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Diversity of the Trichocomaceae in the Katandra Nature Reserve, Central Coast, NSW, Australia
Mycol. Res. 109 (9): 964–973 (September 2005). f The British Mycological Society
964
doi:10.1017/S0953756205003540 Printed in the United Kingdom.
Diversity of the Trichocomaceae in the Katandra Nature Reserve, Central Coast, NSW, Australia
Anne-Laure MARKOVINA1, John I. PITT1, Ailsa D. HOCKING1, Deidre A. CARTER2 and Peter A. McGEE3 1
Food Science Australia, P.O. Box 52, North Ryde, NSW 1670, Australia. School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia. 3 School of Biological Sciences, University of Sydney, NSW 2006, Australia. E-mail : [email protected] 2
Received 18 November 2004; accepted 26 May 2005.
The diversity of the family Trichocomaceae, which includes the major anamorph genera Aspergillus and Penicillium, was studied in the Katandra Nature Reserve, Central Coast, NSW, Australia. Soil, living leaves, leaf litter and detritus were examined by both direct and dilution plating techniques. Fungi were isolated on dichloran Rose Bengal chloramphenicol agar, and dichloran 18 % glycerol agar, media suitable for cultivation of many species within this family. Species of Trichocomaceae were isolated from all sites and all substrates examined. A high diversity was found, with more than 50 known species identified, and an equal number of undescribed species detected. More species of Penicillium were recovered than other genera, with Aspergillus species the next most common. Most of the species recovered were anamorphs, though 16 known and unknown ascosporic species were also isolated from heated and unheated soil. Soils, leaf litter, a scat from a native herbivore and leaves of living native plants yielded higher diversity than insects, worms or introduced plants. More species belonging to the family were isolated from soil in dry sclerophyll forest than in rainforest. Conversely, native rainforest plants harboured more diversity than the dry sclerophyll forest plants examined.
INTRODUCTION The family Trichocomaceae includes the major anamorphic genera Penicillium and Aspergillus. Some members of this family are important causes of food spoilage and biodeterioration, while others are widely used in biotechnology. Some produce mycotoxins and many other diverse secondary metabolites (Pitt & Hocking 1997). Species of Penicillium and Aspergillus grow quickly and produce large numbers of conidia which are easily dispersed. As a result, these fungi occur over a wide range of environments. The taxonomy, biology and diversity of genera and species in the family Trichocomaceae in food commodities and processed foods are well documented (Pitt & Hocking 1997). Species of Trichocomaceae have often been recorded in ecological investigations of soils in North American and European forests (Bissett & Parkinson 1979a, b, c, Yasmeen & Saxena 1990, Bettucci & Roquebert 1995, Christensen, Frisvad & Tuthill 2000, Lumey, Gignac & Currah 2001) and other plant communities (Christensen 1969, 1981, 1989). They have also been found in plant materials and arthropods (Martin 1967, Drummond, De Barro & Pinnock 1991,
Perez et al. 2003). However, relatively little is known about the diversity and distribution of these fungi in natural environments other than soils in North America and Europe. Australian studies on the natural occurrence of species of Trichocomaceae are limited. Twenty five species were recorded from soil in a wheat field (Warcup 1957), while Yip & Weste (1985) isolated 41 species in the rhizosphere of two native Australian plants, and Paulus, Gadek & Hyde (2003) isolated six species of Penicillium and one species of Aspergillus from rainforest litter. Lamb & Brown (1970) recovered four species of Penicillium and one of Aspergillus from the phylloplane of three plant species. In studies of fungal succession in leaf litter from Eucalyptus regnans, Macauley & Thrower (1966) suggested that Penicillium species are not responsible for the primary degradation of plant cellulose, but are secondary decomposers, appearing more frequently on leaves in advanced stages of decomposition. Two introduced plants (Paspalum dilatatum and Salix babylonica) and one Australian native tree (Eucalyptus stellulata) were reported to harbour distinctive fungal populations including some species of Trichocomaceae (Lamb & Brown 1970).
A.-L. Markovina and others The objective of this project was to study the diversity of the family Trichocomaceae in a largely undisturbed natural environment in Australia, with a well documented and wide range of habitats and plant species.
965 Table 1. Collection sites and habitat characteristics in Katandra Nature Reserve, Central Coast, NSW. Site type
Soil type
Dominant plant species
Dry sclerophyll
Sand to sandy-loam
Banksia serrata Acacia spp. Pteridium esculentum (bracken fern) Eucalyptus pilularis (blackbutt) Syncarpia glomulifera (turpentine) Allocasurina torulosa (forest oak)
Wet sclerophyll
Sandy-loam
Eucalyptus paniculata (iron bark) Livistonia australis (cabbage Palm) Pittosporum revolutun (native frangipani) Lianas, Palms
Rain forest
Clay
Acmena smithii (lilly pilly) Archontophoenix cunninghamiana (Bangalow Palm) Ceratopetalum apetalum (coachwood) Livistonia australis (cabbage Palm) Ferns and orchids
Creek bank
Clay
Closed rain forest Bangalow Palm grove Ferns Ficus sp. (fig tree)
Bank of Seymour Pond (artificial)
Sandy-loam
Wet sclerophyll, thick leaf litter
MATERIALS AND METHODS Site description The study site, Katandra Nature Reserve on the Central Coast, NSW, Australia, was chosen because the site is readily accessible, has had limited human impact in most parts, and contains a number of different ecosystems within only 53 ha, thus limiting climatic effects within the diverse vegetation. Ridges within the reserve are capped by sandstones of the Hawkesbury series, while the underlying shales and sandstones belong to the Narrabeen group (Fairley & Moore 2000). A feature of the lower part of the reserve is Seymour Pond, a constructed pond that fills from a spring emanating from a nearby cliff face. The vegetation in the Katandra Nature Reserve can be classified into three main types (Fairley & Moore 2000). First, dry sclerophyll (open forest), with vegetation typically two-layered, with a dominant upper canopy, which allows clear vision through the tree trunks for some distance. Eucalyptus and Angophora are the dominant tree genera in this zone. The trees are usually 10–30 m high with flattish crowns and tall, well-developed trunks. Second, wet sclerophyll (tall open forest), where the vegetation consists of three or more layers, with a continuous tree canopy. Usually, the trees are over 30 m high at maturity. Groups of tall shrubs and small trees form an intermediate layer of vegetation over a low shrub understory. Gullies are frequently dominated by Eucalyptus saligna. Many climbers such as Cissus hypoglauca occur in the understory, while ferns and monocotyledon plants are also common. Third, temperate rainforest (closed forest), found on the moist, shady sides of the ridges where, characteristically, the light intensity is low at the forest floor. The rainforest is typified by vegetation in three layers. Climbing plants are very common, with ferns and orchids equally prominent. Eucalyptus trees do not grow in the rainforest, but are abundant around its perimeter. Details of these habitats are given in Table 1. Pilot study A pilot study aimed to determine whether different species of fungi from the family Trichocomaceae occurred in soils associated with the different vegetation types found in Katandra. Six sampling sites were chosen along a 2 km transect across the reserve and over a 200 m change in altitude. The selected transect encompassed dry sclerophyll
forest, wet sclerophyll forest and temperate rainforest. Samples were collected in midsummer (Feb. 2003) using a garden trowel disinfected with 70 % ethanol between samplings. Samples were stored in resealable plastic bags and kept at 4 xC until processed. Samples were analysed within two days of collection. Soil samples (10 g) were dispensed into 90 ml of sterile 0.1 % (w/w) peptone solution. Suspensions were shaken vigorously for one minute, serially diluted, then aliquots (0.1 ml) were plated in duplicate onto selective media, dichloran Rose Bengal chloramphenicol agar (DRBC) and dichloran 18 % glycerol agar (DG18) (Pitt & Hocking 1997). Plates were incubated at 25 x for 5–7 d. Plates were checked regularly to ensure more slowly growing species were subcultured before plates became overgrown. After incubation, isolation plates were examined under a dissecting microscope, and selected colonies subcultured (three point inoculation) onto Czapek Yeast Extract Agar (CYA) and Malt Extract Agar (MEA) (Pitt & Hocking 1997). Plates were incubated at 25 x for 7 d. Plates were incubated at 25 x for 7 d, then examined macroscopically and microscopically. All types of
Diversity of Trichocomaceae in the environment colonies judged to be different and belonging to the family Trichocomaceae were subcultured and transferred to identification media.
Main study A high degree of diversity was observed at each of the pilot study sampling sites, so two further sites were selected for more intensive study. The first site was located in a tall open dry sclerophyll forest, at approx. 40 m altitude. This site appeared to have a considerable diversity of potential substrates, including soil, leaf litter, and small plants, as well as some invertebrates and other detritus. The soil profile was readily accessible, enabling intensive sampling. The second site was situated in a temperate rainforest area, at approximately 20 m altitude, about 0.5 km from the first site. This site had a very high diversity of plant species, but less leaf litter and detritus.
