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Tuatara: Volume 19, Issue 2, May 1972

Studies on the ‘Kerosene Fungus’ Cladosporium Resinae (Lindau) De Vries — Part II. The Natural Habitat of C. Resinae

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Studies on the ‘Kerosene Fungus’ Cladosporium Resinae (Lindau) De Vries
Part II. The Natural Habitat of C. Resinae

Contents

Introduction
1. The occurrence of C. resinae in hydrocarbons
2. The occurrence of C. resinae on creosoted timbers
3. The occurrence of C. resinae in soil
4. The occurrence of C. resinae in the atmosphere
5. The occurrence of C. resinae in other habitats
6. Summary and conclusions

Introduction

In Part I of these studies the problem of microbial contamination of aviation fuel was discussed and the role of the ‘kerosene fungus’ Cladosporium resinae (Lindau) de Vries (perfect state Amorphotheca resinae Parbery) in contamination and corrosion evaluated. Part II deals with the natural habitat of this interesting fungus.

Cladosporium resinae is not only capable of growing in and utilising kerosene as a source of food, it can grow on wood impregnated with coal tar products such as creosote and utilise these products as food (Christensen, Kaufert, Schmitz and Allison, 1942). In the late 1930's several people (Dr. C. M. Christensen and his colleagues working at the Minnesota Agricultural Experiment Station, Dr. C. A. Richards of Forest Products Laboratory, Madison, Wisconsin, and Dr. J. D. Burns of Page and Hill Co., Robbinsdale, Minnesota) had occasionally observed a brown mould growing on small pieces of wood impregnated with coal tar products when placed in moist chambers or on agar during routine tests for resistance to decay (Christensen et al., 1942). Because of the known toxicity of these preservatives to micro-organisms in general these workers initially more or less disregarded this sporadic appearance of a fungus on treated wood and thought of it only as a laboratory phenomenon. This is understandable when one considers that coal tar products, especially creosote, have been used for over one hundred years to protect wood from attack by fungi, insects and other organisms. According to Christensen et al. (1942), each cubic foot of wood impregnated with these products contains 8 to 20 pounds, or 1 to 2½ gallons, of the preservative and the long service of timbers so treated is evidence of the value of the coal tar products as preservatives. However, in the fall of 1939 an investigation was initiated by page 71 Christensen and his colleagues into the identity of this mould and its occurrence in nature. Their findings were published in 1942. They concluded that the fungus was Hormodendrum resinae (= Cladosporium resinae — see Part III) which Lindau had described in 1907 from the resin of a conifer, Picea excelsa, and that it is a common inhabitant of wood treated with coal tar products including creosote. These workers also isolated the same fungus from resinous wood and from asphalt street pavements.

Some years later Marsden (1954) became interested in this fungus in connection with its possible deleterious effects on the preservative used to protect the wood against decay. He confirmed the findings of Christensen and his colleagues regarding occurrence on creosoted timbers and deep penetration into wood. He found fruiting structures produced within tracheid cavities as well as on the surface of the wood and indications that this mould may deleteriously affect the toxicity of creosote. He described five strains of the fungus (see Part III), and he coined the name ‘creosote fungus’ in 1954. Christensen and colleagues were unable to isolate C. resinae from soil except on one occasion (glasshouse soil) and neither they nor Marsden were able to isolate it from the air. The indications were, therefore, that C. resinae occurred naturally in resinous wood and on creosoted timbers, but not in soil.

With the implicating of C. resinae in contamination of jet aviation fuels and in corrosion of aircraft tanks in the 1960's interest in this fungus revived. Leonard and Klemme (1961) believed that this fungus could be soil-borne because most other Cladosporia could be isolated from the soil. But it was not until the late 1960's that this was confirmed by Parbery (1967). After devising a method which successfully isolated C. resinae from soil he investigated its occurrence in soils from Australia, Britain and Continental Europe. On the basis of his findings he concluded (1969b) that C. resinae is a natural component of the soil microflora and is widely distributed in nature. His conclusions are supported by the results of recent work in New Zealand (Sheridan, Steel and Knox, 1971).

None of the earlier workers were able to isolate C. resinae from the atmosphere yet Christensen et al. (1942) and Hendey (1964) were, according to Parbery (1969b), still of the opinion that it is airborne. Parbery (1969b) referred to three independent trappings of this fungus from the atmosphere, thus supporting this view. Within the last few years Harvey (1971—personal communication) has isolated C. resinae from the air in Wales in very small quantities, but consistently. More recently, using a method which selectively isolates this fungus from the air (Sheridan and Nelson, 1971b), it has been possible to trap it consistently from the atmosphere over Wellington, New Zealand (Sheridan, Sheridan, Hoverd and Nelson, 1971; Sheridan, 1971).

