Contents

Introduction

Introduction

The Sexual (perfect or ascal) state of the ‘kerosene fungus’ was first described by Parbery in 1969. He erected a new family and genus to accommodate it and named this fungus Amorphotheca resinae Parbery (1969a). Almost thirty years earlier Christensen et al. (1942) described what they called ‘sclerotia’ within the agar of six-week-to-two-month old cultures. These were probably sterile sexual fruiting bodies (ascocarps). The occurrence of the sexual state in nature or in pure culture is rare (Parbery, 1969a; Sheridan, Steel and Knox, 1971) but when cultures are stored under mineral oil mature ascocarps often develop (Sheridan and Steel, 1971). Morphology of ascocarps produced on creosoted matchsticks and under mineral oil, and mode of development are apparently unique (Parbery, 1969a; Sheridan and Steel, 1971).

Four forms of the asexual (imperfect or conidial) state have been described (de Vries, 1952; Parbery, 1969a); f. avellaneum which is the most common, f. resinae (= f. viride), f. albidum and f. sterile. De Vries (1952) obtained all four from a single spore culture of the fungus. He prepared monospore cultures of the first three mentioned page 131

Fig. 1: Typical asexual reproductive structures of f. avellaneum (left) and f. resinae.

forms and he found these to be constant over a number of years. Later workers (Parbery, 1969a; Sheridan, Steel and Knox, 1971) have found f. resinae and f. albidum arising from f. avellaneum in culture on a number of occasions; f. resinae may also give rise to f. avellaneum. (Tan, unpublished). The avellaneum and resinae form are morphologically distinct (Fig. 1), but sometimes they arise side by side from the same hypha in slide cultures on malt agar (Ellis, 1971—personal communication). Intermediates occur and the asexual state of this fungus is considered to be very variable (Hendey, 1964; Parbery, 1969a). De Vires (1952) described f. albidum as being morpholo gically similar to f. resinae. Other workers have found an albino morphologically similar to f. avellaneum (Parbery, 1969a; Sheridan, Steel and Knox, 1971). All forms apparently have the same perfect state Amorphotheca resinae Parbery (Parbery, 1969a).

A knowledge of the fine structure of this fungus may throw light on its variability and reproduction. However, nothing has yet been published on this aspect as far as we are aware. We have studied the surface morphology of all forms, except f. sterile, in the scanning electron microscope (Sheridan and Troughton, in preparation) and the internal structure of f. avellaneum and f. resinae in the transmission electron microscope (Tan, 1972). Results of these studies will be discussed in this paper in an attempt to elucidate the problems concerning the variability, reproduction and recognition of this fungus.

De Vries (1955) made the combination Cladosporium resinae (Lindau) de Vries for the asexual state and Hendey (1964) applied the name ‘kerosene fungus’ to this organism. Although the name C. resinae has subsequently been widely used not all research workers have accepted it. The reasons for this appear to arise from its morphological and physiological variability and lack of knowledge of its occurrence and role in nature. This last aspect has been reviewed in Part II of these studies. As a result of an intensive study of this fungus in its natural habitat, the soil, and in pure culture Parbery (1969a) concluded that it is correctly placed in the form-genus Cladosporium. This is in agreement with Nicot and Zakartchenko (1966) and Sheridan, Steel and Knox (1971). Now that soil isolates of the fungus collected in a number of different parts of the world (Australia, England, Wales and parts of Europe by Parbery, 1969a; New Zealand, by Sheridan, Steel and Knox, 1971; England and Ireland by Sheridan, unpublished) and air isolates and feather isolates from New Zealand (Sheridan, 1971) are available for study in addition to fuel isolates also from many parts of the world, new knowledge is rapidly accumulating. In the light of this new knowledge the name of the ‘kerosene fungus’ and its taxonomic position is discussed in this paper.

Apart from reports that kerosene may be used as a sole carbon source and that the optimum temperature for growth in kerosene and in pure culture is close to 30° C. (Hazzard, 1963; Sheridan, Steel and Knox, 1971; Parbery, 1971a) very little has been published in the open scientific literature in relation to the nutrition of the ‘kerosene fungus’ and chemical and physical factors affecting its growth in culture, soil or fuel. There may, however, be valuable contributions to knowledge of this aspect contained in technical reports which we have been unable to procure or the existence of which is unknown to us. In a recent article Parbery (1971a) reports that in pure culture work the shape and size of the flask influences growth and that shaking has a deleterious effect on growth. Different isolates have different temperature requirements. Different isolates also show different growth rates in kerosene (Sheridan and Nelson, 1971a) and many different strains of the fungus possibly exist. Nothing is known about the effect of light on growth and reproduction. Over the past two years an extensive study of the nutrition of local isolates of the fungus has been made in our laboratory, and the growth in kerosene of isolates collected from different parts of the world is currently being studied. Our findings will be discussed in so far as they throw additional light on the physiology of this fungus.

1. Morphology

(a) The sexual (perfect) state

The discovery of the sexual (perfect or ascal) state of the ‘kerosene fungus’ was reported in 1968 (Parbery and Ford, 1968) and a page 133 full description published in 1969 (Parbery, 1969a). Initially, mature ascocarps were produced only in paired cultures of isolates from south-eastern Australia which suggested that the fungus was heterothallic. Subsequently several unpaired isolates collected in Britain, France and Germany, as well as some other Australian isolates, produced fertile ascocarps. On Bushnell-Haas or malt agar 6-8 weeks’ incubation was necessary for the development of mature ascocarps while on modified Weitzman and Silva-Hutner agar some isolates produced fertile ascocarps in 14-20 days (Parbery, 1969a). A few New Zealand isolates produced mature ascocarps on V-8 juice agar in 4-8 weeks at normal laboratory temperatures but this was a rare occurrence (Sheridan, Steel and Knox, 1971) and in general only the asexual state was produced. When cultures on 2% malt extract agar were stored under mineral oil (B.P. quality, Sp.gr. 0.870-0.890) mature ascocarps were present, in many isolates, in the oil after five months (Sheridan and Steel, 1971). V-8 juice agar has also been successfully used (Sheridan, unpublished). Mature ascocarps were found on soil and on creosoted matchsticks in Australia (Parbery, 1969c) but not in New Zealand (Sheridan, Steel and Knox, 1971).