Sampling Samples at the first site were removed from within a 2r2 m quadrat. After a metal rake was used to gather the leaf litter, three soil blocks (20r20 cmr 20 cm) were removed using a clean spade. Each soil block was separated into two sections : topsoil (0–7 cm) and mineral soil (7–20 cm). Topsoil fractions and mineral soil fractions were pooled and stored in clean plastic boxes. Material associated with each soil fraction was stored separately, and included roots, live cockroaches and worms and a macromycete (Pisolithus tinctorius). The leaf litter layer was sorted into leaves, seed pods, live insects, a spider, a cicada exoskeleton, an insect chrysalis, an insect larva and a scat, presumed to be from a native herbivore. Any plants encountered within the quadrat were also collected for analysis. A native bracken fern (Pteridium esculentum) was removed and separated into fronds, root system and rhizosphere. A single seedling of Cinnamomum camphora (camphor laurel, an introduced species) was also collected and analysed for phylloplane fungi. At the second site, the area sampled was a randomly selected 1r1 m quadrat, and only one soil block was removed from the site. Soil fractions, roots and leaf litter were processed and stored as before in individual plastic boxes. In addition, leaves of five native plants, Adiantum hispidulum (rough maidenhair fern), Archontophoenix cunninghamiana (bangalow palm), Synoum glandulosum (scentless rosewood), Stenocarpus salignus (scrub beefwood) and Sloanea australis (maiden’s blush), were collected from plants in the quadrat. All materials were stored individually and kept cool until analysis in the laboratory. Live insects were frozen to kill them before cool storage. All analyses were conducted within four days of sampling.
966 Isolations from soil and leaves Soil attached to the surface of the rhizome of the fern was separated, serially diluted and plated out. Soil was gently removed without washing from roots, which were then cut into 0.5 cm lengths using sterile scissors and gently rolled on the surface of selective media (DRBC and DG18). The same pieces were then surface disinfected in undiluted household bleach (4 % chlorine) for 5 min. After 3 rinses in sterile deionised water, the root pieces were aseptically placed on selective media. Isolations were performed in duplicate. Leaves were gently pressed onto the surface of selective media using flamed forceps. The leaves were then surface-sterilized and incubated in the same manner as the root pieces. Large leaves were cut into smaller pieces prior to isolation. Isolations were performed in duplicate. Isolation from insects and worms Previously killed insects and worms were gently pressed onto duplicate plates of selective media (DRBC and DG18). Worms were additionally surface sterilized in bleach (4 % chlorine) for 5 min and rinsed in sterile deionised water before being plated out on the selective media. Isolation from scat The single scat collected was weighed, broken up, diluted 1 :10 in sterile peptone water, then aliquots (0.1 ml) plated in duplicate onto selective media. Isolation from seed pods Seeds were rolled onto the surface of selective media and incubated at 25 x for 7–10 d. Isolation from insect casings (exoskeletons) These materials were placed on the surface of selective agar and incubated. The surface sterilization step was omitted due to lack of material. All isolations were duplicated. Isolation from leaf litter Leaves from the leaf litter taken from the dry sclerophyll site were sorted into three stages of decomposition : entire leaves, partially decomposed leaves, and fragments that were decomposed, but could still be handled. Four leaves from each stage were removed and gently pressed against the surface of the selective agar, then surface sterilized and plated onto selective media. Each group of leaves was duplicated. From the rainforest site, the leaf litter was sorted into 17 different leaf types based on morphology. Whenever possible, every leaf type was sorted into two leaf stages,
A.-L. Markovina and others
967
fresh and partially decomposed. Each stage of each leaf type was plated out onto selective media.
Table 2. Species of Trichocomaceae isolated from soil in various vegetation stands during the pilot survey, Katandra Nature Reserve.
Heat treatments of soil samples
Fungal species
For the isolation of teleomorph genera from Trichocomaceae, triplicate samples (10 g) of each soil fraction were suspended in 0.1 % peptone (90 ml), heated at 75 x in a waterbath for 15 min and then serially diluted as previously described. Duplicate plates of selective media were inoculated with 0.1 ml of soil suspension.
Aspergillus cervinus A. kanagawaensis Paec. lilacinus Penicillium glabrum P. herquei P. janczewskii P. sclerotiorum P. simplicissimum P. spinulosum P. thomii Penicillium sp. 01 (FRR 5727) Penicillium sp. 02 (FRR 5728) Penicillium sp. 03 (FRR 5729) Talaromyces sp. 01 (FRR 5764) Species richness
Incubation Plates were incubated at 25 x for 7 d. Heated samples were incubated for 14 days.
Dry Wet sclerophyll sclerophyll Rainforest
x
x x x x
x x x x x
x x x x
x
x
x x x 7
9
x 4
Identification of fungi Isolates judged to belong to the family Trichocomaceae by macroscopic and (or) microscopic observations were grown on standard media for identification. Most isolates belonged to Penicillium or Aspergillus : Penicillium species were identified by the methods of Pitt (1980, 2000), and Aspergillus species by those of Klich & Pitt (1988) and Klich (2002). Descriptions of less common newer species were referred to where necessary. Species from other genera were identified using Pitt & Hocking (1997) or specialized texts in a few cases. Isolates which showed characteristics in colony and microscopic morphology judged to differ significantly from known species were designated as putative new species, and assigned numbers as shown in the tables. They were also accessioned into the FRR collection of fungus cultures at Food Science Australia, North Ryde, NSW. More definitive examination by techniques such as secondary metabolites profiles or molecular analysis were not attempted at this stage, but will be used as needed for formal description of the new species.
RESULTS Pilot study transect Fourteen species of Penicillium, Aspergillus and a few other genera of Trichocomaceae were isolated from the pilot study. Seven species were recovered from the dry sclerophyll forest, nine from the wet sclerophyll forest and four from rainforest vegetation (Table 2). The diversity appeared to be quite different at each site, highest in dry sclerophyll and lowest in the rainforest. Undescribed species of Penicillium were isolated from all sites, while one undescribed species of Talaromyces was recovered from the rainforest site. In general, fungi isolated from soil along the transect occurred in low frequencies. P. spinulosum, P. sclerotiorum and P. simplicissimum were all found at more than one site. P. janczewskii was common in dry
sclerophyll forests but was not encountered at other locations. Aspergillus kanagawaensis was isolated on four occasions from wet sclerophyll forest, but occurred only once in dry sclerophyll sites. P. glabrum, usually a common species, was only isolated from one of the wet sclerophyll forest sites. Most species were isolated more than once from an individual site, however such common species as P. paxilli, P. herquei, Paecilomyces lilacinus and Aspergillus cervinus were isolated only once. Main study Rhizosphere and bulk soil Results from the rhizosphere and mineral soil have been pooled. In the dry sclerophyll forest bulk soil, species richness was higher in the topsoil than in the mineral soil. Nineteen species of Trichocomaceae were found in the topsoil including one species of Torulomyces (Tor. laevis) and six undescribed penicillia (Table 3). Sixteen species of Penicillium and Aspergillus were isolated from the mineral soil and the fern rhizosphere, including five undescribed species of Penicillium. Rainforest top soil yielded 12 species of Trichocomaceae including two species of Aspergillus and three undescribed species of Penicillium (Table 2). Fungi isolated from the mineral soil were less numerous than those from the organic layer. Two species of Aspergillus but no new species were isolated from mineral soil. Soils after heat treatment Several Eupenicillium and Neosartorya species were isolated from soils after heating (Table 4). Both mineral and top soils from the dry sclerophyll site showed higher numbers of fungal species than soils from the rainforest habitat.
Diversity of Trichocomaceae in the environment Root materials Many more species of Trichocomaceae were isolated from roots taken from the dry sclerophyll soil than from roots taken from the rainforest site (Table 3). Seven undescribed species of Penicillium and one undescribed species of Torulomyces were isolated from the dry sclerophyll roots, but only two from the rainforest (Table 3). Roots from both top and mineral soils in the dry sclerophyll habitat harboured similar numbers of fungi. At the rainforest site, however, roots isolated from the top soil had a higher fungal diversity than those from the mineral soil. A number of species were obtained from roots after surface disinfection with chlorine, including two undescribed species of Penicillium. Other soil substrates Cockroach and worm surfaces yielded the highest diversity of Trichocomaceae among the substrates collected from the topsoil of dry sclerophyll forest (Table 3). In addition, one undescribed Eupenicillium species was isolated from within the worm taken from the topsoil. Fewer materials were recovered from the mineral soil fraction, and of these, the surface of worms was found to harbour the highest number of fungi (Table 3). Leaf litter Leaves showing most decay (stage 3) among those collected from the dry sclerophyll litter supported the highest number of fungi (14) (Table 3). Similarly, fungal diversity was higher in intact leaves treated with chlorine than in decomposed (stage 3) bleached leaves (data not shown). The lowest fungal diversity and species richness, before and after bleaching, was observed from moderately degraded leaf litter (stage 2, data not shown). Twenty-six species of Trichocomaceae were isolated from the rainforest leaf litter (Table 3). Different types of leaves appeared to have different diversity indices. Diversity indices of entire leaves were within a wide range, 0.45–3.03 (data not shown), suggesting that some leaf types support a more diverse community than others. A similar trend was observed for decomposed leaves, where Gleason indices ranged from 1.44–2.34 (data not shown). Penicillia and aspergilli were recovered from every type of entire leaf ; whilst fungi were isolated from only nine types of decomposed leaves. Materials from within the leaf litter The scat collected from an unknown herbivore yielded a rich fungal biota, i.e. eight species of Penicillium, of which five were undescribed, plus one undescribed species of Aspergillus (Table 3). Fewer fungi were
968 isolated from other substrates. Fungal diversity was similar in seed pods of different native plants. Undescribed species were also isolated from the cicada exoskeleton and the surface of the cockroach (Table 3). Leaves from living plants Only two plant species were collected from the dry sclerophyll forest : a seedling of an introduced plant, camphor laurel (Cinnamomum camphora) and a native braken fern (Pteridium esculentum). Trichocomaceae associated with these plants differed with plant species (data not shown). Only one species of Penicillium, the ubiquitous P. glabrum, was isolated from the leaves of the camphor laurel. Isolates from the leaf surfaces of the native bracken fern were more numerous and diverse. A. niger and P. sclerotiorum were isolated once, P. glabrum occurred on five occasions and an undescribed species of Penicillium (FRR 5631) was isolated twice from the fern leaf surfaces. The difference in fungal population observed in this case is worthy of further investigation. Leaves from each type of native rainforest plant supported diverse communities of Trichocomaceae (Table 5). Seven species including one species of Aspergillus were recovered from the leaf surfaces of Adiantum hispidulum. Nine species, including one undescribed species of Penicillium, were isolated from Archontophoenix cunninghamiana ; six species of Penicillium were found on Synoum glandulosum. Stenocarpus salignus leaves harboured nine species of Penicillia, including one undescribed species of Penicillium. Three species of Penicillium and one species of Eurotium were recovered from the leaves of Sloanea australis.