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Figure 1: Distribution of C. resinae in airfield fuel systems in Australia. (Figures from Hazzard, 1963.) Black dot + ve Hollow dot —ve

Figure 1: Distribution of C. resinae in airfield fuel systems in Australia. (Figures from Hazzard, 1963.) Black dot + ve Hollow dot —ve

From the foregoing it is quite clear that this extraordinary and intriguing, if not treacherous, fungus is very elusive. It has slipped through the fingers of the earlier workers when they have attempted to isolate it from soil and air. Results obtained by the use of new methods have given a better understanding of its distribution and role in nature but we will have to be patient until results from the wider application of these methods become available. In this paper the body of knowledge available at the present time on the occurrence of C. resinae in nature is reviewed in an attempt to determine its natural habitat.

1. The occurrence of C. resinae in hydrocarbons

The first report in the open scientific literature of the occurrence of Cladosporium resinae in hydrocarbon fuels appears to be that of Hendey in 1964. He reviewed the occurrence of this fungus in kerosene-type fuel storage tanks and fuel tanks of aircraft. Hazzard (1963) 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. As stated in Part I of these studies, contamination of kerosene-type fuels with page 73 C. resinae was widespread in the 1960's — until recently it was much easier to isolate this fungus from kerosene than from soil and Hendey's application of the name ‘kerosene fungus’ to this organism is indeed appropriate. The accompanying map (Fig. 1) shows the distribution of C. resinae in airfield fuel systems in Australia. The figures are taken from Hazzard (1963) but the map is incomplete because of the difficulty of locating some of the airfields. Figures are not yet available for New Zealand, hence the distribution of C. resinae cannot be plotted. (For figures for Australia and California see Part I.)

The fungus has been isolated from lighting and aviation kerosene in France (Nicot and Zakartchenko, 1966 — ‘Mazout, kérosène carburants pour l'aviation’) and we have isolated it from both of these in New Zealand. We were not able to confirm from the literature that C. resinae had been isolated from petrol (= gasoline), diesel oil or lubricating oils, but the first sample of petrol we examined yielded five colonies of C. resinae from 250 ml. of the sample. Fuel/water samples of aviation gasoline are at present under test.

C. resinae has been isolated from aviation fuel, from tank filters or aircraft fuel tanks in Australia (Hazzard, 1963), Brazil (Gutheil, 1966), Denmark, England, India, Nigeria, New Zealand, Syria (Anon., 1968), U.S.A. (Engel and Swatek, 1966) and Japan (Inoue, Iwamato and Minoura, 1965).

It has been reported that C. resinae can remain alive for rather a long time in kerosene-type fuels (Hendey, 1964). Nicot and Zakartchenko (1966) found the fungus in their samples to be alive after 30 months but the results of their tests were not consistent. Hazzard (1967) found that it could remain viable for several years in essentially water-free petroleum products. This aspect has not yet been studied in our laboratory.

Four forms of C. resinae are known to exist (see Part III) but only one, j. avellaneum, has apparently been recovered from kerosene-type fuels (Hendey, 1964; Inoue, Iwamoto and Minoura, 1965) (Table 1). C. resinae f. resinae has arisen in culture from f. avellaneum (Hendey, 1964) and been isolated occasionally from soil (Parbery, 1968). An albino has arisen in culture from f. avellaneum (Parbery, 1969a; Sheridan, Steel and Knox, 1971) and is morphologically similar to it (Figs. 2 and 5). De Vries (1955) gave the name f. albidum to an albino which was morphologically similar to f. resinae. For the present purpose we shall refer to both of these as f. albidum (see Part III).

The only form isolated by us directly from kerosene fuels (and soil) is C. resinae f. avellaneum. Both f. albidum and f. resinae have been found in New Zealand as saltants in culture. All three forms have been tested by us for ability to grow in kerosene; all were able to do so (see Fig. 3). Parbery's (1968) isolates of f. resinae from

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Table 1
The Occurrence of C. Resinae in Kerosene-Type Fuels
Authority Date Source Name Form
Prince 1961 Aviation Kerosene (U.S.A.) Cladosporium sp. (?f. avellaneum)
Hazzard 1961, 1962, 1963 " " (Australia) Cladosporium resinae f. avellaneum
Hendey 1964 " " (Britain) C. resinae f. avellaneum
f. resinae as saltant
Inoue, Iwamoto and Minoura 1965 " " (Japan) C. resinae f. avellaneum
Nicot and Zakartchenko 1966 " " (France) (and Lighting Kerosene) C. resinae f. avellaneum
Engel and Swatek 1966 Aviation Kerosene (U.S.A.) Hormodendrum resinae ——
Darby et al. 1968 " " (U.S.A.) C. resinae f. avellaneum
Sheirdan et al. 1971 " " (N.Z.) (and Lighting Kerosene) C. resinae f. avellaneum
f. resinae)
f. albidum)
and intermediates as saltants
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Figure 2: Colonies of C. resinae f. avellaneum on V-8 juice agar containing 0.1% creosote.

Figure 2: Colonies of C. resinae f. avellaneum on V-8 juice agar containing 0.1% creosote.

soil are reported by him as not growing in kerosene. We have not isolated the fungus from pure creosote and none of our isolates so far tested are able to grow in pure creosote.

