Eucalyptus microfungi known from culture. 1. Cladoriella and Fulvoflamma genera nova, with notes on some other poorly known taxa.

A study of microfungi associated with living Eucalyptus leaves and leaf litter revealed several novel and interesting taxa. Cladoriella eucalypti gen. et sp. nov. is described as a Cladosporium-like genus associated with litter collected in South Africa, while Fulvoflamma eucalypti gen. et. sp. nov. is newly described from leaf litter collected in Spain. Beta-conidia are newly reported for species of Pestalotiopsis, namely Pestalotiopsis disseminata in New Zealand, and a Pestalotiopsis sp. from Colombia. Satchmopsis brasiliensis is reported from litter in Colombia and Indonesia, while Torrendiella eucalypti is reported from leaf litter in Indonesia, and shown to have a Sporothrix-like anamorph. Leptospora rubella is reported from living Eucalyptus leaves in Colombia, where it is associated with leaf spots of Mycosphaerella longibasalis, while Macrohilumeucalypti is reported from leaf spots of Eucalyptus in New Zealand.


INTRODUCTION
The genus Eucalyptus (Myrtaceae) contains approximately 700 species (Potts & Pederick 2000), most of which are known to host a range of incredibly diverse and interesting microfungi (Crous et al. 1989, Sankaran et al. 1995. In recent years there have been numerous papers listing and describing the plantpathogenic fungi occurring on eucalypts in the various countries where these trees are grown as ornamentals, or planted in plantations for timber and paper fibre (Old & Davison 2000, Park et al. 2000. As the majority of the plant-pathogenic fungi are known from culture, this has enabled plant pathologists to revise numerous important pathogen complexes such as Mycosphaerella leaf blotch (Crous 1998, 2001, Hunter et al. 2004, Cylindrocladium leaf blight (Crous 2002, Cryphonectria canker (Gryzenhout et al. 2004), Botryosphaeria canker (Slippers et al. 2004ac), Coniella (Van Niekerk et al. 2004), Cytospora (Adams et al. 2005), and Harknessia leaf spots (Lee et al. 2004), to name but a few. In contrast, however, the saprobic microfungi have largely been neglected, and in spite of checklists and descriptions, very few are in fact known from culture, or are represented in freely accessible culture collections. As such, many of these diverse genera will never be represented in international initiatives like Assembling the Tree of Life (AToL), or the Consortium for the Barcoding of Life (CBoL), and biologists will remain ignorant as to their distribution, host range, importance and various ecological roles.
Because the eucalypt microbial community is so rich and diverse, and appears to harbour numerous undescribed and relatively unstudied fungal species, it was decided to focus on this host substrate to obtain cultures for inclusion in larger projects and international initiatives such as those cited above. The current paper represents the first in a series aimed at describing eucalypt microfungi from culture, and recollecting and culturing those already known (Sankaran et al. 1995), to help elucidate their taxonomy, and resolve their phylogenetic relationships.

Isolates
Leaf litter as well as living, symptomatic leaves were chosen for study. Leaves were incubated in moist chambers (Petri dishes with moist filter paper on the laboratory bench), and inspected daily for microfungi. Hyphomycetes and coelomycetes were cultured on 2 % malt extract agar (MEA) plates (Gams et al. 1989) by obtaining single conidial colonies as explained in Crous (2002). Single germinating ascospores were obtained and cultured using the technique as explained in Crous (1998). Colonies were sub-cultured onto fresh MEA, oatmeal agar (OA), cornmeal agar (CMA) and carnation leaf agar (CLA) plates (Gams et al. 1989) and incubated at 25 °C under continuous near-ultraviolet light, to promote sporulation.

DNA amplification and sequence analysis
Genomic DNA was isolated from fungal mycelium grown on malt extract agar plates following the protocol of . The primers ITS1 and ITS4 (White et al. 1990) were used to amplify part (ITS) of the nuclear rRNA operon spanning the 3' end of the 18S rRNA gene (SSU), the first internal transcribed spacer (ITS1), the 5.8S rRNA gene, the second ITS region and the 5' end of the 28S rRNA gene (LSU). PCR conditions and protocols were treated and generated as explained in Crous et al. (2004a). Part of the 18S rRNA gene was amplified and sequenced as explained in Braun et al. (2003) and part of the 28S rRNA gene as explained in Lee et al. (2004). ITS sequences were subjected to a nucleotide-nucleotide BLAST (Altschul et al. 1997) of the NCBI sequence database (BLAST-N 2.2.11; http://www.ncbi.nlm.nih.gov/). The LSU and / or SSU sequences were also used in cases where ITS sequences did not provide adequate BLAST results.