DISCUSSION This paper describes a close study of the biodiversity in Katandra, a small nature reserve on the Central Coast, New South Wales, Australia. The study was limited in scope, being restricted to genera and species within the family Trichocomaceae. Nevertheless, the diversity encountered in Katandra was very high. Although fewer than ten habitats were sampled, more than 100 fungal species belonging to this one family were isolated. The habitats examined were not exceptional and included soil, decaying vegetation and other detritis, living leaves etc. However, the plant flora of Australia is largely unique, and that of the Sydney basin is very diverse, so perhaps this was to be expected also for the fungi. It was nevertheless unexpected that such a high proportion of isolates would be new to science. Considering that the accepted species of Penicillium described throughout the world number less than 250 (Pitt, Samson & Frisvad 2000), it was surprising that we encountered more than 40 new species within the confines of a small nature reserve. One explanation is that the leaves of Eucalyptus and
Dry sclerophyll
FRR number
Stage 1 leaf litter
Stage 2 leaf litter
Stage 3 leaf litter
Associated top soil substrates
Top soil
Associated mineral soil substrates
Mineral soil
Stage 1 leaf litter
Stage 3 leaf litter
Associated top soil substrates
x
Top soil
Associated mineral soil substrates
Mineral soil
x
x
x
x
x x x
x x x
h, i
x
m
x x
x
x
x x x
5765 5760
g i x x x c, d, e, f
x
x x
x
x
h
x
x x
h, i, j, k
x
m, n
x
x x
x x x
h, i, k h
x x
m m
x x x x
x x
x
x
x
x b
x
x
x
a, b, c, d, e, g
x
b
x
x x
x x
x
x
o
x x
x x
x
x
x
x
x
x x
x x c, d, f, g
x
l
x
x x
a, d, e, f, g
x
x
x
h, l
a, b, e, f
x x
x
x x
h, i, j, k,
x x
x x x
m, n
x
x
x
m, n m
x
x x x
x x
o
969
Aspergillus cervinus A. flavus A. kanagawaensis A. niger A. parvulus A. penicilloides A. restrictus Aspergillus sp. 01 Eupenicillium sp. 01 Paecilomyces carneus P. lilacinus Penicillium aurantiogriseum P. bilaiae P. brevicompactum P. chrysogenum P. citreonigrum P. citrinum P. crustosum P. decumbens P. dendriticum P. expansum P. glabrum P. herquei P. italicum P. janczewskii P. janthinellum P. lividum P. loliense P. miczynskii P. minioluteum P. ochrasalmoneum P. paxilli P. quercetorum P. restrictum P. sclerotiorum P. simplicissimum P. soppii P. spinulosum P. thomii P. verruculosum
Associated leaf litter substrates
Rainforest
A.-L. Markovina and others
Table 3. Anamorphs in the Trichocomaceae isolated from soil, litter and other substrates in dry sclerophyll and rainforest vegetation during the main survey, Katandra Nature Reserve.a
5626 5642 5635 5629 5640 5623 5730 5633 5731 5625 5732 5733 5734 5654 5735 5652 5659 5646 5634 5736 5737 5738 5624 5739 5740 5741 5742 5743 5627 5630 5744 5745 5746 5747 5748 5749 5750 5643 5645 5644
Diversity of Trichocomaceae in the environment
Penicillium sp. 04 Penicillium sp. 05 Penicillium sp. 06 Penicillium sp. 07 Penicillium sp. 08 Penicillium sp. 09 Penicillium sp. 10 Penicillium sp. 11 Penicillium sp. 12 Penicillium sp. 13 Penicillium sp. 14 Penicillium sp. 15 Penicillium sp. 16 Penicillium sp. 17 Penicillium sp. 18 Penicillium sp. 19 Penicillium sp. 20 Penicillium sp. 21 Penicillium sp. 22 Penicillium sp. 23 Penicillium sp. 24 Penicillium sp. 25 Penicillium sp. 26 Penicillium sp. 27 Penicillium sp. 28 Penicillium sp. 29 Penicillium sp. 30 Penicillium sp. 31 Penicillium sp. 32 Penicillium sp. 33 Penicillium sp. 34 Penicillium sp. 35 Penicillium sp. 36 Penicillium sp. 37 Penicillium sp. 38 Penicillium sp. 39 Penicillium sp. 40 Torulomyces laevis Torulomyces sp. 01 Torulomyces sp. 02 Species richness
x x x x x x x x
x
x x
x
x x x h h h h
m
o
p
m m o c c, f f g g g g g x x
x x x x x x x x x x m
12
8
14
19
16
20
16
12
9
a
Substrates associated with leaf litter: a, earth ball; b, chrysalis; c, cicada exoskeleton; d, Casuarina seed pod; e, Eucalyptus seed pod ; f, cockroach; g, scat; substrates associated with dry sclerophyll top soil : h, root ; i, worm; j, insect larva; k, cockroach; l, spider; substrates associated with dry sclerophyll mineral soil: m, root; n, worm; substrates associated with rainforest top soil: o, root; substrates associated with rainforest mineral soil: and p, root.
970
A.-L. Markovina and others
971
Table 4. Teleomorph species of Trichocomaceae isolated from soils subjected to heat treatment, Katandra Nature Reserve. Dry sclerophyll Fungal species Eupenicillium alutaceum E. javanicum E. ludwigii Eupenicillium sp. 02 Eupenicillium sp. 03 Eupenicillium sp. 04 Eupenicillium sp. 05 Eupenicillium sp. 06 Eupenicillium sp. 07 Eupenicillium sp. 08 Neosartorya fisheri Neosartorya sp. 01 Neosartorya sp. 02 Species richness
FRR number
Top soil x x x x x x x x x x x
5753 5754 5755 5756 5757 5758 5759 5761 5762
11
Rain forest Mineral soil
Top soil
Mineral soil
x x x
x
x
x x
x
9
2
x x x 4
Table 5. Species from the Family Trichocomaceae associated with leaves of rainforest plants, Katandra Nature Reserve. Adiantum hispidulum Aspergillus niger A. ochraceus Eurotium rubrum Penicillium brevicompactum P. citrinum P. corylophilum P. expansum P. glabrum P. herquei P. janczewskii P. miczynskii P. paxilli P. purpurogenum P. rugulosum P. sclerotiorum P. spinulosum P. thomii Penicillium sp. 41 (FRR 5751) Penicillium sp. 42 (FRR 5752) Species richness
Archontophoenix cunninghamiana
Synoum glandulosum
Stenocarpus salignus
Sloanea australis
x
x x
x x
x
x x x
x x x
x
x x
x
x x
x
x x
x x x x
x x x
x
x
7
x 9
6
other native Australian genera are very high in tannins, oils and other factors which provide resistance to insect and fungal attack, so that the degradation of such leaves can be expected to demand a distinct mycobiota. Alternatively, extensive ecological surveys of the Trichocomaceae are scarce so that any comprehensive survey might result in the discovery of large numbers of undescribed species. In total, 19 different species classifiable in the family Trichocomaceae were isolated from leaves of living plants, 39 species from leaf litter, 30 from topsoils, and 20 from mineral soil layers. Many more species of Penicillium (72) than Aspergillus (9) were recovered from Katandra. This is in keeping with current knowledge of the ecology of these genera, as Katandra is located on the south eastern side of a range of hills which provide a sheltered spot, enjoying cool to temperate climatic conditions throughout most of
x x x x 9
4
the year. Previous research has shown that Aspergillus species are much more likely to be found in regions where temperatures are high throughout the year (Christensen et al. 2000, Klich 2002). The diversity of phylloplane fungi on living plants in the rainforest was slightly lower than that from leaf litter and the soil beneath (Tables 3 and 5). This was to be expected, as few species in the family Trichocomaceae are known to be plant pathogens or commensals. In addition, the natural defenses of Australian native plants may be inhibitory to phylloplane fungi. The leaf litter sustained a higher number of fungi (23, Table 3) than that of rhizosphere and mineral soils (16, Table 3). Variation in fungal diversity was also evident in the different soil horizons analysed. Fungal diversity was highest in the topsoils and lowest in the deeper soil profiles. Warcup (1957) and Bissett &
Diversity of Trichocomaceae in the environment Parkinson (1979a, b, c) reported a decrease in the frequency of fungal occurrence with increasing soil depth. The large number of species isolated from the herbivore scat is also of interest. Herbivores may feed on native vegetation present in their immediate environment but may also travel some distances in search of food and thus would aid in the dispersal of native microfungi across potentially different ecosystems. Only four of the nine species of Trichocomaceae isolated from the scat were found in the adjacent leaf litter (Table 3), and the remaining five were undescribed species of Penicillium. A large number of species from Trichocomaceae have been described from dung (Siefert, Kendrick & Murase 1983). If the undescribed species arose from materials consumed by the herbivore, then this survey may have underestimated diversity on plant substrates at Katandra. If the species represent fungi normally associated with scat, the source of inoculum and means of distribution are unclear. Some substrates such as earthworms, spiders, insect larvae and macrofungi were found to have a similar suite of species of Trichocomaceae as the adjacent soil or leaf litter (Table 3). This is not surprising given that soil particles pass through the digestive system of earthworms and that many insect larvae spend a proportion of their life cycle buried in the soil. Fungi isolated from surface-disinfected roots, however, seemed to differ from those associated with the surrounding soil matrix and included four undescribed species (Table 3). In some studies, the diversity of Trichocomaceae in natural habitats has been reported to be limited (Christensen 1969, Bissett & Parkinson 1979a, b, c, Parungao, Fryar & Hyde 2002, Paulus et al. 2003). However, other studies have reported a high number of species. From the A horizons of soils in four different ecosystems in central Ontario, Keller & Bidochka (1998) recovered 43 species of Trichocomaceae. Lumley et al. (2001) isolated 38 species of Trichocomaceae from decomposing logs in forests in Alberta, while 23 species of Penicillium were isolated from the A horizon soil of an oak and birch forest in Long Island, NY, USA (Gochenaur 1978). In the present survey, 23 species of Trichocomaceae were identified from leaf litter of a dry sclerophyll forest, including five undescribed species, while 25 species were isolated from rainforest litter (Table 3). From the dry sclerophyll site, 19 species of Trichocomaceae were found in the top soil and 16 in the mineral soil. In the rainforest, 12 species were isolated from top soils and nine from mineral soils. Considering that this survey was conducted in one small nature reserve, our results report a large diversity in comparison with some of the previous studies (Christensen 1969, Bissett & Parkinson 1979a, b, c, Parungao, Fryar & Hyde 2002, Paulus et al. 2003). One reason for these differences may relate to isolation techniques. In this study, we chose to use the