2. The occurrence of C. resinae on creosoted timbers

Cladosporium resinae was repeatedly isolated from wood impregnated with creosote and coal tar, e.g. telephone poles and railway ties, by Christensen, Kaufert, Schmitz and Allison (1942). Samples were taken near the ground line from twenty-nine creosoted poles in Minnesota, Wisconsin, Pennsylvania and Delaware. C. resinae was isolated from twenty-seven of these. It was likewise isolated from six of eight creosoted ties sampled in Minnesota, and from all of eleven ties from Ohio, from all of four ties from Louisiana, three from the State of Washington, and from ties sampled in Missouri and Indiana. The fungus was also found on several creosoted southern pine fence posts in service for seventeen years in Iowa. These workers also successfully isolated this fungus from resinous woods.

Their work led the above workers to an investigation of the natural habitat of the fungus. Soil was tested around the University Farm, St. Paul, Minnesota, which included cultivated and uncultivated land, and soil near a creosoting plant but no C. resinae was found. Neither were they able to isolate it from the atmosphere. The reason for this failure to isolate the kerosene fungus from the soil and air was almost certainly due to the use of unsuitable techniques (see 3. The occurrence of C. resinae in soil). Marsden (1954) isolated

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Table 2
The Occurrence of C. Resinae on Resinous Wood and Creosoted Timbers
AuthorityDateSourceNameForm
Lindau1907Resin of Pices excelsa (Europe)Hormodendrum resinae (= Hormodendron)——
Christensenet al.1942Resonous Wood (U.S.A.) Creosoted TimbersH. resinae(f. f. avellaneum)
Marsden1954Creosoted Timbers (U.S.A.)H. resinae(f. avellaneum)
(f. resinae)
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Figure 3: Growth of C. resinae in aviation turbine kerosene. Left: f. avellaneum isolated from soil. Right: f. avellaneum isolated from air. Centre: f. albidum.

Figure 3: Growth of C. resinae in aviation turbine kerosene. Left: f. avellaneum isolated from soil. Right: f. avellaneum isolated from air. Centre: f. albidum.

C. resinae from creosoted wood by placing chips of wood from creosoted poles on to malt extract agar containing 1% creosote. In this way C. resinae was obtained from several treated and partially decayed poles from Georgia, Massachusetts, New York, Pennsylvania and Santo Domingo. Christensen et al. (1942) and Marsden (1954) were of the opinion that creosoted timbers or resinous wood constituted the natural habitat of this fungus. Although we have not attempted to isolate C. resinae from creosoted timber in New Zealand we have consistently isolated it from soil collected around the base of creosoted telegraph and electric poles. Using a creosoted matchstick method of isolation (Sheridan, Steel and Knox, 1971) we often found the fungus growing and sporulating on the matchsticks as well as on the soil (Fig. 4).

De Vries (1955) has studied various strains of Cladosporium resinae including two of Marsden's (1954). He concluded that Marsden's New York strain agreed with C. resinae f. avellaneum and his New York saltant strain with f. resinae (Fig. 5). The fungus isolated by Christensen et al. (1942) appears to be f. avellaneum (Table 2). De Vries tested the growth of five strains of the fungus on media containing 4% coal tar or 1% creosote. The strains tested were: 1, Enola; 2, ‘Christensen’ (received from Marsden); 3, C. resinae f. avellaneum; 4, C. resinae f. resinae; 5, C. resinae f. albidum. Contrary to De Vries's expectations, the last three strains did not grow on these media while the Enola and Christensen isolates made good growth. More recent work confirms that this fungus produces a wide variety of morphological forms and these vary in their ability to tolerate and grow in kerosene and creosote (Hendey, 1964; Parbery, 1969b). This aspect is dealt with more fully in Part III of these studies.

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3. The occurrence of C. resinae in soil

Cladosporium resinae was first isolated successfully from soil by Parbery (1967), working in Australia. He devised the ‘creosoted matchstick method’ in which two decapitated matchsticks, sterilised and soaked in sterile creosote, were added to a Petri dish containing moistened soil (See Fig. 4). The plates were incubated at 25° C. along with control plates containing non-creosoted matchsticks for up to three weeks. In Victoria, Parbery took soil samples from the base of, and some distance from, test creosoted poles and from virgin bushland, 50 metres from the testing site. C. resinae appeared in twelve out of fourteen soil samples tested. Since the fungus appeared in four samples of soil from the virgin bushland, this was considered by Parbery (1967) to be a strong indication that the fungus occurs naturally in soils. This observation led Parbery to further studies on the distribution of C. resinae in soil (Parbery, 1969b). He collected sixty isolates of C. resinae from soils in Australia. Britain, France and Sweden, proving the fungus to be widely distributed and indicating that it is a natural component of the soil microflora. The collection sites of soil yielding C. resinae were:

In England — fallow soil, under pines and poplar, by a creosoted pole, from soil under a larch and under Norway spruce.

In Wales — garden soil, the bases of creosoted poles, grass tussock and soil under firs and pines.

In France — soil under juniper, Norway spruce, oak, beech, Picea pungens, Cedrela sinensis and a soil bitumen mixture near base of pole.

In Sweden — there were two positive isolations from Stockholm, under Picea pungens and under oak and elm.