In culture ascocarps appear as very small, black, more or less spherical bodies, 71-128 micron high by 43-86 micron wide, usually immersed in the medium (Sheridan, Steel and Knox, 1971) whereas on soil and creosoted matchsticks in some cases they produce flanged or funnel-shaped outgrowths from their apical regions (Parbery, 1969c). Parbery (1969c) has suggested that this funnel-shaped apex is not normally produced by the ascocarp in nature but appears as a result of development at high humidity. The majority of ascocarps, in any one isolate, produced under mineral oil possess funnel-shaped apices (Fig. 2). These appear to originate as blown-out, spherical portions of the ascocarp, the weakest point being that furthest from the ascocarp body. They rupture at this point, giving a funnel-shaped appearance. The internal wall of the funnel appears to be ornamented (Fig. 3). In many cases asci containing ascocarps can be seen through the semi-transparent wall of the immature ascocarp (Fig. 4).

Mature ascocarps measured 139 micron high by 80 micron wide. The funnel-shaped apex ranged from 27 to 110 micron wide by 24 to 71 micron high. The narrowest part between apex and body of the ascocarp ranged from 20 to 68 micron wide. An isolate producing spherical ascocarps in culture has produced funnel-shaped apices on ascocarps in mineral oil. It appears, therefore, that ascocarps with funnel-shaped apices are the natural form. No other fungus is known to produce this type of ascocarp. Some of our New Zealand isolates have produced atypical ascocarp-like bodies which were very much elongated and some of which carried coarse, black hairs in the early stages of development (Fig. 5). No asci or ascospores have been found inside these bodies.

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Fig. 2: Ascocarps. Top: from agar medium. Bottom from mineral oil.

Gametangia consist of a spirally coiled ascogonium enveloping a simple, cylindric, antheridium (Fig. 6). Parbery (1969a) has followed the development of the ascocarp. He describes an amorphous peridium composed of a melanoid substance apparently deposited around the sphere of hyphae within which the asci develop. He has not observed the development of the ascogenous hyphae from the page 135

Fig. 3: Pits inside apical funnel of ascocarp.
Fig. 4: Asci within immature ascocarp.
Fig. 5: Atypical, sterile, ascocarp-like body.

page 136 ascogonium. There does not appear to be a true ostiole in the mature ascocarp. The mode of ascocarp development is believed to be quite unlike that of any other ascomycete (Parbery, 1969a).

Fig. 6: a. Ascogonium. b. Radiating hyphae around ascogonia.

Asci are scattered within the ascocarp and contain 8 one-celled, ellipsoid, hyaline to light-brown, ascospores. Asci from New Zealand isolates measured 15-26 micron by 10-15 micron and ascospores measured 5-10 micron by 3.2-5.4 micron (Fig. 7).

Ascospores appear to be reluctant to germinate. Parbery (1969a) reports that of 21 single spore isolations only 4 germinated. We have also obtained poor germination (Sheridan, Steel and Knox, 1971). The reason for this reluctance to germinate is unknown and this warrants further study. Those that germinated, in the above-mentioned case, produced colonies which gave rise to the asexual state.

Ascocarp-like bodies and appressorium-like bodies have been observed by us in old cultures of the fungus in aviation kerosene but neither asci nor ascospores have been seen. The demonstration of mature, fertile, ascocarps in kerosene would be of considerable interest. Parbery's diagnosis of Amorphotheca resinae is reproduced below.

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Fig. 7: Asci and ascospores.

Amorphotheca resinae Parbery (Parbery, 1969a)

‘Ascocarps in culture usually embedded in medium, sometimes superficial, on finger nails and snake skin superficial, dark brown to black, globose to subglobose, 52-112 micron high by 40-90 micron wide; peridium a melanoid membrane, dark brown, amorphous, sometimes incorporating some hyphae, 5-20 micron thick, often uneven in thickness, aerial portions of superficial ascocarps thinner than embedded portions; ostiole absent; asci up to 8-spored, subglobose to pyriform, hyaline, lyse at maturity; 10-15 micron wide by 12-27 micron long; ascospores 1-celled, ellipsoid to slightly naviculoid, hyaline becoming light brown, occasionally dark, often biguttulate, inner membrane constricted, 3.5-5.5 micron wide by 7-9.5 micron long (most commonly 4 by 8 micron). Conidial state polymorphic and variable. Extreme types apparently genetically fixed.

‘Type specimen: From soil, south-eastern Australia, MELU 7130 (= IMI, 129861 X 129862) (= D.G.P. W1.1 X V2.0).’

(b) The asexual (imperfect) state

Four forms of the asexual state have been described (de Vries, 1952, 1955). Two of these are morphologically distinct (see Fig. 1); f. avellaneum is a typical Cladosporium, f. resinae is very similar to a paniculate Septonema or Xylohypha but differs from the former in having one-celled conidia and from the latter in the lighter colour of the conidia (see Hughes, 1953). According to Parbery (1969a) both forms produce the same perfect state Amorphotheca resinae. We have also found gametangia and mature ascocarps in our New Zealand isolates of both forms. The albino, f. albidum, was described by de Vries (1955) as being of the resinae form; our New Zealand isolates are of the avellaneum form (Sheridan, Steel and Knox, 1971) while Parbery (1969a) states that the albino can be of either morphology. Recently, however, we have found one albino saltant in culture morphologically similar to f. resinae (unpublished). A fourth page 138 form, f. sterile, was described by de Vries (1955) as being a pigmented sterile saltant. Parbery (1969a) found both pigmented and white sterile forms. The forms found by us are pigmented.