972 dilution and direct plating techniques developed in seed and food mycology (Pitt & Hocking 1997) to isolate species of Trichocomaceae. The media used were specifically developed to assist growth of food spoilage fungi, and are favourable to the growth of Trichocomaceae. Previous surveys used methods that may have reduced the chances of isolating Trichocomaceae. Warcup (1957) compared isolation techniques for soil microfungi and found that a high proportion of fungi obtained from hyphal isolation were not recovered from soil dilution or soil plate methods. Fungi such as Penicillium species, although recovered in high numbers from dilution methods, were never isolated from hyphal propagules. Other studies of leaf litter fungi also relied on different isolation techniques. Paulus et al. (2003) isolated leaf endophytes, while excluding phylloplane species, by washing leaves to remove surface fungi before plating onto a selective medium. They also incubated entire leaves in a moist chamber for six weeks or until fungal fruiting bodies were visible. Both methods failed to isolate any Penicillium species from the surface of leaves. In a study of leaf litter fungi of two rainforest trees, Polishook, Bills & Lodge (1996) used the leaf particle technique (Bills & Polishook 1994), which yielded 18 species of Trichocomaceae. Conversely, the same authors were unsuccessful in isolating Penicillia or Aspergilli from the same litter samples using the direct method of incubating leaves in moist chambers. A high diversity of Trichocomaceae was isolated from a small reserve in eastern Australia, after sampling from a restricted number of sites. Only a few species were found widely, indeed most species were isolated from one or only a few substrates. While the reasons for this diversity are unclear, the results indicate the importance of using appropriate methods and sampling protocols in studies of this type. The mechanisms underlying this diversity remain to be explored, and specificity between substrate and fungus remains to be examined.
ACKNOWLEDGEMENTS Our thanks to Nick Charley (Food Science Australia, North Ryde) and Christine Newman (School of Biological Sciences, University of Sydney) for technical assistance, and Su-lin Leong (Food Science Australia, North Ryde) for providing helpful comments and suggestions. We also wish to thank the Gosford City Council for permission to take samples from Katandra Nature Reserve for this study.
REFERENCES Bills, G. F. & Polishook, J. D. (1994) Abundance and diversity of microfungi in leaf litter of a lowland rainforest in Costa Rica. Mycologia 86 : 187–198. Bisset, J. & Parkinson, D. (1979a) The distribution of fungi in some alpine soils. Canadian Journal of Botany 57: 1609–1629. Bisset, J. & Parkinson, D. (1979b) Fungal community structure in some alpine soils. Canadian Journal of Botany 57: 1630–1641.
A.-L. Markovina and others Bisset, J. & Parkinson, D. (1979c) Functional relationships between soil fungi and environment in alpine tundra. Canadian Journal of Botany 57 : 1642–1659. Bettucci, L. & Roquebert, M.-F. (1995) Microfungi from a tropical rainforest litter and soil: a preliminary study. Nova Hedwigia 61: 111–118. Christensen, M. (1969) Soil microfungi of dry to mesoic coniferhardwood forests in Northern Wisconsin. Ecology 50: 9–27. Christensen, M. (1981) Species diversity and dominance in fungal communities. In The Fungal Community: its organization and role in the ecosystem (D. T. Wicklow & G. C. Carrol, eds): 201–232. Marcel Dekker, New York. Christensen, M. (1989) A view of fungal ecology. Mycologia 81: 1–19. Christensen, M., Frisvad, J. C. & Tuthill, D. E. (2000) Penicillium species diversity in soil and some taxonomic and ecological notes. In Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification (R. A. Samson & J. I. Pitt, eds): 309–320. Harwood Academic Publishers, London. Drummond, J., De Barro, P. J. & Pinnock, D. E. (1991) Field and laboratory studies on the fungus Aspergillus parasiticus, a pathogen of the pink sugar cane mealybug Saccharicoccus sacchari. Biological Control 1: 288–292. Fairley, A. & Moore, P. (2000) Native Plants of the Sydney District: an identification guide. Kangaroo Press, Sydney. Gochenaur, S. E. (1978) Fungi of a long island oak-birch forest I. Community organization and seasonal occurrence of the opportunistic decomposers of the A horizon. Mycologia 70 : 975–994. Keller, L. & Bidochka, M. J. (1998) Habitat and temporal differences among soil microfungal assemblages in Ontario. Canadian Journal of Botany 76: 1798–1805. Klich, M. A. (2002) Biogeography of Aspergillus species in soil and litter. Mycologia 94 : 21–27. Klich, M. A. & Pitt, J. I. (1988) A Laboratory Guide to common Aspergillus Species and their Teleomorphs. CSIRO Division of Food Science, North Ryde, NSW. Lamb, R. J. & Brown, J. F. (1970) Non-parasitic microflora on leaf surfaces of Paspalum dilatatum, Salix babylonica and Eucalyptus stellulata. Transactions of the British Mycological Society 55: 383–390. Lumey, T. C., Gignac, D. & Currah, R. S. (2001) Microfungus communities of white spruce and trembling aspen logs at different stages of decay in disturbed and undisturbed sites in the boreal mixedwood region of Alberta. Canadian Journal of Botany 79: 76–92. Macauley, B. J. & Thrower, L. B. (1966) Succession of fungi in leaf litter of Eucalyptus regnans. Transactions of the British Mycological Society 49: 509–520.
973 Martin, J. H. D. (1967) Records of entomogenous fungi from Queensland. Queensland Journal of Agriculture and Animal Science 24: 109–112. Parungao, M. M., Fryar, S. C. & Hyde, K. D. (2002) Diversity of fungi on rainforest litter in North Queensland, Australia. Biodiversity and Conservation 11: 1185–1194. Paulus, B., Gadek, P. & Hyde, K. D. (2003) Estimation of microfungal diversity in tropical rainforest leaf litter using particle filtration: the effects of leaf storage and surface treatments. Mycological Research 107: 748–756. Perez, J., Infante, F., Vega, F. E., Holguin, F., Macias, J., Valle, J., Nieto, G., Peterson, S. W., Kurtzman, C. P. & O’Donnell, K. (2003) Mycobiota associated with the coffee berry borer (Hypothenemus hampei) in Mexico. Mycological Research 107: 879–887. Pitt, J. I. (1980) [‘ 1979’] The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. Academic Press, London. Pitt, J. I. (2000). A Laboratory Guide to common Penicillium Species, 3rd edn. Food Science Australia, North Ryde, NSW. Pitt, J. I. & Hocking, A. D. (1997) Fungi and Food Spoilage, 2nd edn. Blackie Academic and Professional, London. Pitt, J. I., Samson, R. A. & Frisvad, J. C. (2000) List of accepted species and their synonyms in the family Trichocomaceae. In Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification (R. A. Samson & J. I. Pitt, eds): 9–49. Harwood Academic Publishers, London. Polishook, J. D., Bills, G. F. & Lodge, D. J (1996) Microfungi from decaying leaves of two rainforest trees in Puerto Rico. Journal of Industrial Microbiology 17: 284–294. Raper, K. B. & Fennell, D. I. (1965) The Genus Aspergillus. Williams & Wilkins, Baltimore, MD. Seifert, K., Kendrick, B. & Murase, G. (1983) A Key to Hyphomycetes on Dung. University of Waterloo Press, Waterloo, Ontario. Yasmeen & Saxena, S. K. (1990) Rhizosphere and rhizoplane mycoflora of Adiantum incisum. Indian Fern Journal 7 : 21–23. Yip, H. Y. & Weste, G. (1985) Rhizoplane mycoflora of two understorey species in the dry sclerophyll forest of the Brisbane ranges. Sydowia 38 : 383–399. Warcup, J. H. (1957) Studies on the occurrence and activity of fungi in a wheat-field soil. Transactions of the British Mycological Society 40: 237–262.