In Australia — isolates from Victoria were collected from burned hilltop forest, alluvial soil, beside a creosoted pole, skeletal hillside, roadside under eucalypt, petrol station, virgin bush, mountain soil of virgin bush, sandy arid soil and under Pinus radiata.

In New Zealand this fungus was first isolated from a garden soil in Wellington, near a creosoted fowl house, in November, 1969 (Sheridan and Knox, 1970). A modification of Parbery's ‘creosoted matchstick method’ was used. The matchsticks were steeped in creosote for 48 hours, transferred to a clean beaker covered with aluminium foil, and then autoclaved for 30 minutes at 103 kNm-2. This modification ensured that very little liquid creosote was transferred to the soil. This is an advantage because none of our isolates would tolerate as much as 3% creosote and the presence of excess creosote could suppress growth. C. resinae has subsequently been found to be widespread in soil in this country (Figs. 6 and 7), occurring most frequently in and near built-up areas but also occurring occasionally in soils remote from human habitation.

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Figure 4: Cresoted matchsticks on soil contained in Petri dishes.

Figure 4: Cresoted matchsticks on soil contained in Petri dishes.

Figure 5: Conidiophores and conidia of C. resinae. Left: f. avellaneum. Right: f. resinae.

Figure 5: Conidiophores and conidia of C. resinae. Left: f. avellaneum. Right: f. resinae.

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We have isolated C. resinae in New Zealand from cultivated garden soils, from soils at the base of creosoted poles and fence posts and soils at the edge of a tar spillage on a roadside, from the top of sawdust mounds on old saw mill sites (Ketetahi Mills, near Taurewa, and near spiral railway, Raurimu), from soil under grass, gorse, conifers, scrub and native bush and from sandy soil in the Rangipo Desert. It has been found from sea level up to 5,000 feet. The collection sites are shown in Table 3.

Table 3
Sites of Collection of Soil Samples
A.
Classification No. of Samples No. yielding C. resinae
Gardens 57 26
Parks and reserves 48 24
Roadsides 446 206
Open country 57 1
Unclassified 100 32
Total 708 289
B.
Classification No. of Samples No. yielding C. resinae
Cultivated soil 38 22
Under grass 197 82
Under conifers 38 20
Under scrub 73 23
Near creosoted poles and posts 47 32
On sand dunes, beach and desert 7 3
Total 400 182

Parbery (1969b) reports that there does not appear to be any correlation between isolation of C. resinae and any soil or vegetation type but there does appear to be some positive correlation between frequency of isolation and the annual average rainfall. This correlation could, in his opinion, be related to soil mosture or to organic matter content. This aspect warrants further study. In both Parbery's and our own studies not all soil samples taken beside creosoted poles yielded C. resinae, and several isolates were obtained from what is regarded as virgin soil.

C. resinae does not compete actively with other micro-organisms in a soil plate in the absence of creosote but this does not exclude it from being a soil inhabitor. Jones and Edington (1968) have demonstrated that there is an ecological niche available to hydrocarbon-utilising micro-organisms in soil. According to Parbery (1968) C. resinae can possibly utilise oils, waxes, steroids, terpenes, hydrocarbon and other biochemically related compounds in the soil for which it is able to compete against other micro-organisms. More recently Jones (1970) has reported studies on the origin and distribution page break
Figure 6: Sampling sites for C. resinae in soils in New Zealand. Red spot +ve Black spot —ve

Figure 6: Sampling sites for C. resinae in soils in New Zealand. Red spot +ve Black spot —ve

page break
Figure 7: Sampling sites for C. resinae in soils in Wellington area. Note position of Hirst spore trap. Red spot +ve, black spot —ve.

Figure 7: Sampling sites for C. resinae in soils in Wellington area. Note position of Hirst spore trap. Red spot +ve, black spot —ve.

page 83 of hydrocarbons in an upland moorland soil and underlying shale in Britain. There is a need for studies on the relationship between the occurrence of C. resinae in soils and the nature of hydrocarbons presents. While the distribution of this fungus in New Zealand suggests that the spread of C. resinae is associated with the construction of tar-sealed roads and the use of creosoted timbers in fencing and as power poles, the fungus has been found from time to time in areas remote from roads and from creosoted timbers. In some places at least, C. resinae must have been a natural component of the soil microflora for a very long time. In the near future it is hoped to examine soils from the old kauri forests of New Zealand for the presence of C. resinae. If it is found there this would be evidence to support the suggestion that C. resinae was present in this country before the advent of the jet age; perhaps before the arrival of the white man. This fungus grows readily on kauri gum so it is possible that it will be found in the old kauri forest areas.

Parbery (1969b) reports that of seventeen isolates tested, seven were able to grow in medium with kerosene as the only carbon source. All of the New Zealand soil isolates tested by us were able to grow in medium with aviation turbine kerosene as sole source of carbon but not all isolates from kerosene fuel could do so (Sheridan and Nelson 1971a).

Data on the occurrence of C. resinae in soil and the forms of the fungus found is presented in Table 4.