Fig. 8: Colonies of three forms of C. resinae growing on V-8 juice agar.
Fig. 9: Colony of f. resinae showing sectoring.

De Vries (1952) described colonies of f. avellaneum as greyish brown and colonies of f. resinae as dark olivaceous green. However, according to Parbery (1969a) there is no correlation between form and colour. Our two basic forms are distinct in colour and colony appearance on V-8 juice agar medium (Fig. 8). Colonies of f. avellaneum are hazel to brown, sometimes with an orange tint developing with age; a narrow white margin is often present, there is little or no aerial mycelium and the colonies sporulate profusely. Sometimes coremia are present. Colonies of f. resinae are darker brown in colour generally with an olivaceous green tint; they produce copious mycelium, never have a white margin to the colony, sometimes grow more slowly than f. avellaneum, sector frequently and sporulate less profusely (Fig. 9).

The colour of colonies of these two forms depends to some extent on culture medium and is generally darker on malt agar than on V-8 juice agar. Colonies of f. albidum are white on all media.

Typically f. avellaneum produces unbranched or sparsely branched, dark, septate, warted, conidiophores each with a mass of one-celled, ellipsoid to oval, blastospores (up to 1,000) borne on ramoconidia (Fig. 10a). The sporulating structure at the apex of the conidiophore is very compact. In contrast f. resinae produces branched conidiophores page 139

Fig. 10 a. Typicial f. avellaneum. b. Intermediate. c. Typical f. resinae.

page 140 (dark and warted) which themselves may become divided into spore-like cells, and long chains of one-celled, ellipsoid to elongate blastospores (Fig. 10c). No true ramoconidia are produced. The whole structure is very open and has been described as paniculate although dendritic is probably a better term.

Fig. 11: ‘Foot-cell’ produced by f. avellaneum on creosoted matchsticks.
Fig. 12: Hyphae fusion in f. avellaneum.
Fig. 13: Hyphae developing in place of spores in f. resinae.

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Intermediates in morphology and colour have been reported by Parbery (1969a). When we examined de Vries's type of f. avellaneum on which Parbery (1969a) has also based his description, we found that in colour and other colony characters it resembled our f. resinae. It produced the open, dendritic, asexual reproductive structures typical of this form but also both ramoconidia and blastospores typical of f. avellaneum (Fig. 10b and c). It is considered by us to be intermediate but very close to f. resinae. On passage through kerosene this isolate changed back to a form close to f. avellaneum. Hendey (1964) has found a variant which corresponds very closely to f. resinae but differs in colour, being light fawn with paler margins, almost identical in colour with f. avellaneum. We have also found one such isolate. Because of this variability Hendey is of the opinion that it is unlikely that these forms have any real taxonomic significance and that the subspecific epithets are of use only to facilitate reference to the different growth forms (see 3 Taxonomy).

Most of our soil isolates of f. avellaneum produced a ‘foot-cell’ when growing on creosoted matchsticks and sometimes on agar (Fig. 11). Parbery (1969a) has also observed this but states that it is not common. The significance of the foot-cell is unknown. Hyphal fusion is of common occurrence in both f. avellaneum and f. resinae (Fig. 12).

The majority of our New Zealand isolates, which are morphologically f. avellaneum, also produce conidia directly on undifferentiated hyphae in pure culture; f. resinae has not been observed to do this. No setae have been seen in f. resinae as reported by de Vries (1952), but in both forms a hypha may sometimes develop in place of a spore (Fig. 13).

The form most commonly encountered in soils, fuels and air is f. avellaneum. Parbery (1969a) gives the frequency of isolation from soil of the various forms as follows: f. avellaneum 80%, f. resinae 4% f. albidum 2% (not directly but as a saltant from f. avellaneum), f. sterile 2% (one culture as a saltant from f. albidum) and intermediate types 12%. We have isolated only f. avellaneum directly from soils, fuels, air and feathers in New Zealand: all the other forms have arisen in culture.

Because the type material of f. avellaneum (IMI 49620) is no longer true to type and because Parbery (1969a) based his description on it we have redescribed this form together with f. resinae and f. albidum from material isolated in our laboratory and originating from soil. We should point out that there is no guarantee that any of the forms described here will be stable for long periods of time. Our stocks are stored on V-8 juice agar under mineral oil.

C. resinae f. avallaneum (description based on Cl, = IMI 145195)

Cultural characters, on V-8 juice agar plates, after 5 days at 25° C. Colony powdery, due to profuse sporulation, with little aerial page 142 mycelium, hazel to brown in colour with a white margin, mean diam. 33 millimetres. Sometimes pointed coremia are produced. Reverse darker brown with whitish margin. When 0.1% creosote is added to the medium the colonies are ashy-brown with a less distinct margin, and a strong characteristic smell is produced. Sporulating structures are very compact.

Conidiophores. On creosoted matchsticks, erect, unbranched, stout, dark, septate, warted often with a distinct ‘foot-cell’, 112-225 micron long but sometimes up to 1 millimetre, X 4.8-6.4 micron wide at the base and 3.0-4.0 micron at the apex. On V-8 juice agar similar but sometimes not warted and only occasionally with a foot-cell’. Ramoconidia numerous, and blastospores up to 1.000 in short chains. Conidial heads ashy-grey to brown. On soil and in culture often more slender conidiophores are produced which appear little different from hyphae.