Corresponding Editor: S. W. Peterson
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doi:10.1017/S0953756205003540 Printed in the United Kingdom.
Diversity of the Trichocomaceae in the Katandra Nature Reserve, Central Coast, NSW, Australia
Anne-Laure MARKOVINA1, John I. PITT1, Ailsa D. HOCKING1, Deidre A. CARTER2 and Peter A. McGEE3 1
Food Science Australia, P.O. Box 52, North Ryde, NSW 1670, Australia. School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia. 3 School of Biological Sciences, University of Sydney, NSW 2006, Australia. E-mail : [email protected] 2
Received 18 November 2004; accepted 26 May 2005.
The diversity of the family Trichocomaceae, which includes the major anamorph genera Aspergillus and Penicillium, was studied in the Katandra Nature Reserve, Central Coast, NSW, Australia. Soil, living leaves, leaf litter and detritus were examined by both direct and dilution plating techniques. Fungi were isolated on dichloran Rose Bengal chloramphenicol agar, and dichloran 18 % glycerol agar, media suitable for cultivation of many species within this family. Species of Trichocomaceae were isolated from all sites and all substrates examined. A high diversity was found, with more than 50 known species identified, and an equal number of undescribed species detected. More species of Penicillium were recovered than other genera, with Aspergillus species the next most common. Most of the species recovered were anamorphs, though 16 known and unknown ascosporic species were also isolated from heated and unheated soil. Soils, leaf litter, a scat from a native herbivore and leaves of living native plants yielded higher diversity than insects, worms or introduced plants. More species belonging to the family were isolated from soil in dry sclerophyll forest than in rainforest. Conversely, native rainforest plants harboured more diversity than the dry sclerophyll forest plants examined.
INTRODUCTION The family Trichocomaceae includes the major anamorphic genera Penicillium and Aspergillus. Some members of this family are important causes of food spoilage and biodeterioration, while others are widely used in biotechnology. Some produce mycotoxins and many other diverse secondary metabolites (Pitt & Hocking 1997). Species of Penicillium and Aspergillus grow quickly and produce large numbers of conidia which are easily dispersed. As a result, these fungi occur over a wide range of environments. The taxonomy, biology and diversity of genera and species in the family Trichocomaceae in food commodities and processed foods are well documented (Pitt & Hocking 1997). Species of Trichocomaceae have often been recorded in ecological investigations of soils in North American and European forests (Bissett & Parkinson 1979a, b, c, Yasmeen & Saxena 1990, Bettucci & Roquebert 1995, Christensen, Frisvad & Tuthill 2000, Lumey, Gignac & Currah 2001) and other plant communities (Christensen 1969, 1981, 1989). They have also been found in plant materials and arthropods (Martin 1967, Drummond, De Barro & Pinnock 1991,
Perez et al. 2003). However, relatively little is known about the diversity and distribution of these fungi in natural environments other than soils in North America and Europe. Australian studies on the natural occurrence of species of Trichocomaceae are limited. Twenty five species were recorded from soil in a wheat field (Warcup 1957), while Yip & Weste (1985) isolated 41 species in the rhizosphere of two native Australian plants, and Paulus, Gadek & Hyde (2003) isolated six species of Penicillium and one species of Aspergillus from rainforest litter. Lamb & Brown (1970) recovered four species of Penicillium and one of Aspergillus from the phylloplane of three plant species. In studies of fungal succession in leaf litter from Eucalyptus regnans, Macauley & Thrower (1966) suggested that Penicillium species are not responsible for the primary degradation of plant cellulose, but are secondary decomposers, appearing more frequently on leaves in advanced stages of decomposition. Two introduced plants (Paspalum dilatatum and Salix babylonica) and one Australian native tree (Eucalyptus stellulata) were reported to harbour distinctive fungal populations including some species of Trichocomaceae (Lamb & Brown 1970).
A.-L. Markovina and others The objective of this project was to study the diversity of the family Trichocomaceae in a largely undisturbed natural environment in Australia, with a well documented and wide range of habitats and plant species.
965 Table 1. Collection sites and habitat characteristics in Katandra Nature Reserve, Central Coast, NSW. Site type
Soil type
Dominant plant species
Dry sclerophyll
Sand to sandy-loam
Banksia serrata Acacia spp. Pteridium esculentum (bracken fern) Eucalyptus pilularis (blackbutt) Syncarpia glomulifera (turpentine) Allocasurina torulosa (forest oak)
Wet sclerophyll
Sandy-loam
Eucalyptus paniculata (iron bark) Livistonia australis (cabbage Palm) Pittosporum revolutun (native frangipani) Lianas, Palms
Rain forest
Clay
Acmena smithii (lilly pilly) Archontophoenix cunninghamiana (Bangalow Palm) Ceratopetalum apetalum (coachwood) Livistonia australis (cabbage Palm) Ferns and orchids
Creek bank
Clay
Closed rain forest Bangalow Palm grove Ferns Ficus sp. (fig tree)
Bank of Seymour Pond (artificial)
Sandy-loam
Wet sclerophyll, thick leaf litter
MATERIALS AND METHODS Site description The study site, Katandra Nature Reserve on the Central Coast, NSW, Australia, was chosen because the site is readily accessible, has had limited human impact in most parts, and contains a number of different ecosystems within only 53 ha, thus limiting climatic effects within the diverse vegetation. Ridges within the reserve are capped by sandstones of the Hawkesbury series, while the underlying shales and sandstones belong to the Narrabeen group (Fairley & Moore 2000). A feature of the lower part of the reserve is Seymour Pond, a constructed pond that fills from a spring emanating from a nearby cliff face. The vegetation in the Katandra Nature Reserve can be classified into three main types (Fairley & Moore 2000). First, dry sclerophyll (open forest), with vegetation typically two-layered, with a dominant upper canopy, which allows clear vision through the tree trunks for some distance. Eucalyptus and Angophora are the dominant tree genera in this zone. The trees are usually 10–30 m high with flattish crowns and tall, well-developed trunks. Second, wet sclerophyll (tall open forest), where the vegetation consists of three or more layers, with a continuous tree canopy. Usually, the trees are over 30 m high at maturity. Groups of tall shrubs and small trees form an intermediate layer of vegetation over a low shrub understory. Gullies are frequently dominated by Eucalyptus saligna. Many climbers such as Cissus hypoglauca occur in the understory, while ferns and monocotyledon plants are also common. Third, temperate rainforest (closed forest), found on the moist, shady sides of the ridges where, characteristically, the light intensity is low at the forest floor. The rainforest is typified by vegetation in three layers. Climbing plants are very common, with ferns and orchids equally prominent. Eucalyptus trees do not grow in the rainforest, but are abundant around its perimeter. Details of these habitats are given in Table 1. Pilot study A pilot study aimed to determine whether different species of fungi from the family Trichocomaceae occurred in soils associated with the different vegetation types found in Katandra. Six sampling sites were chosen along a 2 km transect across the reserve and over a 200 m change in altitude. The selected transect encompassed dry sclerophyll
forest, wet sclerophyll forest and temperate rainforest. Samples were collected in midsummer (Feb. 2003) using a garden trowel disinfected with 70 % ethanol between samplings. Samples were stored in resealable plastic bags and kept at 4 xC until processed. Samples were analysed within two days of collection. Soil samples (10 g) were dispensed into 90 ml of sterile 0.1 % (w/w) peptone solution. Suspensions were shaken vigorously for one minute, serially diluted, then aliquots (0.1 ml) were plated in duplicate onto selective media, dichloran Rose Bengal chloramphenicol agar (DRBC) and dichloran 18 % glycerol agar (DG18) (Pitt & Hocking 1997). Plates were incubated at 25 x for 5–7 d. Plates were checked regularly to ensure more slowly growing species were subcultured before plates became overgrown. After incubation, isolation plates were examined under a dissecting microscope, and selected colonies subcultured (three point inoculation) onto Czapek Yeast Extract Agar (CYA) and Malt Extract Agar (MEA) (Pitt & Hocking 1997). Plates were incubated at 25 x for 7 d. Plates were incubated at 25 x for 7 d, then examined macroscopically and microscopically. All types of
Diversity of Trichocomaceae in the environment colonies judged to be different and belonging to the family Trichocomaceae were subcultured and transferred to identification media.
Main study A high degree of diversity was observed at each of the pilot study sampling sites, so two further sites were selected for more intensive study. The first site was located in a tall open dry sclerophyll forest, at approx. 40 m altitude. This site appeared to have a considerable diversity of potential substrates, including soil, leaf litter, and small plants, as well as some invertebrates and other detritus. The soil profile was readily accessible, enabling intensive sampling. The second site was situated in a temperate rainforest area, at approximately 20 m altitude, about 0.5 km from the first site. This site had a very high diversity of plant species, but less leaf litter and detritus.
