillium expansum. No P. carneum isolates were found. The three species produce different mycotoxins, but no obvious relationship between mold and animal disease was detected, based on medical records. P. roqueforti appears to dominate in silage, but the ecological and toxicological importance of P. carneum and P. paneum as feed spoilage fungi is not clear. This is the first report of P. expansum in silage.

FIG. 1

RAPD fingerprinting using either NS2 (a) or NS7 (b) as a primer. Lanes: 1, P. roqueforti SVA 3631/1996, having a T in position 180 of the ITS1 region; 2, P. roqueforti SVA 8176/1995, isolate 5 (A in position 180); 3, P. roqueforti SVA 2986/1992 (T in position 180); 4, P. roqueforti type strain (IBT 6754); 5, P. paneum SVA 494/1990; 6, P. paneum SVA 7023/1994, isolate 2; 7, P. paneum type strain (IBT 12407); 8, P. expansum SVA 2294/1994, isolate 5; 9, P. expansum SVA 6180/1994 isolate 1; 10, P. carneum type strain (IBT 6884); M, molecular weight marker (1-kb DNA ladder; GIBCO BRL, Gaithersburg, Md.).

FIG. 2

Schematic illustration of secondary metabolite profiles. TLC plates were eluted in toluene-ethyl acetate-formic acid (TEF) and chloroform-acetone-isopropanol (CAP) and evaluated under UV light (365 nm). Lanes: 1, P. roqueforti SVA 3631/1996, having a T in position 180 of the ITS1 region; 2, P. roqueforti SVA 8176/1995, isolate 5 (A in position 180); 3, P. roqueforti SVA 2986/1992 (T in position 180); 4, P. roqueforti type strain (IBT 6754); 5, P. paneum SVA 494/1990; 6, P. paneum SVA 7023/1994, isolate 2; 7, P. paneum type strain (IBT 12407); 8, P. expansum SVA 2294/1994, isolate 5; 9, P. expansum SVA 6180/1994, isolate 1; 10, P. carneum type strain (IBT 6884). Lanes 11 to 14 are standards: griseofulvin (11), patulin (12), PR toxin (13), and roquefortine C (14). Dotted lines around spots indicate weak spots.