Ramoconidia. One-celled, smooth or warted, 9.7-12.9 micron long X 3.2-3.8 micron wide, usually with 3 projections.

Blastospores. Ellipsoid to oval, one-celled, smooth, brown en masse, 6.4 X 3.2 micron.

C. resinae f. resinae (description based on R1, = IMI 159585)

Cultural characters, on V-8 juice agar plates, after 5 days at 25° C. Colony woolly with much aerial mycelium, dark brown with olivaceous green tint, lacking white margin, few spores, diam. 33 millimetres. Coremia absent. Reverse similar in colour to surface. When 0.1% creosote is added to the medium the same characteristic smell as that produced by f. avellaneum is noticeable — after a number of weeks the smell is very strong. Sporulating structures very open.

Conidiophores. On V-8 juice agar, dendritic or paniculate, horizontal to erect, several times branched towards the apex, dark, septate, smooth or warted, up to 1 or 2 millimetres long by 2-4 micron wide. Conidia blastospores, produced in long chains, one-celled, ellipsoid to elongate-cylindric, smooth, brown, 3.5-15 micron X 2.5 micron. Also large, brown, warted, spore-like cells towards top of conidio-phore, 10-37 micron X 2.5 micron. Where these give rise to first blastospores, the cell resembles a ramoconidium. Sporulating portions of colony lighter in colour than rest of colony.

C. resinae f. albidum

(1) avellaneum form (description based on C31W — 1M1 159589) Cultural characters, on V-8 juice-agar plates, after 5 days at 25° C.: colony powdery due to profuse sporulation, little aerial mycelium, pure white, diam. 33 millimetres, strong characteristic smell produced.

Conidiophores and conidia as for f. avellaneum.

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(2) resinae form (description based on ‘albino-resinae form,) Cultural characters, on V-8 juice-agar plates, and smell, similar to the avellaneum form.

Conidiophores and conidia as for f. resinae.

The conidiophores and conidia of both (1) and (2) appear to be more fragile than those of the pigmented forms. No surface ornamentation has been seen in the light microscope; in the scanning eelctron microscope rather indefinite wart-like structures have been seen but further studies are needed.

(c) Fine structure

Apart from de Vries's (1952) statement that hyphae of f. avellaneum are uninucleate little is known about the nuclear state of hyphae or asexual spores and nothing about the nuclear state of the sexual spores (ascospores) of the ‘kerosene fungus’. Parbery (1969a) thinks it is likely that the asexual spores and the parent mycelium can be heterokaryotic. Our initial attempts to clearly demonstrate the nuclear condition of mycelium and spores using the usual nuclear staining techniques have failed. However, in electron microscope studies we have invariably found only one nucleus in asexual spores of f. avellaneum (blastospores) and it appears from these studies that each blastospore has only one nucleus, many mitochondria and, in most cases, a vacuole (Fig. 14). Unidentified membrane-bound bodies have been seen in some electron micrographs (Fig. 14a—arrowed). The cell wall and the outer membrane appear to be unusually thick. Much more work needs to be done on the fine structure of this fungus especially in view of recent reports that all methane-utilising bacteria so far examined possess a complex internal membrane system (Davies and Whittenbury, 1970; Whittenbury, 1969).

If all spores are uninucleate the occurrence of other forms of the fungus in single spore cultures cannot be due to heterokaryon formation but would appear to be due to a mutation. One would, therefore, expect these forms to be stable.

The external morphology of three forms isolated in New Zealand, f. avellaneum, f. resinae and f. albidum, has been studied in the scanning electron microscope (stereoscan at P.E.L., D.S.I.R.: this work carried out by Dr. John Troughton). Mature conidiophores and ramoconidia of all isolates of f. avellaneum so far examined are coarsely warted while the blastospores show no surface ornamentation at all (Fig. 15). Although f. resinae has proved more difficult to handle, blastospores appear to be smooth while the conidiophores, and spore-like cells of the conidiophores, are warted as in f. avellaneum (Sheridan and Troughton — in preparation). The albinos of both forms do not possess conspicuous warts. The lack of any surface ornamentation on the blastospores indicates that little help will page 144 be obtained from these studies in elucidating taxonomic problems (see 3 Taxonomy). The ascospores are currently being studied.

Fig. 14: Electron micrograph showing single nucleus and many mitochondria in blastospore of f. avellaneum.

2. Taxonomy

Lindau (1907) described a fungus which he isolated from resin of Picea excelsa and named Hormodendrum resinae Lindau. De Vries page 145 (1952) named a fungus which he isolated from Nivea ointment Cladosporium avellaneum; later (1955) he found that H. resinae and C. avellaneum were identical and made the new combination Cladosporium resinae (Lindau) de Vries.

Fig. 14a: Note zigzag membrane, nucleus, vacuole, many mitochondria, microtubules and two unidentified bodies (arrowed).

Since its introduction in 1955, the name C. resinae has been widely accepted but the earlier name Hormodendrum resinae has continued to appear in the literature. De Vries himself had noted that this page 146 fungus differs in some respects from other species of Cladosporium and placed some reserve on his decision. These differences are both morphological (two distinct forms produced by C. resinae and setae produced by one of these forms) and physiological.

Fig. 15: Ornamentation on conidiophore and ramoconidum of f. avellaneum as seen in the scanning electron microscope.