Sampling Samples at the first site were removed from within a 2r2 m quadrat. After a metal rake was used to gather the leaf litter, three soil blocks (20r20 cmr 20 cm) were removed using a clean spade. Each soil block was separated into two sections : topsoil (0–7 cm) and mineral soil (7–20 cm). Topsoil fractions and mineral soil fractions were pooled and stored in clean plastic boxes. Material associated with each soil fraction was stored separately, and included roots, live cockroaches and worms and a macromycete (Pisolithus tinctorius). The leaf litter layer was sorted into leaves, seed pods, live insects, a spider, a cicada exoskeleton, an insect chrysalis, an insect larva and a scat, presumed to be from a native herbivore. Any plants encountered within the quadrat were also collected for analysis. A native bracken fern (Pteridium esculentum) was removed and separated into fronds, root system and rhizosphere. A single seedling of Cinnamomum camphora (camphor laurel, an introduced species) was also collected and analysed for phylloplane fungi. At the second site, the area sampled was a randomly selected 1r1 m quadrat, and only one soil block was removed from the site. Soil fractions, roots and leaf litter were processed and stored as before in individual plastic boxes. In addition, leaves of five native plants, Adiantum hispidulum (rough maidenhair fern), Archontophoenix cunninghamiana (bangalow palm), Synoum glandulosum (scentless rosewood), Stenocarpus salignus (scrub beefwood) and Sloanea australis (maiden’s blush), were collected from plants in the quadrat. All materials were stored individually and kept cool until analysis in the laboratory. Live insects were frozen to kill them before cool storage. All analyses were conducted within four days of sampling.
966 Isolations from soil and leaves Soil attached to the surface of the rhizome of the fern was separated, serially diluted and plated out. Soil was gently removed without washing from roots, which were then cut into 0.5 cm lengths using sterile scissors and gently rolled on the surface of selective media (DRBC and DG18). The same pieces were then surface disinfected in undiluted household bleach (4 % chlorine) for 5 min. After 3 rinses in sterile deionised water, the root pieces were aseptically placed on selective media. Isolations were performed in duplicate. Leaves were gently pressed onto the surface of selective media using flamed forceps. The leaves were then surface-sterilized and incubated in the same manner as the root pieces. Large leaves were cut into smaller pieces prior to isolation. Isolations were performed in duplicate. Isolation from insects and worms Previously killed insects and worms were gently pressed onto duplicate plates of selective media (DRBC and DG18). Worms were additionally surface sterilized in bleach (4 % chlorine) for 5 min and rinsed in sterile deionised water before being plated out on the selective media. Isolation from scat The single scat collected was weighed, broken up, diluted 1 :10 in sterile peptone water, then aliquots (0.1 ml) plated in duplicate onto selective media. Isolation from seed pods Seeds were rolled onto the surface of selective media and incubated at 25 x for 7–10 d. Isolation from insect casings (exoskeletons) These materials were placed on the surface of selective agar and incubated. The surface sterilization step was omitted due to lack of material. All isolations were duplicated. Isolation from leaf litter Leaves from the leaf litter taken from the dry sclerophyll site were sorted into three stages of decomposition : entire leaves, partially decomposed leaves, and fragments that were decomposed, but could still be handled. Four leaves from each stage were removed and gently pressed against the surface of the selective agar, then surface sterilized and plated onto selective media. Each group of leaves was duplicated. From the rainforest site, the leaf litter was sorted into 17 different leaf types based on morphology. Whenever possible, every leaf type was sorted into two leaf stages,
A.-L. Markovina and others
967
fresh and partially decomposed. Each stage of each leaf type was plated out onto selective media.
Table 2. Species of Trichocomaceae isolated from soil in various vegetation stands during the pilot survey, Katandra Nature Reserve.
Heat treatments of soil samples
Fungal species
For the isolation of teleomorph genera from Trichocomaceae, triplicate samples (10 g) of each soil fraction were suspended in 0.1 % peptone (90 ml), heated at 75 x in a waterbath for 15 min and then serially diluted as previously described. Duplicate plates of selective media were inoculated with 0.1 ml of soil suspension.
Aspergillus cervinus A. kanagawaensis Paec. lilacinus Penicillium glabrum P. herquei P. janczewskii P. sclerotiorum P. simplicissimum P. spinulosum P. thomii Penicillium sp. 01 (FRR 5727) Penicillium sp. 02 (FRR 5728) Penicillium sp. 03 (FRR 5729) Talaromyces sp. 01 (FRR 5764) Species richness
Incubation Plates were incubated at 25 x for 7 d. Heated samples were incubated for 14 days.
Dry Wet sclerophyll sclerophyll Rainforest
x
x x x x
x x x x x
x x x x
x
x
x x x 7
9
x 4
Identification of fungi Isolates judged to belong to the family Trichocomaceae by macroscopic and (or) microscopic observations were grown on standard media for identification. Most isolates belonged to Penicillium or Aspergillus : Penicillium species were identified by the methods of Pitt (1980, 2000), and Aspergillus species by those of Klich & Pitt (1988) and Klich (2002). Descriptions of less common newer species were referred to where necessary. Species from other genera were identified using Pitt & Hocking (1997) or specialized texts in a few cases. Isolates which showed characteristics in colony and microscopic morphology judged to differ significantly from known species were designated as putative new species, and assigned numbers as shown in the tables. They were also accessioned into the FRR collection of fungus cultures at Food Science Australia, North Ryde, NSW. More definitive examination by techniques such as secondary metabolites profiles or molecular analysis were not attempted at this stage, but will be used as needed for formal description of the new species.
RESULTS Pilot study transect Fourteen species of Penicillium, Aspergillus and a few other genera of Trichocomaceae were isolated from the pilot study. Seven species were recovered from the dry sclerophyll forest, nine from the wet sclerophyll forest and four from rainforest vegetation (Table 2). The diversity appeared to be quite different at each site, highest in dry sclerophyll and lowest in the rainforest. Undescribed species of Penicillium were isolated from all sites, while one undescribed species of Talaromyces was recovered from the rainforest site. In general, fungi isolated from soil along the transect occurred in low frequencies. P. spinulosum, P. sclerotiorum and P. simplicissimum were all found at more than one site. P. janczewskii was common in dry
sclerophyll forests but was not encountered at other locations. Aspergillus kanagawaensis was isolated on four occasions from wet sclerophyll forest, but occurred only once in dry sclerophyll sites. P. glabrum, usually a common species, was only isolated from one of the wet sclerophyll forest sites. Most species were isolated more than once from an individual site, however such common species as P. paxilli, P. herquei, Paecilomyces lilacinus and Aspergillus cervinus were isolated only once. Main study Rhizosphere and bulk soil Results from the rhizosphere and mineral soil have been pooled. In the dry sclerophyll forest bulk soil, species richness was higher in the topsoil than in the mineral soil. Nineteen species of Trichocomaceae were found in the topsoil including one species of Torulomyces (Tor. laevis) and six undescribed penicillia (Table 3). Sixteen species of Penicillium and Aspergillus were isolated from the mineral soil and the fern rhizosphere, including five undescribed species of Penicillium. Rainforest top soil yielded 12 species of Trichocomaceae including two species of Aspergillus and three undescribed species of Penicillium (Table 2). Fungi isolated from the mineral soil were less numerous than those from the organic layer. Two species of Aspergillus but no new species were isolated from mineral soil. Soils after heat treatment Several Eupenicillium and Neosartorya species were isolated from soils after heating (Table 4). Both mineral and top soils from the dry sclerophyll site showed higher numbers of fungal species than soils from the rainforest habitat.
Diversity of Trichocomaceae in the environment Root materials Many more species of Trichocomaceae were isolated from roots taken from the dry sclerophyll soil than from roots taken from the rainforest site (Table 3). Seven undescribed species of Penicillium and one undescribed species of Torulomyces were isolated from the dry sclerophyll roots, but only two from the rainforest (Table 3). Roots from both top and mineral soils in the dry sclerophyll habitat harboured similar numbers of fungi. At the rainforest site, however, roots isolated from the top soil had a higher fungal diversity than those from the mineral soil. A number of species were obtained from roots after surface disinfection with chlorine, including two undescribed species of Penicillium. Other soil substrates Cockroach and worm surfaces yielded the highest diversity of Trichocomaceae among the substrates collected from the topsoil of dry sclerophyll forest (Table 3). In addition, one undescribed Eupenicillium species was isolated from within the worm taken from the topsoil. Fewer materials were recovered from the mineral soil fraction, and of these, the surface of worms was found to harbour the highest number of fungi (Table 3). Leaf litter Leaves showing most decay (stage 3) among those collected from the dry sclerophyll litter supported the highest number of fungi (14) (Table 3). Similarly, fungal diversity was higher in intact leaves treated with chlorine than in decomposed (stage 3) bleached leaves (data not shown). The lowest fungal diversity and species richness, before and after bleaching, was observed from moderately degraded leaf litter (stage 2, data not shown). Twenty-six species of Trichocomaceae were isolated from the rainforest leaf litter (Table 3). Different types of leaves appeared to have different diversity indices. Diversity indices of entire leaves were within a wide range, 0.45–3.03 (data not shown), suggesting that some leaf types support a more diverse community than others. A similar trend was observed for decomposed leaves, where Gleason indices ranged from 1.44–2.34 (data not shown). Penicillia and aspergilli were recovered from every type of entire leaf ; whilst fungi were isolated from only nine types of decomposed leaves. Materials from within the leaf litter The scat collected from an unknown herbivore yielded a rich fungal biota, i.e. eight species of Penicillium, of which five were undescribed, plus one undescribed species of Aspergillus (Table 3). Fewer fungi were
968 isolated from other substrates. Fungal diversity was similar in seed pods of different native plants. Undescribed species were also isolated from the cicada exoskeleton and the surface of the cockroach (Table 3). Leaves from living plants Only two plant species were collected from the dry sclerophyll forest : a seedling of an introduced plant, camphor laurel (Cinnamomum camphora) and a native braken fern (Pteridium esculentum). Trichocomaceae associated with these plants differed with plant species (data not shown). Only one species of Penicillium, the ubiquitous P. glabrum, was isolated from the leaves of the camphor laurel. Isolates from the leaf surfaces of the native bracken fern were more numerous and diverse. A. niger and P. sclerotiorum were isolated once, P. glabrum occurred on five occasions and an undescribed species of Penicillium (FRR 5631) was isolated twice from the fern leaf surfaces. The difference in fungal population observed in this case is worthy of further investigation. Leaves from each type of native rainforest plant supported diverse communities of Trichocomaceae (Table 5). Seven species including one species of Aspergillus were recovered from the leaf surfaces of Adiantum hispidulum. Nine species, including one undescribed species of Penicillium, were isolated from Archontophoenix cunninghamiana ; six species of Penicillium were found on Synoum glandulosum. Stenocarpus salignus leaves harboured nine species of Penicillia, including one undescribed species of Penicillium. Three species of Penicillium and one species of Eurotium were recovered from the leaves of Sloanea australis.
