Pyrolysis-Gas-Liquid Chromatography of Fungi: Differentiation of Species and Strains of Several Members of the Aspergillus flavus Group

Abstract

Four fungi of the Aspergillusflavus group were differentiated to the species level and strain level by pyrolysis-gas-liquid chromotography. Comparisons of pyrochromatograms revealed more similarities than dissimilarities among both species and strains in the pyrolytic elution patterns. Quantitative analysis was made by comparing the number of peaks in which two strains or reference species agreed or disagreed, the degree of superimposability between the pyrolytic elution patterns of strains and reference species, and the presence or absence of peaks for strain pairs within each species. The accuracy and precision of these techniques suggest that pyrolysis-gas-liquid chromatography may have wide application in the detection, enumeration, and identification of fungi by nonmycologically trained personnel. Methods which have been used to characterize microorganisms include analysis of volatile products in head space (6), fatty acid composition (3, 8, 12), deoxyribonucleic acid base composition (5, 11), infrared spectroscopy (20), electrophoresis (19), and gas chromatography (1, 2). However, results from studies with these methods give an incomplete picture of the genotypic expression of microorganisms and are usually too tedious and time consuming for routine analysis. A comparatively new method for identifying and characterizing microorganisms combines pyrolytic degradation of cells or cell fragments with gas-liquid chromatographic analysis (15). Resulting pyrochromatograms show elution patterns characteristic of genus, species, and, in some instances, strains (16). Pyrolysis-gas-liquid chromatography (PGLC) has previously been used to characterize bacteria (14), fungi (13), and nucleotides or nucleosides (22). Differentiation of species or strains of fungi by PGLC has not been comprehensively described, although Oyama and Carle (13) examined one taxon from each of several fungal genera. Moreover, no workers, with the exception of Cone and Lechowich (4) who were unsuccessful in differ-957 Vol. 20, No. 6

Pyrolysis Gas Chromatography of Pseudomonas and Acinetobacter Species

“…Early reports on Py-GC suggested that differences between organisms could be related to the presence or absence of pyrogram peaks, and that visual analysis was adequate for identification (Reiner 1965;Vincent & Kulik 1970). However, closely related organisms produce similar peaks that differ only quantitatively.…”

The Application of Pyrolysis Techniques to the Identification of Micro‐organisms

“…have been constructed either from glass (e.g. Vincent & Kulik 1970) or copper (e.g. Reiner 1965) and have varied in length from 1-8 m (Cone & Lechowich 1970) to 6 m (e.g.…”

“…Reiner 1967), the latter figure reflecting the capacity of most chromatograph ovens. Bore sizes for packed columns have varied between 0.76 mm (Reiner 1967) and 4.0 mm (Vincent & Kulik 1970). Since p.g.1.c.…”

“…Fungi represent a special problem for pyrolysis as there are a number of possible ways of collecting samples. Vincent & Kulik (1970) and harvested the conidia from mycelial mats with methanol, concentrated and then lyophilized them. Sekhon & Carmichael (1 972) grew their strains on cellophane membranes placed on an agar culture plate and then harvested whole colonies and dried the samples.…”

“…to fungi. Vincent & Kulik (1970) applied the technique to the Aspergillus fIuuus group of fungi imperfecti, pyrolysing three strains each of A . parasiticus, A .…”