The genus Cladosporium was founded by Link in 1816, and was studied by Fries (1829) and Corda (1837). However, the original Latin descriptions of these earliest workers are too brief and indefinite for precision. Janczewski (1894) reported that in C. herbarum Fries after the terminal formation of conidial chains, the conidiophore continued its growth to form a sympodium. Later this condition was referred to as the ‘Cladosporium type’ (see de Vries, 1952; pp. 34-5). The conidiophore of Hormodendrum cladosporioides (Fres) Sacc. did not continue its growth after the appearance of conidia and was referred to as ‘Hormodendrum type’. Gilman (1945) stated that the conidia of Cladosporium are at first one-celled and then usually a cross-wall is formed. Bisby (1944) noted that in a week-old culture 90% of the spores of this genus are produced acropetally and are oval or globose; most of the remaining 10% are larger and one-celled but about 0.1% of all spores are 1-septate.

The genus Hormodendrum was founded by Bonorden in 1851. Harz (1871) gave a fairly clear description: conidiophore erect, simple or unequally branched towards the apex; conidia heterogenous, page 147 in chains, easy to break up, 2-celled, oval or elliptic. Lindau (1907) noted that in his material there are two different ways in which spores were formed, acrogenous budding and fragmenting of the branches into conidia. He placed Hormodendrum in the unicellular Phaeo-sporae, and Cladosporium in the bicellular Phaeodidymae. Brooks and Hansford (1922-23) associate Hormodendrum with short conidiophores and chains of smaller spores while Cladosporium was always represented by larger conidia. Gilman (1945) stated that conidial chains in Cladosporium are developed acrogenously on branches and that all the branches are borne on a single main stipe. In general it appears that Hormodendrum species have been associated with human skin diseases and Cladosporium with plant diseases.

The essential fundamental characters of Cladosporium and Hormodendrum as described by various authors are summarised below.

Schostakowitsch (1895) had shown that in a medium with 10% potassium nitrate H. cladosporioides (Fres.) Sacc. produced conidiophore prolongation, while on agar medium, C. herbarum Fres. produced no prolongation. De Vries (1952) has made the most thorough investigation of Cladosporium species. He clearly demonstrated how the conidiophore of C. macrocarpum Preuss developed from a ‘Hormodendrum type’ into ‘Cladosporium type’ through conidiophore prolongation (1952, pp. 35-36, 77). In fact, the ‘kerosene fungus’, an accepted ‘Hormodendrum type’ has quite often produced conidiophore prolongation on malt slide cultures (Fig. 16). Because of this it is considered that conidiophore prolongation cannot be regarded as a criterion to separate the two genera.

Fig. 16: Camera lucida drawings to show conidiophore prolongation in f. avellaneum.

In Cladosporium, conidia are considered as larger and bi- to pluricellular while in Hormodendrum they are smaller and one-celled. This is not true in all or even in most cases. Septation, unicellular and bi- to pluricellular conidia generally occur in both Cladosporium and Hormodendrum.

Since almost all the essential characters of both genera are overlapped, and there are no definite criteria to separate them, it is only natural that the two merge into one. Mycologists such as Laurent (1888). Costantin (1889), Bennett (1928), Brett (1948), Bisby (1944), de Vries (1952), Smith (1969) and von Arx (1970) generally regard H. cladosporioides as synonymous with C. herbarum. Since Cladosporium is preferred, and predates Hormodendrum, the name Hormodendrum should be dropped.

We believe that the fundamental characters of the ‘kerosene fungus’ such as acrogenous formation of blastospores and ramoconidia, and shape and size of conidiophores and conidia are in one way or the other related to Cladosporium species. The whole morphology is such that it cannot form a new genus or be assigned page 149 to any other existing genus, but is correctly placed within Cladosporium. This is in agreement with de Vries (1952, 1955), Nicot and Zakartchenko (1966) and Parbery (1969a).

Cladosporium resinae as known at the present time exists as f. avellaneum, f. resinae, f. albidum, f. sterile and intermediate forms The last two forms occur rarely. Forma albidum can be morphologically identical to f. resinae (de Vries, 1955) or to f. avellaneum (Sheridan et al., 1971): f. resinae and f. avellaneum are morphologically distinct (Figs. 1 and 10). Forma avellaneum gives rise to f. resinae in cultures but in general is relatively stable. Parbery (1969a) reported that no reversion occurred, but only recently we have observed reversion to occur in our subcultures of de Vries (1955) type of f. avellaneum (which changed to f. resinae and back again) and two of our New Zealand isolates. Ellis (personal communication, 1971) has observed both forms to exist side by side being produced by the same hyphae. He suggested that it is best to refer all forms to the sexual state Amorphotheca resinae.

Because the four asexual forms are relatively stable, we are of the opinion that the subspecific epithet should be retained as an aid for recognition until such times as further cytological and genetical information is available and more is known about the sexual state.

The perfect state of some Cladosporium species has been described and all of them belong to different families. Mycosphaerella tassiana (de Not.) Johanson, is the sexual state of C. herbarum (Pers.) Link. (von Arx, 1949). Most Mycosphaerella species are reported as having functional male organs producing spermatia and the ascocarps are ostiolate. The perfect state of Cladosporium ladina Muller is Leptosphaeria ladina Muller which is characterised by the extrusion of the ascus and release of ascospores on maturation (Muller, 1950). Microascus pedrosoi (Fuentes and Wolf, 1956) is the perfect state of Hormodendrum pedrosoi Brumpt, and is placed in a group, i.e. Haerangiomycetes, characterised by ostiolate ascocarps and the absence of a definite ascal wall.

The perfect state of C. resinae as described here (see (a) sexual state) obviously shows no affinity towards Mycosphaerella, Leptosphaeria or Haerangiomycetes. The formation of gametangia (Fig. 6a), the presence of radiating ground tissue in the early stage (Fig. 6b) and peridium in later stage shows it to be closely related to various members of Eurotiales. However, its unique character, the amorphous peridium, make it difficult to fit into any of the previously existing families of this order. Parbery (1969a) erected a new family, Amorphothecaceae, with one genus Amorphotheca and named the perfect stage of Cladosporium resinae, Amorphotheca resinae. Since its introduction in 1969, the name has been widely accepted by mycologists interested in this fungus. In his book ‘Dematiaceous Hyphomycetes’ Ellis (1971) describes Cladosporium resinae as the asexual state of Amorphotheca resinae Parbery.