DISCUSSION This paper describes a close study of the biodiversity in Katandra, a small nature reserve on the Central Coast, New South Wales, Australia. The study was limited in scope, being restricted to genera and species within the family Trichocomaceae. Nevertheless, the diversity encountered in Katandra was very high. Although fewer than ten habitats were sampled, more than 100 fungal species belonging to this one family were isolated. The habitats examined were not exceptional and included soil, decaying vegetation and other detritis, living leaves etc. However, the plant flora of Australia is largely unique, and that of the Sydney basin is very diverse, so perhaps this was to be expected also for the fungi. It was nevertheless unexpected that such a high proportion of isolates would be new to science. Considering that the accepted species of Penicillium described throughout the world number less than 250 (Pitt, Samson & Frisvad 2000), it was surprising that we encountered more than 40 new species within the confines of a small nature reserve. One explanation is that the leaves of Eucalyptus and
Dry sclerophyll
FRR number
Stage 1 leaf litter
Stage 2 leaf litter
Stage 3 leaf litter
Associated top soil substrates
Top soil
Associated mineral soil substrates
Mineral soil
Stage 1 leaf litter
Stage 3 leaf litter
Associated top soil substrates
x
Top soil
Associated mineral soil substrates
Mineral soil
x
x
x
x
x x x
x x x
h, i
x
m
x x
x
x
x x x
5765 5760
g i x x x c, d, e, f
x
x x
x
x
h
x
x x
h, i, j, k
x
m, n
x
x x
x x x
h, i, k h
x x
m m
x x x x
x x
x
x
x
x b
x
x
x
a, b, c, d, e, g
x
b
x
x x
x x
x
x
o
x x
x x
x
x
x
x
x
x x
x x c, d, f, g
x
l
x
x x
a, d, e, f, g
x
x
x
h, l
a, b, e, f
x x
x
x x
h, i, j, k,
x x
x x x
m, n
x
x
x
m, n m
x
x x x
x x
o
969
Aspergillus cervinus A. flavus A. kanagawaensis A. niger A. parvulus A. penicilloides A. restrictus Aspergillus sp. 01 Eupenicillium sp. 01 Paecilomyces carneus P. lilacinus Penicillium aurantiogriseum P. bilaiae P. brevicompactum P. chrysogenum P. citreonigrum P. citrinum P. crustosum P. decumbens P. dendriticum P. expansum P. glabrum P. herquei P. italicum P. janczewskii P. janthinellum P. lividum P. loliense P. miczynskii P. minioluteum P. ochrasalmoneum P. paxilli P. quercetorum P. restrictum P. sclerotiorum P. simplicissimum P. soppii P. spinulosum P. thomii P. verruculosum
Associated leaf litter substrates
Rainforest
A.-L. Markovina and others
Table 3. Anamorphs in the Trichocomaceae isolated from soil, litter and other substrates in dry sclerophyll and rainforest vegetation during the main survey, Katandra Nature Reserve.a
5626 5642 5635 5629 5640 5623 5730 5633 5731 5625 5732 5733 5734 5654 5735 5652 5659 5646 5634 5736 5737 5738 5624 5739 5740 5741 5742 5743 5627 5630 5744 5745 5746 5747 5748 5749 5750 5643 5645 5644
Diversity of Trichocomaceae in the environment
Penicillium sp. 04 Penicillium sp. 05 Penicillium sp. 06 Penicillium sp. 07 Penicillium sp. 08 Penicillium sp. 09 Penicillium sp. 10 Penicillium sp. 11 Penicillium sp. 12 Penicillium sp. 13 Penicillium sp. 14 Penicillium sp. 15 Penicillium sp. 16 Penicillium sp. 17 Penicillium sp. 18 Penicillium sp. 19 Penicillium sp. 20 Penicillium sp. 21 Penicillium sp. 22 Penicillium sp. 23 Penicillium sp. 24 Penicillium sp. 25 Penicillium sp. 26 Penicillium sp. 27 Penicillium sp. 28 Penicillium sp. 29 Penicillium sp. 30 Penicillium sp. 31 Penicillium sp. 32 Penicillium sp. 33 Penicillium sp. 34 Penicillium sp. 35 Penicillium sp. 36 Penicillium sp. 37 Penicillium sp. 38 Penicillium sp. 39 Penicillium sp. 40 Torulomyces laevis Torulomyces sp. 01 Torulomyces sp. 02 Species richness
x x x x x x x x
x
x x
x
x x x h h h h
m
o
p
m m o c c, f f g g g g g x x
x x x x x x x x x x m
12
8
14
19
16
20
16
12
9
a
Substrates associated with leaf litter: a, earth ball; b, chrysalis; c, cicada exoskeleton; d, Casuarina seed pod; e, Eucalyptus seed pod ; f, cockroach; g, scat; substrates associated with dry sclerophyll top soil : h, root ; i, worm; j, insect larva; k, cockroach; l, spider; substrates associated with dry sclerophyll mineral soil: m, root; n, worm; substrates associated with rainforest top soil: o, root; substrates associated with rainforest mineral soil: and p, root.
970
A.-L. Markovina and others
971
Table 4. Teleomorph species of Trichocomaceae isolated from soils subjected to heat treatment, Katandra Nature Reserve. Dry sclerophyll Fungal species Eupenicillium alutaceum E. javanicum E. ludwigii Eupenicillium sp. 02 Eupenicillium sp. 03 Eupenicillium sp. 04 Eupenicillium sp. 05 Eupenicillium sp. 06 Eupenicillium sp. 07 Eupenicillium sp. 08 Neosartorya fisheri Neosartorya sp. 01 Neosartorya sp. 02 Species richness
FRR number
Top soil x x x x x x x x x x x
5753 5754 5755 5756 5757 5758 5759 5761 5762
11
Rain forest Mineral soil
Top soil
Mineral soil
x x x
x
x
x x
x
9
2
x x x 4
Table 5. Species from the Family Trichocomaceae associated with leaves of rainforest plants, Katandra Nature Reserve. Adiantum hispidulum Aspergillus niger A. ochraceus Eurotium rubrum Penicillium brevicompactum P. citrinum P. corylophilum P. expansum P. glabrum P. herquei P. janczewskii P. miczynskii P. paxilli P. purpurogenum P. rugulosum P. sclerotiorum P. spinulosum P. thomii Penicillium sp. 41 (FRR 5751) Penicillium sp. 42 (FRR 5752) Species richness
Archontophoenix cunninghamiana
Synoum glandulosum
Stenocarpus salignus
Sloanea australis
x
x x
x x
x
x x x
x x x
x
x x
x
x x
x
x x
x x x x
x x x
x
x
7
x 9
6
other native Australian genera are very high in tannins, oils and other factors which provide resistance to insect and fungal attack, so that the degradation of such leaves can be expected to demand a distinct mycobiota. Alternatively, extensive ecological surveys of the Trichocomaceae are scarce so that any comprehensive survey might result in the discovery of large numbers of undescribed species. In total, 19 different species classifiable in the family Trichocomaceae were isolated from leaves of living plants, 39 species from leaf litter, 30 from topsoils, and 20 from mineral soil layers. Many more species of Penicillium (72) than Aspergillus (9) were recovered from Katandra. This is in keeping with current knowledge of the ecology of these genera, as Katandra is located on the south eastern side of a range of hills which provide a sheltered spot, enjoying cool to temperate climatic conditions throughout most of
x x x x 9
4
the year. Previous research has shown that Aspergillus species are much more likely to be found in regions where temperatures are high throughout the year (Christensen et al. 2000, Klich 2002). The diversity of phylloplane fungi on living plants in the rainforest was slightly lower than that from leaf litter and the soil beneath (Tables 3 and 5). This was to be expected, as few species in the family Trichocomaceae are known to be plant pathogens or commensals. In addition, the natural defenses of Australian native plants may be inhibitory to phylloplane fungi. The leaf litter sustained a higher number of fungi (23, Table 3) than that of rhizosphere and mineral soils (16, Table 3). Variation in fungal diversity was also evident in the different soil horizons analysed. Fungal diversity was highest in the topsoils and lowest in the deeper soil profiles. Warcup (1957) and Bissett &
Diversity of Trichocomaceae in the environment Parkinson (1979a, b, c) reported a decrease in the frequency of fungal occurrence with increasing soil depth. The large number of species isolated from the herbivore scat is also of interest. Herbivores may feed on native vegetation present in their immediate environment but may also travel some distances in search of food and thus would aid in the dispersal of native microfungi across potentially different ecosystems. Only four of the nine species of Trichocomaceae isolated from the scat were found in the adjacent leaf litter (Table 3), and the remaining five were undescribed species of Penicillium. A large number of species from Trichocomaceae have been described from dung (Siefert, Kendrick & Murase 1983). If the undescribed species arose from materials consumed by the herbivore, then this survey may have underestimated diversity on plant substrates at Katandra. If the species represent fungi normally associated with scat, the source of inoculum and means of distribution are unclear. Some substrates such as earthworms, spiders, insect larvae and macrofungi were found to have a similar suite of species of Trichocomaceae as the adjacent soil or leaf litter (Table 3). This is not surprising given that soil particles pass through the digestive system of earthworms and that many insect larvae spend a proportion of their life cycle buried in the soil. Fungi isolated from surface-disinfected roots, however, seemed to differ from those associated with the surrounding soil matrix and included four undescribed species (Table 3). In some studies, the diversity of Trichocomaceae in natural habitats has been reported to be limited (Christensen 1969, Bissett & Parkinson 1979a, b, c, Parungao, Fryar & Hyde 2002, Paulus et al. 2003). However, other studies have reported a high number of species. From the A horizons of soils in four different ecosystems in central Ontario, Keller & Bidochka (1998) recovered 43 species of Trichocomaceae. Lumley et al. (2001) isolated 38 species of Trichocomaceae from decomposing logs in forests in Alberta, while 23 species of Penicillium were isolated from the A horizon soil of an oak and birch forest in Long Island, NY, USA (Gochenaur 1978). In the present survey, 23 species of Trichocomaceae were identified from leaf litter of a dry sclerophyll forest, including five undescribed species, while 25 species were isolated from rainforest litter (Table 3). From the dry sclerophyll site, 19 species of Trichocomaceae were found in the top soil and 16 in the mineral soil. In the rainforest, 12 species were isolated from top soils and nine from mineral soils. Considering that this survey was conducted in one small nature reserve, our results report a large diversity in comparison with some of the previous studies (Christensen 1969, Bissett & Parkinson 1979a, b, c, Parungao, Fryar & Hyde 2002, Paulus et al. 2003). One reason for these differences may relate to isolation techniques. In this study, we chose to use the
