Introduction

The First Report in the open scientific literature of the occurrence of Cladosporium resinae (Lindau) de Vries in hydrocarbon fuels appears to be that of Hendey in 1964. Hazzard (1963) positively identified C. resinae in aviation kerosene in Australia in 1961 but his results are contained in a technical report which has not been widely circulated. In the same year Prince (1961) reported finding a Cladosporium sp. in jet fuel in the United States of America and his photographs indicate that it was probably C. resinae. Contamination of aviation fuels with C. resinae has subsequently been shown to be widespread. The fungus has been isolated from aviation fuels, aircraft tanks or filters in Australia (Hazzard, 1963), Brazil (Gutheil, 1966), Denmark, England, India, Japan, Nigeria, New Zealand, Syria (Anon., 1968) and U.S.A. (Engel and Swatek, 1966).

Parbery (1967) first reported consistently isolating C. resinae from Australian soils in 1967. He was later (1969) able to show that this fungus is widespread in soils in Australia, Europe and Britain. The results of a soil survey in New Zealand support his findings (Sheridan, Steel and Knox. 1971).

Four forms of C. resinae are known to exist (De Vries, 1955) but only one, f. avellaneum, has apparently been recovered from kerosene-type fuels (Hazzard, 1963; Hendey, 1964). C. resinae f. resinae has arisen in culture from f. avellaneum (Hendey, 1964) and been isolated occasionally from soil (Parbery, 1968). An albino, f. albidum, has also arisen in culture from f. avellaneum (Parbery, 1968; Sheridan, Steel and Knox, 1971). The only form isolated from kerosene fuels and soils in New Zealand is C. resinae f. avellaneum (Sheridan, Steel and Knox, 1971). The other two forms have been found here as saltants in culture — the fourth form (f. sterile) has not been seen by us (Sheridan — unpublished). All of the above forms (except f. sterile) have been tested in our laboratory for ability to grow in kerosene. Parbery's (1968) isolates of f. resinae from soils are reported by him as not growing in kerosene. As far as we are aware no one has tested isolates of C. resinae from the air for their ability to grow in kerosene.

This paper compares the growth of New Zealand soil, fuel and air isolates of the ‘kerosene fungus’ C. resinae in aviation turbine and lighting kerosene.

Figure 1. Growth of soil isolates of C. resinae f. avellaneum in kerosene after six weeks. Left: Aviation turbine kerosene. Right: lighting kerosene. Centre: Uninoculated control.

Materials and Methods

Results

The fungus grew at the interface between the mineral salts medium and kerosene (Fig. 1) producing a dark brown fungal mat of mycelium in the case of f. avellaneum and a white fungal mat in the case of f. albidum (Fig. 2). After six weeks when the growths were harvested, black sclerotial-type bodies were present in some cases adhering firmly to the side of the bottle. These were possibly immature ascocarps. The fungus produced asexual spores within the kerosene.

Figure 2. Growth of C. resinae in aviation turbine kerosene after six weeks.
Left: Soil isolates) f avellaneum
Right: Air isolates)
Centre: Soil isolates — f. albidum

All isolates tested were able to grow on both aviation turbine and lighting kerosene. Table 1 shows the results of three separate page 15 tests where the growth of soil, fuel and air isolates of C. resinae was compared under similar conditions. The figures in the table refer to dry weight of growth harvested from two bottles. Before harvesting the fungus in the first test, V-8 juice agar plates were inoculated with each isolate. The resulting growth was used to inoculate kerosene in the second test. The process was repeated for the third test (note that in the third test 8oz. bottles were used containing 100ml. of mineral salts medium and 25ml. kerosene). Thus the requirements of ‘Koch's Postulates’ were satisfied.

Figure 3. Dry weight of soil, fuel and air isolates of C. Resinae after six weeks’ growth in aviation turbine kerosene.

In Third Test used 8oz. medicine flats with 100ml. B/H + 25ml. kerosene.

isolates was greater in lighting kerosene than in aviation turbine kerosene. The opposite was true of the air isolates. However, in the second test (see Table 1) the amount of growth produced by air isolates in aviation turbine and lighting kerosene was very similar. Visual observation from the time of inoculation indicated that the growth of all isolates was initially slower in lighting kerosene. By the third or fourth week it had caught up and overtaken that in aviation turbine kerosene.

The indications from the above results are that soil and air isolates of C. resinae grow better and faster in kerosene than do fuel isolates. Because of the small number of isolates involved it was decided to include a greater number of soil and fuel isolates in another experiment. Results are shown in Fig. 5. The greatest amount of growth was produced by a soil isolate; some fuel isolates, although producing visible growth, did not produce enough for harvesting. It is obvious from Fig. 5 that there is a wide range of ability of isolates of the fungus to grow in aviation turbine kerosene.

Some isolates of f. avellaneum produced a reddish brown pigmentation of the mineral salt medium. The nature of this pigment is being investigated.

C. resinae f. resinae has been shown to grow in aviation turbine kerosene but has not yet been tested in lighting kerosene. The white form, f. albidum, was recovered from two bottles containing aviation turbine kerosene which had been inoculated with the fuel isolates of f. avellaneum (C94, C95) but it was not tested further.

Discussion

All soil and air isolates and the majority of fuel isolates tested by us were able to grow in aviation turbine and lighting kerosene in the absence of any other source of carbon. Isolates varied considerably in the amount of growth produced after six weeks, indicating that many strains of the fungus exist, differing in their ability to grow in and utilise kerosene. All isolates grew well on V-8 juice agar without creosote, no difference being noted in the rate of growth. Further evidence for the existence of a number of strains of this fungus is obtained from the amount of reddish brown pigment produced by isolates and present in the aqueous phase when growing in kerosene. Some isolates produced an appreciable amount of this pigment, others none at all.

The amount of growth of soil and fuel isolates in general decreased on successive transfers through kerosene while that of air isolates increased. The reason for this is not known. Further work is necessary in relation to air isolates of the fungus because contamination of fuel by airborne spores is likely to occur.

All three forms of C. resinae tested, i.e. f. avellaneum, f. albidum and f. resinae, were able to grow in kerosene. The question may be page 18

Figure 4. Comparison of growth of soil, air and fuel isolates in aviation turbine and lighting kerosene. (ATK and LK.)

asked why only f. avellaneum is isolated from samples of aviation fuel. The reason for this appears to be related to the predominance of this form in the soil (Parbery, 1968; Sheridan, Steel and Knox, 1971) and in the air (Sheridan, 1971). We have not so far isolated f. albidum or f. resinae directly from soil or air. C. resinae f. resinae does not sporulate as profusely as f. avellaneum in culture and the spores are much more difficult to dislodge. In two instances we recovered f. albidum from kerosene after inoculating with f. avellaneum page 19

Figure 5. Growth of soil and fuel isolates of C. resinae f. avellaneum in aviation turbine kerosene.

page 20 (C94 and C95). This supports our belief that this form occurs from time to time in fuel supplies but is not recognised as being C. resinae.

Acknowledgments

The authors wish to thank Mr. W. Freitag and Mr. W. Johnston of B.P. New Zealand Ltd. for their help and interest in this work, particularly for supplying us with samples of kerosene. Financial assistance provided by the Victoria University of Wellington, Internal Research Committee, is gratefully acknowledged. Finally, our thanks are due to all those within the Botany Department of this university, and elsewhere, who have assisted us in so many ways.