4. The occurrence of C. resinae in the atmosphere

Christensen et al. (1942) exposed in their laboratory and out-of-doors, creosoted wood blocks and agar containing creosote but failed to recover C. resinae from the air. Nevertheless, they concluded that this did not exclude the possibility of spores being airborne or being present at certain times and places. Neither Marsden (1954) nor Hendey (1964) reported successful isolation from the air. According to Parbery (1969b), Christensen et al. (1942) and Hendey (1964) were of the opinion that spores of the fungus are airborne even though they failed to demonstrate this. Parbery (1969b) reports knowledge of three independent trappings of this mould from the atmosphere which support this view. These are: Anon. (1961) refers to a culture of C. resinae collected from an air filter, Chabert (1968) reports isolating it several times from the atmosphere over Rabat, Morocco, and Dr. H. J. Stewart, University of Melbourne, has given Dr. D. G. Parbery a culture of C. resinae collected from air over Johannesburgh, South Africa. Parbery (1969b) concludes that it is probable that the general failure to trap this mould from the air demonstrates the inadequacy of technique rather than the absence of spores of this mould in the air. Other experiments carried out page 84
Figure 8: Diagrams of Hirst spore trap and air slit sampler. Left: Impactor unit on larger scale; approximately one-third natural size. Arrows indicate how the air circulates.

Figure 8: Diagrams of Hirst spore trap and air slit sampler. Left: Impactor unit on larger scale; approximately one-third natural size. Arrows indicate how the air circulates.

at Rothamsted, England, by J. M. Hirst indicated that freely released spores of C. resinae did not remain viable when they were exposed to light (Hirst, 1971—personal communication).

Harvey (1967; 1971 — personal communication) has isolated C. resinae from the air in Wales in very small quantities, but consistently over a period of years. Parbery (1969b) was apparently not aware of Harvey's work.

We became interested in the possibility that C. resinae was a significant component of the airspora in New Zealand because of its widespread occurrence in soils and profuse sporulation, but all our initial attempts to trap it failed. After some ten months work we eventually perfected a technique which consistently isolated the fungus from the air of our laboratory. The application of the same technique out-of-doors has resulted in numerous trappings. A Hirst spore trap (see Fig. 8) is in use which is capable of continuous operation for twenty-four hours unattended.

Because C. resinae grows readily on a wide variety of agar media containing creosote (Sheridan, Steel and Knox, 1971) and few other fungi (or bacteria) will tolerate the creosote, we experimented with the use of a selective medium containing creosote for the isolation of this fungus. The method described here is a simple one and has proved suitable for isolating C. resinae from the air in New Zealand

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Table 4
The Occurrence of C. Resinae in Soil
Authority Date Source Name Form
Parbery 1967, 1968, 1969 Soils (Australia, Europe, Britain) Cladosporium resinae
(= Amorhpotheca resinae)
f. resinae (occasionally)
f. avellaneum
*
f. albidum) as saltant
f. sterile)
and intermediates
Sheridan et al. 1970, 1971 Soils (New Zealand) C. resinae
(= Amorphotheca resinae)
f. avellaneum
f. resinae)
*
f. albidum) as saltant
f. sterile)
and intermediates

* morphologically similar for f. avellaneum

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(Sheridan and Nelson, 1971b). The selective medium is prepared as follows. Forty grams of Davis agar (other brands should also be suitable) is added to 1,500 ml. tap water in a glass vessel which is heated in a boiling water bath until the agar melts. To this is added 350ml. of V-8 juice (Campbell's Soups Ltd.). The medium is mixed gently, distributed into medicine flats and autoclaved at 103 kNm−2 (15 p.s.i.) for 10 minutes. When the medium has cooled to about 50°C. sterile creosote (sterilised by autoclaving) is added aseptically to give a final concentration of 0.1%. The concentration is not critical since all strains of the fungus studied by us will tolerate creosote up to 1%. However, a lower concentration is undesirable because the volatile components of the creosote disappear rather rapidly when plates containing the selective medium are exposed to the air.

Plates of this selective medium are exposed in an Air Slit Sampler (see Fig. 8) with an intake of 18 litres of air per minute, for periods of up to 30 minutes. An exposure of less than 30 minutes is uneconomical; a longer exposure results in growth of contaminants and unreliable results. After exposure, plates are incubated at 25° C. for 5 days. Colonies of the fungus can be recognised visually and by the characteristic smell produced on this medium and confirmed by microscopic examination (see Figs. 2 and 5).

For continuous monitoring of the air we use a Hirst Spore Trap (see Fig. 8) operating 2 metres above the ground with an intake of 10 litres/min., carrying a microscope slide coated with vaseline. When the slide is removed after exposure it is placed into a sterile Petri dish and sterile selective medium at 50° C. is gently poured over the slide. These preparations are incubated as above and colonies identified after 5 days.

Because the volume of air taken into the trap and the rate of movement of the slide past the orifice is known it is possible (assuming that each colony develops from a single spore) to determine the concentration of C. resinae in the air and the time at which the spore landed on the slide (Fig. 9).

For selective isolation of the fungus from air at oil installations, airports and on aircraft during flight, trappings are made on a sterile glass fibre paper in a Personal Dust Sampler (Figs. 10 and 11). After each run (1-8 hrs.) the paper is removed aseptically and placed inside a sterile disposable plastic universal container for transport to the laboratory. On arrival it is placed inside a sterile Petri dish, correct side up, and selective medium at 50° C. is poured over it. Alternatively, 5 ml. of the medium may be added to the universal container. Incubation is as above.