Taxonomy
Fungal structures were mounted in lactic acid or in water when stated. The extremes of spore measurements (30 observations) are given in parentheses. Colony colours (surface and reverse) were rated after 7-14 d on MEA and OA at 25 °C in the dark, using the colour charts of Rayner (1970). All cultures obtained in this study are maintained in the culture collection of the Centraalbureau voor Schimmelcultures (CBS) in Utrecht, the Netherlands (Table 1), and type specimens in the mycology herbarium (PREM) at the Biosystematics Division of the Plant Protection Research Institute, Agricultural Research Council of South Africa.
Cultural characteristics: Colonies on MEA producing abundant amounts of diffusing red pigment that changes the colour of the medium to red; colonies irregular,   Notes: The genus Cladosporium Link contains 772 names (Dugan et al. 2004), many of which represent elements not congeneric with the type species, C. herbarum (Pers.: Fr.) Link, which is an anamorph of Davidiella Crous & U. Braun (Braun et al. 2003). The recent description of Devriesia Seifert & N.L. Nickerson (Seifert et al. 2004) for a group of heatresistant, chlamydospore forming species with slightly thickened conidial scars proves this point. Cladoriella resembles Devriesia in general morphology, but lacks chlamydospores, forms a distinct red pigment in culture, and clusters apart from the Cladosporium complex (Mycosphaerellaceae), the Cladophialophora Borelli complex (Herpotrichiellaceae), or the Pseudocladosporium U. Braun complex (Venturiaceae). BLAST results of the ITS sequence of this species had an E-value of 1e-90 with ITS sequences of
Etymology: Named after its characteristic conidiomata and spore masses that appear as orange candle flames once plant material is incubated in moist chambers.
Mycelium immersed, consisting of smooth, hyaline, branched, septate hyphae, 1-1.5 µm wide; aggregating in the epidermis to form a pale to dark brown stroma, up to 50 µm wide, which gives rise to a conidioma. Conidiomata sporodochial, appearing as erect, orange, fusoid structures on the leaf surface (like the flame of a candle), up to 100 µm diam and 200 µm high; basal region consisting of pale brown cells of textura angularis to textura epidermoidea, 3-7 × 2-3 µm, giving rise to thick-walled, pale brown cells of textura porrecta, 6-15 × 2-3 µm, becoming thin-walled, hyaline, and radiating outwards from the narrower, semi- Notes: As far as we could establish, M. eucalypti has not previously been known from culture (Swart 1988  Cultural characteristics: Colonies on OA reaching 52-54 mm diam in 7 d with an even, glabrous, colourless margin; immersed mycelium colourless, aerial mycelium pure white, fluffy, covering most of the colony surface, and very dense and high in the centre and in concentric zones after 7 d; reverse in the centre buff. Colonies on CMA reaching 52-55 mm diam after 7 d, as on OA, but aerial mycelium less well-developed, and reverse colourless. Colonies on MEA reaching 56 mm diam in 7 d, with an even or slightly undulating colourless margin; immersed mycelium colourless, but surface of the colony completely covered by a high, dense mat of pure white, in the centre yellowish, fluffy aerial mycelium, the margin also covered by a diffuse layer of aerial hyphae; reverse with a faint cinnamon tinge.