3.Physiology

(a) Growth in kerosene

The form of the ‘kerosene fungus’ usually recovered from aviation kerosene is f. avellaneum (Hazzard, 1963; Hendey, 1964), Parbery's (1968) isolates of f. resinae from soils are reported by him as not growing in kerosene but New Zealand isolates of this form and f. albidum have grown readily in kerosene (Sheridan and Nelson, 1971a). When the growth of New Zealand soil, fuel and air isolates of the ‘kerosene fungus’ in aviation turbine and lighting kerosene was compared (Sheridan and Nelson, 1971a) it was found that some soil and air isolates of f. avellaneum grew better and faster in kerosene than fuel isolates (Fig. 17). This is somewhat surprising because association with kerosene would be expected to select in favour of increased tolerance. However, it seems possible that naturally occurring hydrocarbons in the soil may select in favour of these vigorous strains, and the sexual state, if it occurs in nature, may allow new strains to arise.

During these studies it was observed that the amount of growth produced by many isolates decreased on successive passage through kerosene. It would be interesting to know whether vigour is restored on passage through soil, and whether the sexual state occurs in kerosene.

Further evidence for the existence of different strains comes from the fact that some isolates produce a brown pigment in the mineral salts medium while others do not. Pigment production in culture has also been observed by us and by de Vries (1952). Fig. 18 shows five isolates, and a mixture of isolates growing in aviation turbine and lighting kerosene after four weeks at 25°C. All are soil isolates from the United Kingdom, except A89/71 which is an air isolate from New Zealand and ‘mixed’. From the left: the first is from soil collected in a garden at Hayley Green, near Birmingham—note the presence of pigment in the mineral salts medium in both bottles; the second is from soil collected in Dixon Park, Belfast — note the presence of pigment in aviation turbine kerosene only; the third is from a soil collected in Wales and the fourth from a soil collected in the Lickey Hills near Birmingham — neither have produced pigment. Growth is in every case greater in aviation than in lighting kerosene. After six weeks the amount of growth in lighting kerosene is often greater than that in aviation kerosene indicating that the rate of growth in the former speeds up after three to four weeks.

The optimum temperature for growth in kerosene appears to depend on the strain of the fungus and the type of kerosene. In one experiment where the growth was harvested after six weeks, an isolate of f. avellaneum had an optimum of 25°C., while f. resinae had an optimum of 20°C. in aviation turbine kerosene. The optimum for both was 30°C. in lighting kerosene. It would be interesting to know page 151 whether this difference in optimum temperature is of general occurrence among isolates of the fungus.

Fig. 17: Growth of isolates of C. resinae f. avellaneum in aviation turbine kerosene after six weeks. Left: Four soil isolates. Centre: Three fuel isolates. Right: Four air isolates.
Fig. 18: Isolates of C. resinae f. avellaneum growing in kerosene: note the pigment present in the mineral salts medium (lower layer) in some of the bottles. See text for explanation.

No attempt has been made here to review the literature on pathways of carbon assimilation or enzymes produced by the ‘kerosene fungus’ and no work is being done in our laboratory in this connection at the present time. Recent work elsewhere, however, has shown that methane-utilising bacteria use one of two pathways of carbon assimilation, the serine pathway or the ribose phosphate cycle of formaldehyde fixation. Lawrence and Quayle (1970) have examined the distribution of two key enzymes each of which appears to be specifically involved in one of the assimilation pathways.

It is hoped that the application of gas chromatographic techniques will throw some light on the components of kerosene utilised by this fungus and on the relationship between naturally occurring hydrocarbons and the occurrence of the ‘kerosene fungus’ in soil.

(c) Growth on different culture media

The choice of culture media for growth of the ‘kerosene fungus’ is very important because the medium influences the production of ascocarps, ability to grow subsequently in kerosene, rate of growth, colony chracteristics and pigment production (Parbery, 1969; Sheridan. Steel and Knox, 1971; de Vries, 1952). The constituents of a number of useful media are given here together with brief notes on their suitability. Parbery (1969a) found that isolates do not lose their ability to grow in kerosene when kept on Bushnell-Haas agar with 2% glucose but some do lose it on other agar media such as page 153 malt. Bushnell-Haas medium, without agar, is generally the basalmineral salts medium of choice in experiments on growth in kerosene. The kerosene is layered on top of the medium (see for example Fig. 17). For the production of the sexual state (ascocarps) Parbery (1969a) found cherry agar, which is the standard medium of the Centraalbureau Voor Schimmelcultures, very useful. On this medium and on modified Weitzman and Silva-Hutner agar some isolates produced fertile ascocarps in 14-20 days. On most other media two or three times this period was required. Ascocarps develop frequently in mineral oil layered on top of V-8 juice agar or malt agar slopes of the fungus (Sheridan and Steel, 1971). Czapek-Dox agar has been also used for maintenance and culture studies. Sucrose should be replaced with some other sugar: Parbery and de Vries used glucose (see (d) Nutrition. 1. Carbon source requirements).

Fig. 19: Growth of isolates of C. resinae f. avellaneum on V-8 juice agar and Bushnell-Haas agar containing different concentrations of creosote (after 11 days at 25° C.).