972 dilution and direct plating techniques developed in seed and food mycology (Pitt & Hocking 1997) to isolate species of Trichocomaceae. The media used were specifically developed to assist growth of food spoilage fungi, and are favourable to the growth of Trichocomaceae. Previous surveys used methods that may have reduced the chances of isolating Trichocomaceae. Warcup (1957) compared isolation techniques for soil microfungi and found that a high proportion of fungi obtained from hyphal isolation were not recovered from soil dilution or soil plate methods. Fungi such as Penicillium species, although recovered in high numbers from dilution methods, were never isolated from hyphal propagules. Other studies of leaf litter fungi also relied on different isolation techniques. Paulus et al. (2003) isolated leaf endophytes, while excluding phylloplane species, by washing leaves to remove surface fungi before plating onto a selective medium. They also incubated entire leaves in a moist chamber for six weeks or until fungal fruiting bodies were visible. Both methods failed to isolate any Penicillium species from the surface of leaves. In a study of leaf litter fungi of two rainforest trees, Polishook, Bills & Lodge (1996) used the leaf particle technique (Bills & Polishook 1994), which yielded 18 species of Trichocomaceae. Conversely, the same authors were unsuccessful in isolating Penicillia or Aspergilli from the same litter samples using the direct method of incubating leaves in moist chambers. A high diversity of Trichocomaceae was isolated from a small reserve in eastern Australia, after sampling from a restricted number of sites. Only a few species were found widely, indeed most species were isolated from one or only a few substrates. While the reasons for this diversity are unclear, the results indicate the importance of using appropriate methods and sampling protocols in studies of this type. The mechanisms underlying this diversity remain to be explored, and specificity between substrate and fungus remains to be examined.
ACKNOWLEDGEMENTS Our thanks to Nick Charley (Food Science Australia, North Ryde) and Christine Newman (School of Biological Sciences, University of Sydney) for technical assistance, and Su-lin Leong (Food Science Australia, North Ryde) for providing helpful comments and suggestions. We also wish to thank the Gosford City Council for permission to take samples from Katandra Nature Reserve for this study.
REFERENCES Bills, G. F. & Polishook, J. D. (1994) Abundance and diversity of microfungi in leaf litter of a lowland rainforest in Costa Rica. Mycologia 86 : 187–198. Bisset, J. & Parkinson, D. (1979a) The distribution of fungi in some alpine soils. Canadian Journal of Botany 57: 1609–1629. Bisset, J. & Parkinson, D. (1979b) Fungal community structure in some alpine soils. Canadian Journal of Botany 57: 1630–1641.
A.-L. Markovina and others Bisset, J. & Parkinson, D. (1979c) Functional relationships between soil fungi and environment in alpine tundra. Canadian Journal of Botany 57 : 1642–1659. Bettucci, L. & Roquebert, M.-F. (1995) Microfungi from a tropical rainforest litter and soil: a preliminary study. Nova Hedwigia 61: 111–118. Christensen, M. (1969) Soil microfungi of dry to mesoic coniferhardwood forests in Northern Wisconsin. Ecology 50: 9–27. Christensen, M. (1981) Species diversity and dominance in fungal communities. In The Fungal Community: its organization and role in the ecosystem (D. T. Wicklow & G. C. Carrol, eds): 201–232. Marcel Dekker, New York. Christensen, M. (1989) A view of fungal ecology. Mycologia 81: 1–19. Christensen, M., Frisvad, J. C. & Tuthill, D. E. (2000) Penicillium species diversity in soil and some taxonomic and ecological notes. In Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification (R. A. Samson & J. I. Pitt, eds): 309–320. Harwood Academic Publishers, London. Drummond, J., De Barro, P. J. & Pinnock, D. E. (1991) Field and laboratory studies on the fungus Aspergillus parasiticus, a pathogen of the pink sugar cane mealybug Saccharicoccus sacchari. Biological Control 1: 288–292. Fairley, A. & Moore, P. (2000) Native Plants of the Sydney District: an identification guide. Kangaroo Press, Sydney. Gochenaur, S. E. (1978) Fungi of a long island oak-birch forest I. Community organization and seasonal occurrence of the opportunistic decomposers of the A horizon. Mycologia 70 : 975–994. Keller, L. & Bidochka, M. J. (1998) Habitat and temporal differences among soil microfungal assemblages in Ontario. Canadian Journal of Botany 76: 1798–1805. Klich, M. A. (2002) Biogeography of Aspergillus species in soil and litter. Mycologia 94 : 21–27. Klich, M. A. & Pitt, J. I. (1988) A Laboratory Guide to common Aspergillus Species and their Teleomorphs. CSIRO Division of Food Science, North Ryde, NSW. Lamb, R. J. & Brown, J. F. (1970) Non-parasitic microflora on leaf surfaces of Paspalum dilatatum, Salix babylonica and Eucalyptus stellulata. Transactions of the British Mycological Society 55: 383–390. Lumey, T. C., Gignac, D. & Currah, R. S. (2001) Microfungus communities of white spruce and trembling aspen logs at different stages of decay in disturbed and undisturbed sites in the boreal mixedwood region of Alberta. Canadian Journal of Botany 79: 76–92. Macauley, B. J. & Thrower, L. B. (1966) Succession of fungi in leaf litter of Eucalyptus regnans. Transactions of the British Mycological Society 49: 509–520.
973 Martin, J. H. D. (1967) Records of entomogenous fungi from Queensland. Queensland Journal of Agriculture and Animal Science 24: 109–112. Parungao, M. M., Fryar, S. C. & Hyde, K. D. (2002) Diversity of fungi on rainforest litter in North Queensland, Australia. Biodiversity and Conservation 11: 1185–1194. Paulus, B., Gadek, P. & Hyde, K. D. (2003) Estimation of microfungal diversity in tropical rainforest leaf litter using particle filtration: the effects of leaf storage and surface treatments. Mycological Research 107: 748–756. Perez, J., Infante, F., Vega, F. E., Holguin, F., Macias, J., Valle, J., Nieto, G., Peterson, S. W., Kurtzman, C. P. & O’Donnell, K. (2003) Mycobiota associated with the coffee berry borer (Hypothenemus hampei) in Mexico. Mycological Research 107: 879–887. Pitt, J. I. (1980) [‘ 1979’] The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. Academic Press, London. Pitt, J. I. (2000). A Laboratory Guide to common Penicillium Species, 3rd edn. Food Science Australia, North Ryde, NSW. Pitt, J. I. & Hocking, A. D. (1997) Fungi and Food Spoilage, 2nd edn. Blackie Academic and Professional, London. Pitt, J. I., Samson, R. A. & Frisvad, J. C. (2000) List of accepted species and their synonyms in the family Trichocomaceae. In Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification (R. A. Samson & J. I. Pitt, eds): 9–49. Harwood Academic Publishers, London. Polishook, J. D., Bills, G. F. & Lodge, D. J (1996) Microfungi from decaying leaves of two rainforest trees in Puerto Rico. Journal of Industrial Microbiology 17: 284–294. Raper, K. B. & Fennell, D. I. (1965) The Genus Aspergillus. Williams & Wilkins, Baltimore, MD. Seifert, K., Kendrick, B. & Murase, G. (1983) A Key to Hyphomycetes on Dung. University of Waterloo Press, Waterloo, Ontario. Yasmeen & Saxena, S. K. (1990) Rhizosphere and rhizoplane mycoflora of Adiantum incisum. Indian Fern Journal 7 : 21–23. Yip, H. Y. & Weste, G. (1985) Rhizoplane mycoflora of two understorey species in the dry sclerophyll forest of the Brisbane ranges. Sydowia 38 : 383–399. Warcup, J. H. (1957) Studies on the occurrence and activity of fungi in a wheat-field soil. Transactions of the British Mycological Society 40: 237–262.
Corresponding Editor: S. W. Peterson