Substrate and distribution: Eucalyptus botryoides, New
Zealand (North Island).  to ruffled, and aerial mycelium less well-developed. Colonies on MEA reaching 49-55 mm diam in 7 d, with an irregularly undulating, colourless, glabrous margin; immersed mycelium colourless, but surface of the colony completely covered by a moderately high, densely woolly mat of pure white, locally faintly sulphuryellow, aerial mycelium; reverse ochreous to fulvous, brown where conidiomata develop.
Substrate and distribution: Eucalyptus eurograndis?, Colombia. Notes: BLASTn results of the ITS sequence of this species had an E-value of 0.0 with ITS sequences of Pestalotiopsis spp., including Pestalotiopsis disseminata and Pestalotiopsis uvicola (Speg.) Bissett (both 99 % similar). The primary reason for the inclusion of these Pestalotiopsis spp. in the present paper is the presence of a synanamorph, which has never before been reported for species of Pestalotiopsis in the literature (Nag Raj 1993). According to unpublished notes in the CBS database, this has once before been observed for a culture of a Pestalotiopsis sp. in the collection. Conidiomata were observed in host tissue to exude a mixture of black and hyaline spores in a typical cirrhus associated with Pestalotiopsis conidiomata. The cirrhus consisted of two conidial types, namely typical Pestalotiopsis conidia (alpha), and long, narrow, bent, needle-like cylindrical conidia (beta) resembling the beta conidia observed in species of Phomopsis, or the conidia typically associated with Libertella anamorphs. Conidia were 25-30 ×1-1.5 µm, widest in the middle, tapering to a subobtuse apex, and a truncate base. Conidia were formed on slightly tapering, hyaline, subcylindrical conidiogenous cells that terminated in an apex with 1-2 loci which gave rise to conidia in a sympodial arrangement. In some cases the conidiogenous cells were situated on 1-3-septate conidiophores that were 10-20 × 2-3 µm.
Beta-conidia were initially observed in the collection obtained from Colombia. Although they occurred in the same conidioma, none could be induced to germinate on MEA (observed over 2 wk), while all alpha conidia germinated within 1-2 d. The second collection which had a mixture of both conidial types was obtained from New Zealand. Again, the beta-conidia could not be induced to germinate, and thus their ecological role as potential conidia, or spermatia, still needs to be resolved. None of the colonies derived from alpha conidia could be induced to form beta conidia on MEA, OA or CLA. In this regard it is interesting to note that, contrary to common opinion, it has only recently been proven that beta-conidia of Phomopsis spp. do, in fact, germinate in culture (Sergeeva et al. 2003). B. Sutton & Hodges, Nova Hedwigia 26: 3. 1975. Fig. 6. Conidiomata cupulate, superficial, up to 180 µm wide and 100 µm deep, dark brown, attached centrally to a stroma of dark brown cells that occupy the stomatal chamber; wall consisting of two regions, the lower region having thick-walled dark-brown cells, up to 5 layers thick, the upper region consisting of thin-walled, paler cells, up to 5 layers thick. Conidiogenous cells restricted to the lower part of the basal wall, 3-7 × 2-3 µm, doliiform to lageniform, phalidic with periclinal thickening, hyaline, with an indistinct collarette. Conidia hyaline, aseptate, guttulate, subcylindrical, predominantly straight, with obtuse ends, 11-17 × 1-1.5 µm.