Growth of 141 New Zealand soil isolates was compared on four different media: Czapek-Dox agar minus sucrose (C/D), Bushnell-Haas agar (B/H), modified Weitzman and Silva-Hunter agar (WS/H) and V-8 juice agar (V-8) (Sheridan, Steel and Knox, 1971). All page 154 media contained 0.1% creosote, and V-8 juice agar without creosote was included for comparison. All isolates behaved similarly. Fig. 20 shows results after 10 days at 25°C. Best growth was obtained on V-8 juice agar without added creosote indicating that creosote of this concentration depressed growth. Since Czapek-Dox and Bushnell-Haas media had no carbon source other than creosote all isolates were able to utilise creosote. The pure white form, f. albidum, behaved similarly. Sporulation was good on all media but the perfect state was not seen in these experiments.

Fig. 20: Mean diameter of 141 isolates of C. resinae f. avellaneum after 10 days at 25° C. on different media (all contain 0.1% creosote except V-8—).

Bushnell-Haas medium (Bushnell and Haas, 1941)

Magnesium sulphate (7H2O)	0.20 g.
Potassium dihydrogen phosphate	1.00 g.
Dipotassium hydrogen phosphate	1.00 g.
Ammonium nitrate	1.00 g.
Calcium chloride	0.02 g.
Ferric chloride (6H2O)	2 drops concentrated soln.
Distilled water	1,000 ml.

To this mineral salts medium 2% glucose may be added as a carbon source, and the medium may be solidified with 2% agar. The pH is adjusted to 7-7.2 with dilute sodium hydroxide.

Modified Weitzman and Silva-Hutner agar (Weitzsman and Silva-Hutner, 1966)

Magnesium sulphate (7H2O)	1.0 g.
Potassium dihydrogen phosphate	1.0 g.
Sodium nitrate	1.0 g.
‘Pomodoro’ tomato paste	10 g.
‘Thistle’ oat meal	10 g.
Agar	18 g.
Distilled water	1,000 ml.

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The pH is adjusted to 5.6 with dilute sodium hydroxide. Czapek-Dox agar

Magnesium sulphate (7H2O)	0.5 g.
Potassium dihydrogen phosphate	1.0 g.
*Sodium nitrate	2.0 g.
Potassium chloride	0.5 g.
Ferrous sulphate (7H2O)	0.01 g.
**Sucrose	30.0 g.
Agar	20.0 g.
Distilled water	1,000 ml.
V-8 juice agar V-8 vegetable juice (Campbell Soup Co. Ltd.)	350 ml.
Tap water	1,500 ml.
Agar	40 g.

The V-8 juice is added to the melted agar. This is a a very acid medium and does not withstand prolonged autoclaving. Malt extract agar

Plain maltexo (Wilson's)	20 g.
Tap water	1,000 ml.
Agar	20 g.

Glucose and peptone may be added at 2% and 1% respectively to give a richer medium for the production of ascocarps.

All media are autoclaved at 103kNm^-2 (121° C.) for 15 minutes. When required sterile creosote is added aseptically to melted media just before pouring plates.

(d) Nutrition

1.Carbon source requirements. The carbon source requirements of a New Zealand isolate of the ‘kerosene fungus’ were studied in our laboratory (Tan, unpublished). Carbon sources tested included mono-, di-, tri- and polysaccharides — all were used at 1.5% by weight. Ammonium chloride was used as nitrogen source. Results are presented in Fig. 21. Best growth (measured as dry weight) was obtained with xylose followed by maltose and cellobiose. Sucrose and glucose did not give such good growth. Other workers have found glucose to be a better carbon source than sucrose (Parbery, 1969a; de Vries, 1952); we found glucose to be only slightly better than sucrose. Arabinose, casein and glycogen depressed growth below that of the control — the growth in the control indicates that impurities were present as contaminants in sufficient amount to allow growth without any added carbon.

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Fig. 21: Comparison of different carbon sources for growth of C. resinae.

Cochrane (1958) states that xylose has been reported to be superior to glucose for some organisms. However, he points out that xylose is known to be broken down to furfural during autoclaving so that reports of its non-utilisability should be regarded with some reserve until confirmed with filter-sterilised xylose. The xylose used in the work reported here was sterilised by autoclaving.

2. Nitrogen source requirements. Eight different nitrogen sources were compared at 0.15% nitrogen level (Tan, unpublished). Glucose at 2.5% was used as carbon source. Results are presented in Fig. 22. Ammonium salts gave better growth (as dry weight) than nitrite or nitrate. This is in agreement with Marsden's findings (1954). Urea appears to be toxic to this fungus. Asparagine has been reported as a good nitrogen source for other fungi (Papavizas, 1970). On ammonium nitrate medium the pH decreased during growth from over 6 to around 3. This decrease has also been noted by Parbery (1971a).

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However, the optimum pH for growth of C. resinae is low so that this decrease, which adversely affects fungi with higher optima, has little or no effect on C. resinae.

Fig. 22: Comparison of different nitrogen sources for growth of C. resinae.

(e) Effect of pH on growth

Christensen et al. (1942) found that their isolates of C. resinae could grow between pH 3 and 9.6. Parbery (1971a) considers it hard to evaluate their results because the final pH of most of their media was about 7. All New Zealand isolates of the fungus grew well between pH 2 and 6.5 (Tan, unpublished). At pH 1.5 growth stopped abruptly and no growth occurred beyond pH 10. The optimum pH page 158 was close to 3 (Fig. 23). This is extremely low. Among 34 species listed by Cochrane (1958) only two had an optimum pH of 4.0 or below. Dothistroma pini, the causal agent of pine needle blight, is reported to have an optimum pH of 3.5 (Ivory, 1967).

Fig. 23: The effect of pH on growth of C. resinae.