Results of Air Trappings Over Wellington

1. Using the Air Slit Sampler
Results are shown in Table 5 for trappings during November and December, 1970. During 216 hours trapping. 11 colonies were page 87
Figure 9: Colonies of C. resinae (arrowed), trapped from the air, after five days on V-8 juice agar containing 0.1% creosote. Hirst spore trap.

Figure 9: Colonies of C. resinae (arrowed), trapped from the air, after five days on V-8 juice agar containing 0.1% creosote. Hirst spore trap.

Figure 10: Colonies of C. resinae, trapped from the air, after five days on V-8 juice agar containing 0.1% creosote. Personal dust sampler.

Figure 10: Colonies of C. resinae, trapped from the air, after five days on V-8 juice agar containing 0.1% creosote. Personal dust sampler.

page 88 obtained. No colonies were obtained during 92 hours trapping at the University Field Station, Taurewa, National Park, from January 5, 1971, until January 20, 1971, nor during 67 hours at Brooklyn, between March 10 and March 16, 1971. This method is laborious because the trap requires attention at approximately half-hour intervals, is not suitable for wet-weather operation, and night work is tedious. The Hirst spore trap is far superior for continuous monitoring of the air.
Table 5
Data on Spore Trappings From Air: Air Slit Sampler
Location Date Time No. of Spores of C. resinae trapped Weather
1970
Brooklyn Wellington Nov. 21 9.00-9.30 p.m. 1 Dry, mild, It. sthly.
Nov. 21 9.30-10.00 p.m. 1 Dry, mild, It. sthly.
Dec 6 9.15-9.45 a.m. 1 (Dry, very warm
9.45—10.15 a.m. 1 (Gusty, nthly.
Dec. 7 9.45-11.30 a.m. 1 Dry, mild, calm
Dec. 8 8.45-9.00 p.m. 1 Dry, mild, It. sthly.
Dec. 25 12.30-2.00 p.m. 3 Dry, overcast, mild, gusty nthly.
Dec. 29 5.30-6.00 p.m. 1 Dry, very hot, calm
Dec. 30 1.20-2.00 p.m. 1 Dry, hot, calm
2. Using the Hirst Spore Trap

This spore trap has been operating continuously, day and night, in Brooklyn since March 14, 1971 (see Fig. 7 for location). Up to July 21, 1971, 26 isolations of C. resinae have been made. Eleven were trapped during dry weather, 4 of the remaining 15 during heavy rain; 12 were trapped during daylight, 14 were trapped during darkness (Table 6). There is thus little evidence for the deleterious effect of light or for a preference for wet weather in dispersal. Presumably the spores trapped originated from the soil since the trap is operating in a region where the fungus is known to exist in the soil. Seagulls and other birds may also be implicated since the fungus has been isolated from feathers. (See 5. Occurrence of C. resinae in other habitats.) This work is continuing, but there is now little doubt that C. resinae forms a significant component of the air spora over Wellington.

3. Using the Personal Dust Sampler

Monitoring of the air of our laboratory at intervals from April to June, 1971, using this sampler has shown a concentration of C. resinae from 0 to 12 per cu. metre with an average of 6 (Table 7). The higher concentration occurred when we were harvesting C. resinae from kerosene in experiments to determine dry weights. Only one trapping has been made with this sampler from the air at

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Table 6
Data on Spore Trappings From the Air: Hirst Spore Trap
Location Date Time No. of Spores of C. resinae trapped Weather
1971
Brooklyn March 23 11.00 p.m. 1 Dry
Wellington March 26 7.00 a.m. 1 Showery
March 30 9.00 a.m. 1 Dry
March 31 3.00 a.m. 1 Showery
April 1 8.00 p.m. 1 Dry
April 3 11.00 p.m. 1 Dry
April 6 2.00 a.m. 1 Dry
April 12 (8.00 a.m. 2 Dry
(10.00 a.m.
April 22 7.00 a.m. 1 Heavy rain
April 24 2.30 p.m. 2 Dry
7.00 p.m.
May 3 2.30 p.m. 2 Drizzle
May 5 9.00 p.m. 1 Heavy rain
May 9 9.00 a.m. 1 Showery, stormy
May 12 12.30 a.m. 1 Dry
May 14 3.30 p.m. 1 Showery
May 16 8.00 a.m. 1 Heavy rain
May 19 9.00 p.m. 1 Showery
May 24 6.30 p.m. 1 Heavy rain
May 28 7.00 p.m. 1 Dry
June 7 11.00 p.m. 1 Rain
June 12 8.00 p.m. 1 Showery
July 2 5.00 a.m. 1 Showery, very cold
July 4 12.00 midnight 1 Showery, very cold
July 7 8.00 p.m. 1 Showery, very cold

Karori (23/5/71) and one at the University Field Station, Taurewa, National Park (20/5/71). No fungus has been isolated yet from Wellington Airport, on aircraft or at oil installations in the few tests so far run. Further work is in progress.