Satchmopsis brasiliensis
Cultural characteristics: Colonies spreading on MEA, flat with sparse aerial mycelium and smooth margins; surface sienna to umber with patches of white, and dark brown conidiomata; reverse umber (centre) to sienna (margins); on OA umber with no aerial mycelium, and dark brown conidiomata.
Substrate and distribution: Eucalyptus spp., Colombia, Indonesia. Notes: The collections from Indonesia and Colombia are morphologically similar. Colonies appear similar on MEA, and conidia of the Indonesian collection (11-17 × 1-1.5 µm) are similar to those of the Colombian collection (12-14 × 1-1.5 µm), and fit within the range given for the species, namely 11.5-15.5 × 1-1.5 µm (Sutton 1975). However, from the sequence data (data not shown) it is clear that there are some base pair differences between these isolates, suggesting that these strains may in fact represent different species. The only obvious morphological difference observed was that conidiomata of the Colombian collection were pale brown, with cells at the margin of the wall being up to 5 µm wide. In contrast, conidiomata from the Indonesian collection were darker brown, with cells at the margins being narrower, namely 3-4 µm wide. Whether these morphological differences can be related to the differences observed in the DNA sequences, can only be resolved once further collections have been obtained. BLASTn results of the ITS sequence of this species has E-values of 5e-167 to 1e-115 with ITS sequences of unidentified leaf litter and mycorrhizal ascomycetes. The closest known species include Pezicula frangulae (Pers.) Fuckel (2e-110), Pezicula ocellata (Pers.) Seaver (9e-107; Helotiales), and Cryptosporiopsis spp. Apothecia on host scattered or gregarious in large groups, erumpent, stipitate, arising from a subepidermal stroma visible around the stipe as a dark discoloration. Disc plane to convex, greyish brown to olivaceous, smooth, 0.4-1.5 mm diam. Receptacle cupulate, concolorous but usually darker than the hymenium, bearing dark brown to reddish brown setae. Stipe central, smooth and dark brown, 0.4-1.8 mm high. Setae mostly 20-50 per apothecium, (150-)200-250 µm long, smooth, with dark brown walls thickened up to 1.5 µm, septate, paler at the blunt top, attenuated and bent at the base. Asci cylindrical-clavate, apex conicalrounded, the apical apparatus blueing in Melzer's reagent, croziers present, 8-spored, 75-100 × 7-9 µm; ascospores fusoid, 0-septate, narrowly rounded at both ends, contents guttulate, hyaline, each end provided with a central, everted (umbrella-shaped) mucelaginous appendage, 17-25 × 3-4 µm; sometimes producing ellipsoid microspores 3-5.5 × 1.5-2 µm directly from apertures at one or both ends. Paraphyses simple or branched near the base, obtuse, hyaline, somewhat inflated and up to 3.5 µm wide at the top.
Cultural characteristics: Colonies on OA reaching a diam of 15-20(-30) mm in 14 d, with an even to slightly ruffled, glabrous and colourless margin; immersed mycelium at first colourless, then very faintly yellowish (primrose) or reddish (apricot), after 10-20 d gradually developing a mixture of several tinges, pale hazel, ochreous and amber, in the centre sometimes also greyish to olivaceous buff, most of the surface almost glabrous and without aerial mycelium, locally with patches of woolly, pure-white aerial mycelium. Colonies on MEA reaching 33-37 mm diam in 14 d, with a ruffled, glabrous, colourless margin; most of the colony surface covered by a fairly dense, woolly but low mat of pure white aerial mycelium; reverse in centre ochreous to umber, fading to the colourless margin.
Apothecia formed on OA after about 10 wk, mostly on the agar surface, most very similar in shape and size to those formed in planta, but with less setae; however, large abnormally shaped apothecia are also formed: hymenium convex, protruding from the agar surface as a greyish-black, globular mass with a smooth surface, 1-2.5 mm diam, receptacle reduced, hairs present or absent, lacking a stipe.
Anamorph in vitro: Conidiophores developing on the surface of globular ascomatal initials after 2-3 wk, smooth-walled, variable, simple, but mostly branched near the base, 15-30 × 2-4(-5) µm thick, hyaline or somewhat yellowish brown, conidiogenesis blastic, sympodial, sometimes seemingly retrogressive, apertures mostly terminal but also immediately below septa (acropleurogenous), scars visible but not thickened or protruding; conidia hyaline, ellipsoid, broadly rounded at the top, slightly attenuated into a blunt base, with one or two small guttules, 4-5.2(-6) × (1.5-)1.8-2 µm. Notes: The material used in this study generally agrees well with the description given by Spooner (1987). There are, however, some additional observations that were not reported by this author, particularly, the presence of apical appendages on the ascospores, and the production of microspores from liberated ascospores. We observed 3-8 large guttules in ascospores of T. eucalypti, while Spooner reported only two or three guttules per spore. After drying, the guttules in our material often merged into larger bodies, and this could explain the difference between our obervations and those of Spooner, which were based on herbarium specimens. After drying of our specimen, the  appendages of the ascospores were barely visible. The present study is also the first to report on observtions in pure culture. The anamorph was only observed in culture, and showed plasticity in conidiogenesis making it very difficult to assign it to a particular anamorph genus. It could be circumscribed as Sporothrix-like, although it lacks the denticles characteristic of that anamorph, and it also differs by branched and septate conidiophores. Sporothrix schenkii Hektoen & C.F. Perkins, the type species of Sporothrix Hektoen & C.F. Perkins, is commonly isolated from Eucalyptus wood, but is linked to Ophiostoma Syd. & P. Syd. BLASTn results of the ITS sequence of this species had an E-value of 0.0 with ITS sequences of