(f) Effect of temperature on growth

The optimum temperature for growth of the kerosene fungus on an agar medium is reported to be around 30°C with a range from 5-40°C. (Hendey, 1964; Parbery, 1971a; Sheridan, Steel and Knox, 1971). There is one report of an optimum at 37° C. (in Parbery, 1971a). None of the New Zealand isolates so far studied would grow below 5° C. or above 45° C. (Tan, unpublished). The optimum on an agar medium lay somewhere between 20° C. and 35° C.; in general it was close to 30° C. but this appeared to depend on isolate and medium (Figs. 24 and 25). In lighting kerosene the optimum for an

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Fig. 24: The effect of temperature on the growth of C. resinae on V-8 juice agar. X — X f. avellaneum. O — O f. albidum.

Fig. 25: Effect of different temperatures on the growth rate of C. resinae.

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isolate of f. avellaneum and f. resinae was 30° C. while in aviation turbine kerosene it was 25° C. and 20° C. respectively. On an agar medium the optimum for growth of f. albidum was 30° C. Further work is clearly desirable since some strains may have a lower optimum temperature in kerosene than hitherto realised.

4. Summary and Conclusions

The recent discovery of the sexual or perfect state of the ‘kerosene fungus’ was a major contribution both to knowledge of the life cycle of this unusual and interesting fungus and to a better understanding of its phylogenetic and taxonomic position. Parbery's (1969 a and b) observations for Australian isolates have been confirmed for New Zealand isolates (Sheridan, Steel and Knox, 1971; Sheridan and Steel, 1971). Most mycologists familiar with this fungus have accepted Parbery's (1969a) name Amorphotheca resinae in the new family Amorphothecaceae even though a considerable amount of work still needs to be done on the cytology and genetics of this fungus and the relationship between the many asexual forms and the sexual state.

There are two distinct morphological, asexual, forms of the ‘kerosene fungus’ and three distinct colony forms. In this paper the morphologically and culturally distinct f. avellaneum and f. resinae have been redescribed, and it is pointed out that intermediates are of frequent occurrence. The albino, which is pure white on all culture media, can be of either morphology. A fourth form, f. sterile, may be either pigmented or white. None of these forms appear to remain stable for long periods; although de Vries (1952) found his forms to be constant, his type of f. avellaneum (IMI 49620) is no longer true to type. In view of this variability, we would agree with Hendey (1964) that it is unlikely that the various forms have any real taxonomic significance and that ‘the subspecific epithets are of use only to facilitate reference to the different growth forms’. All forms of the ‘kerosene fungus’ should be referred to Amorphotheca resinae.

The avellaneum form is a good Cladosporium; the resinae form is not. Nevertheless, because both have the same perfect state, one can change into the other and vice versa and intermediates exist, and both appear to be physiologically similar, it is undesirable to place them in separate genera. The name Cladosporium resinae should be retained for the asexual state of the ‘kerosene fungus’. Cladosporium predates Hormodendrum.

It appears that isolates of the ‘kerosene fungus’ differ in their ability to utilise kerosene and creosote and that this is dependent to some extent on past history and genetical make-up. As already pointed out cytological and genetical studies are needed and much more needs to be done on its physiology because of the significance of this fungus as a fuel contaminant. A large number of isolates from different parts of the world should be studied. Very recently Parbery (1971b) has published a review of biological problems in jet aviation fuel and the biology of Amorphotheca resinae in which he points out some of the research problems associated with studies on this fungus and the need for these to be overcome in future work.

The ‘kerosene fungus’ is a most unusual organism; it grows in the presence of substances normally considered to be fungistatic or fungicidal (kerosene and creosote) and can utilise these as a source of carbon, it produces an unusual brown pigment, has an unusually thick membrane and spore wall, is very variable in asexual morphology and produces a unique sexual form. Yet very few mycologists have shown interest in it in the past. It can only be hoped that the publication of these papers will arouse and stimulate interest in this fungus and its activities, and that the many problems mentioned here will be solved in the near future.

Acknowledgments

The authors wish to express their appreciation to the following: Professor H. D. Gordon, head of the Botany Department, Victoria University of Wellington, New Zealand, for his constant encouragement and helpful advice during the course of our studies, and the technical staff of our department, Mr. Ron Hoverd, Mrs. Ila Labone, Miss Margaret Priday and Mr. Herbert Christophers for competent technical assistance; the director of the Commonwealth Mycological Institute, Kew, England, and his staff, particularly Dr. M. B. Ellis, Dr. B. C. Sutton and Dr. Agnes H. S. Onions, for identifications and supplying isolates of Amorphotheca resinae and for helpful discussions; Professor R. K. McKee, head of the Department of Mycology and Plant Pathology, the Queen's University of Belfast, Northern Ireland, who kindly allowed the senior author the use of laboratory facilities during a recent visit, and Mr. J. C. Taylor and Dr. J. P. Malone of the same department for assisting with collection of soil samples and air monitoring; Mr. Walter Freitag and Mr. Walter Johnston of B.P. (N.Z.) Ltd. for supplying samples of kerosene and page 162 cultures of fungi isolated from aviation fuel, and Mr. Ken Chater of B.P. Research Centre, Sunbury-on-Thames, England, for providing us with a fuel isolate of A. resinae and for useful discussions; and Dr. John Troughton of Physics and Engineering Laboratory, D.S.I.R., New Zealand, for Stereoscan studies, and Mr. Mervyn Loper of the Electron Microscope Unit of Victoria University for electron microscope studies.

The costs of research were met by our department and grants from the Internal Research Fund of this university. One of us (Y.L.T.) gratefully acknowledges financial help from the Lee Foundation of Singapore. The maps are reproduced by permission of the Lands and Survey Department of the New Zealand Government.

Finally it is a pleasure to thank Mrs. Mary Sheridan for supervising the air samplers on many occasions and for reading and criticising the manuscripts, Mrs. Shona Greer for typing the manuscripts, and the staff of the library for help in procuring many of the publications referred to in these studies.

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