Discussion

The key factor in the isolation of C. resinae from the air is the use of a selective medium. V-8 juice agar containing 0.1% creosote has proved ideal. The acid medium suppresses growth of bacteria, and the creosote suppresses growth of most fungi. Certain precautions must be taken to avoid contamination with C. resinae from within the laboratory. Where possible, preparations of materials, pouring of media and incubation should be done in a place remote from the laboratory. In recent tests the air of our laboratory yielded an average of six spores (assuming each colony arises from a single spore) per cubic metre. The air above Wellington (at Brooklyn) during a trapping schedule in November and December, 1970, yielded 1 spore per 20 cu. m.

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Table 7
Data on Spore Trappings From Air: Personal Dust Sampler
Location Date Duration of trapping Time at which C. resinae trapped No. of Spores of C. resinae trapped
1971
Our Laboratory April 5 8 hours 9.30-10.30 a.m. 1
" April 22 8 hours
" April 27 4 hours
" April 28 9 hours 11.45 a.m.-2.00 p.m. 2
2.00-4.45 p.m. 3
" May 25 5 hours 12 noon-2.00 p.m. 1
2.00-5.00 p.m. 1
" May 26 9 hours 8.00-10.00 a.m. 5
10.00 a.m.-1.00 p.m. 4
1.00-3.30 p.m. 1
3.30 p.m.-5.00 p.m. 2
" May 27 7 hours 9.00-11.00 a.m. 4
11.00 a.m.-1. p.m. 8
3.30-4.00 p.m. 1
" May 31 8 hours 8.00-11.45 a.m. 4
11.45 a.m.-2.30 p.m. 2
2.30-4.30 p.m. 4
" June 1 8½ hours 8.30-10.30 a.m. 1
10.30a.m.-12.30 p.m. 3
12.30-2.00 p.m. 1
2.00-5.00 p.m. 2
" June 2 5 hours 9.00-11.00 a.m. 1
11.00 a.m.-1.00 p.m. 1
1.00-2.00 p.m. 2
" June 9 7½ hours 8.45-10.00 a.m. 3
10.00 a.m.-12.00 noon 1
12.00 noon-2.00 p.m. 1
2.00-4.00 p.m. 1
Totals 78¾ hours 60

Each of the spore traps serves a different purpose. The Hirst spore trap is ideal for continuous monitoring. The personal dust sampler has proved useful for monitoring air inside aircraft, at airports and at oil installations. The air slit sampler is used as a standard against which the others are compared periodically to check on their efficiency.

Only f. avellaneum has been so far isolated from the atmosphere by us. This is not surprising since f. albidum has not been found in soil and f. resinae is only occasionally isolated from soil (Parbery, 1968). We have not isolated f. resinae directly from soil in New Zealand. Further, this form sporulates sparsely and spores are difficult to dislodge.

The ease and regularity with which C. resinae is isolated from the air over Wellington makes one wonder how this fungus has eluded page 91
Figure 11: The personal dust sampler.

Figure 11: The personal dust sampler.

the earlier workers in their attempts to isolate it from the air. There would appear to be two reasons: firstly spores of C. resinae are not readily distinguishable from those of other species of Cladosporium and secondly in the absence of creosote vapour contaminating fungi quickly over-run C. resinae. With suitable techniques now available the distribution of C. resinae in the air throughout the world should soon be found. Data at present available on the occurrence of C. resinae in the atmosphere is collected in Table 8.

How to Make a Simple, Efficient Sampler for Trapping C. Resinae from the Air

Where none of the three traps mentioned here is available a simple, cheap and efficient substitute can be made using an old page 92
Figure 12: Apparatus for air sampling for C. resinae constructed from a vacuum cleaner and pieces of plastic water pipe (Belinda’) Approximately one-quarter natural size.

Figure 12: Apparatus for air sampling for C. resinae constructed from a vacuum cleaner and pieces of plastic water pipe (Belinda’) Approximately one-quarter natural size.

vacuum cleaner and pieces of plastic water pipe. A wire grid is cemented into the pipe to support a fibre or filter paper disc (= collecting pad), 47 cm. diameter (see Fig. 12). Air is sucked through the collecting pad for preselected periods. After exposure the disc is placed inside a sterile Petri dish and V-8 juice agar containing 0.1% creosote is poured over it as described above for the personal dust sampler. The volume of air per minute sucked into the trap can be measured, using a domestic gas meter. Our sampler, ‘Belinda’, has an intake of approximately 160 litres/min.

5. The occurrence of C. resinae in other habitats

Apart from its occurrence in kerosene, petrol, creosoted timbers, asphalt, soils, and in the air, C. resinae has been isolated from a cosmetic face cream (de Vries, 1952) bituminised cardboard (Anon., 1968), the female sex hormone, progesterone (Fonken, Murray and Reineke, 1964), methyl-p-hydroxybenzoate (Sokolski, Chichester and Honeywell, 1965) and feathers of birds (Sheridan, 1971) (Table 9). In connection with this last source we have made two isolations from chicken feathers and ten from seagull feathers collected in Wellington. The feathers were picked up from the ground or on the beach, cut to size and placed on V-8 juice agar containing 0.1% creosote, in Petri dishes, and incubated at 25° C. for five days. In studies on the dissemination of fungi by migratory birds, Warner and French (1970) have recovered viable spores of C. resinae as long as forty-five days after being applied to birds. Further, Parbery (1969) has found feathers to be an excellent substrate for growth of some of his soil isolates of the fungus. It is anticipated, therefore, that C. resinae will be found, on investigation, to occur on feathers in many countries.

It has been estimated that more than 150 gallons of jet fuel are released into the atmosphere each day from aircraft operating over London (Anon., 1970). This ‘Heathrow Dew’ could conceivably

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Table 8
The Occurrence of C. Resinae in The Atmosphere
Authority Date Source Name Form
Anon. 1961 * air filter C. resinae
Harvey 1967 atmosphere (over Cardiff, Wales) C. resinae
Chabert 1968 atmosphere (over Rabat, Morocco) C. resinae
(Parbery) 1969 * atmosphere (over Johannesburg, S. Africa) C. resinae
Sheridan et al. 1971 atmosphere (over Wellington, New Zealand) C. resinae f. avellaneum

* One isolate only

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Table 9
The Occurrence of C. Resinae in Habitats Other Than Creosoted Timbers, Kerosene-Type Fuels, Soil and Air
Authority Date Source Name Form
De Vries 1952 Cosmetic Face Cream ** Cladosporium resinae f. avellaneum
f. resinae
f. albidum
f. sterile
from a single culture spore
Fonken, Murray and Reineke 1960 Female Sex Hormone, Progesterone Cladosporium resinae
Anon. 1964 Bituminised Cardboard Cladosporium resinae f. albidum
Sokolski, Chidester and Honeywel 1965 Methyl-p-hydroxybenzoate Cladosporium resinae
* Warner and French 1970 Feathers (North America) Cladosporium resinae
Sheridan 1971 Feathers (New Zealand) Cladosporium resinae f. avellaneum

** Called Cladosporium avellaneum in 1952: later renamed C. resinae.

* Applied to birds and then recovered 45 days later.

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contaminate birds, particularly seagulls which are often present in great numbers near airports, and C. resinae might be thus deposited on their feathers. Also these birds might pick up spores of C. resinae from the air (See 4. The occurrence of C. resinae in air) and the kerosene select in its favour. Birds may, therefore, be of some significance in spreading C. resinae from place to place.

The forms isolated from the cosmetic face cream (De Vries, 1955) were C. resinae f. avellaneum and C. resinae f. resinae; from bituminised cardboard, from a gold mine in South Africa, C. resinae f. albidum. Only f. avellaneum has been isolated by us from feathers.

When the white settlers first arrived in New Zealand last century they were struck by the majesty of the magnificent kauri forests (Agathis australis) in the north of the North Island. The trees proved very valuable as a source of durable timber with the result that only a few thousand acres of trees remain. C. resinae grows readily on kauri gum and it is possible that the fungus occurs naturally in soils in the old kauri forests. We are looking into this but have not yet been able to visit these areas to procure soil samples.

6. Summary and conclusions

The ‘kerosene fungus’, Cladosporium resinae, is widespread as a contaminant of kerosene-type fuels, occurs frequently on creosoted timbers, is widely distributed in soil, occurs on feathers and in the air and has been reported from other habitats. There is little doubt that the soil constitutes the natural habitat of this rather extraordinary fungus. From here the fungus can contaminate power poles and fence posts, being selected for by creosote, and enter fuel supply systems with contaminating soil. Airborne spores can, no doubt, find their way into fuel storage and aircraft tanks. The form most frequently isolated from soil, f. avellaneum, is the one most frequently isolated from creosoted timbers, fuels and air. This is to be expected because of its profuse sporulation and ease with which these spores become airborne. The albino, f. albidum, has only been found in culture, with one exception (Anon., 1968), but there appears to be no reason why it should not occur in nature since it can grow readily in kerosene and in the presence of creosote, and sporulates as profusely as f. avellaneum.

The other form, f. resinae, is only occasionally isolated from soil but because it does not sporulate as freely as the other two forms and because the spores are difficult to dislodge from the conidiophores its spread will be restricted. C. resinae f. sterile and intermediate forms have been found in culture by Parbery (1969a) and by us.

Now that reliable methods are available for isolating C. resinae from soil and air new knowledge about the distribution of this fungus will accumulate rapidly.

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Acknowledgments

The work which was carried out in our laboratories was supported in part by grants from the Victoria University of Wellington Internal Research Committee. The Hirst spore trap was purchased with a U.G.C. grant. We are grateful to the Chief Superintendent of the Australian Defence Standards Laboratories for permission to quote data contained in D.S.L. Report No. 252 and to the Operations Manager of N.A.C. for permission to sample the air at airports and on aircraft in New Zealand. Thanks are also due to members of the Botany and Zoology Department of this university and to everyone else who assisted in the soil survey. Mr. Ron Hoverd constructed the apparatus shown in Fig. 13.

(References will be combined with those for Part III.)