Phylogenetic reassessment of Mycosphaerella spp. and their anamorphs occurring on Eucalyptus. II.

Species of Eucalyptus are widely planted as exotics in the tropics and Southern Hemisphere and to some extent in southern Europe, for timber and fibre production. Species of Mycosphaerella are commonly associated with leaves and twigs of Eucalyptus and can result in defoliation, dieback, and even tree death. In the present study, numerous isolates of Mycosphaerella species were collected from leaf litter, living leaves exhibiting leaf spot symptoms or severe Mycosphaerella leaf blotch symptoms. Isolates were compared based on DNA sequence data for the internal transcribed spacer region (ITS1 & ITS2) and the 5.8S gene. These data, together with characteristics of the fungal growth on three different media, morphology of the anamorph and teleomorph structures as well as ascospore germination patterns were used to describe 21 new species.

INTRODUCTION

Eucalyptus spp. are widely planted in the tropics and Southern Hemisphere, providing important sources of structural timber and fibre. Fungal diseases have, however, had a negative impact on their cultivation in many parts of the world (Wingfield ). Mycosphaerella leaf blotch (MLB) was one of the first diseases to seriously damage plantations of Eucalyptus outside their native range, leading to the abandonment of some species for plantation development (Lundquist & Purnell 1987).

Mycosphaerella leaf blotch has been associated with severe defoliation, shoot die-back, and even tree death. This damage has mostly been attributed to M. cryptica (Cooke) Hansf. and M. nubilosa (Cooke) Hansf. (Carnegie , Crous & Wingfield 1996, Wingfield , Cheah 1977, Dungey ). In recent years, it has become apparent that there are many more species of Mycosphaerella Johanson occurring on eucalypts than previously realised. While some of these fungi cause serious disease problems, others cause minor leaf spots, rarely resulting in severe disease (Crous 1998, Crous ). Little is known regarding some of these less important species but some could become more important in genetically uniform plantations of susceptible clonal hybrids or where trees are exposed to conditions of stress.

The genus Mycosphaerella Johanson includes more than 2000 species names (Corlett 1991), and several thousand anamorphs that lack known teleomorphs (Crous & Braun 2003). Of these, 55 species from eucalypts were treated by Crous (1998) and several additional species have been described more recently (Carnegie & Keane 1998, Braun & Dick 2002, Maxwell , Crous , Hunter ). Species of Mycosphaerella are usually assumed to be host-specific, and presently there are little data available that can be used to refute this supposition. Although some taxa have been found to infect other, secondary hosts (Crous , Groenewald ), most seem to have narrow host ranges. Interestingly, where species have been reported to have wider host ranges within a plant family, e.g. as reported for Ramularia Unger anamorphs by Braun (1998), DNA-based techniques have clearly shown that in most cases these morphologically similar taxa are phylogenetically quite distinct (Crous & Groenewald, unpubl. data). Further confusion could result from species colonising atypical host tissue in an attempt to jump to an ideal host when this becomes available. Crous & Groenewald (2005) have referred to this unusual behavioural pattern as the “pogo stick hypothesis”. In Mycosphaerella it has been observed to be true for teleomorph as well as anamorph states. When isolates of these fungi colonising atypical substrates are collected without proving their pathogenicity, incorrect conclusions pertaining to host range could arise.

The genus Mycosphaerella includes species that are pathogens (primary, secondary or opportunistic), saprobes, endophytes (saprobic or plant-pathogenic), or have mutualistic (in lichen) associations (Crous et al. 2000, 2001). Several taxa have low levels of virulence, and appear to be secondary colonists of lesions caused by other pathogens including species of Mycosphaerella (Crous 1998). Some species of Ramularia also appear to be hyperparasites on pustules of various rust species (Braun 1998). Because several species can co-inhabit the same lesion, either as primary or secondary pathogens, saprobes or endophytes (Crous 1998, Crous ), species identification based on the host can be extremely difficult. Although ascospore germination patterns, anamorph morphology and cultures greatly facilitate species identification, co-inhabitancy (Crous & Groenewald 2005) makes it difficult to link these cultures and anamorphs to their correct teleomorphs (Crous 2002).

The present study presents the second in a series characterising the Mycosphaerella species occurring on eucalypts. A major aim of this study was to use comparisons of DNA sequence data to clarify as many as possible of the formerly published host and distribution records (Crous 1998). Furthermore, while previous descriptions focused on species associated with leaf spots, this study also includes species from eucalypt leaf litter.

MATERIALS AND METHODS

Isolates

Eucalyptus leaves bearing Mycosphaerella ascomata, or with Mycosphaerella leaf spots were chosen for study. Excised lesions were soaked in water for approximately 2 h, after which they were placed in the bottom of Petri dish lids, with the top half of the dish containing 2 % malt extract agar (MEA) (Biolab, Midrand, South Africa). Ascospore germination patterns were examined after 24 h, and single-ascospore and conidial cultures established as described by Crous (1998). Colonies were sub-cultured onto carnation leaf agar (CLA) [1 % water agar (Biolab) with autoclaved carnation leaves placed onto the surface of the solidified medium] and incubated at 25 °C under continuous near-ultraviolet light to promote sporulation.

DNA phylogeny

The protocol of Lee & Taylor (1990) was used to isolate genomic DNA from fungal mycelium, grown on MEA in Petri dishes. The primers ITS1 and ITS4 (White ) were used to amplify part of the nuclear rRNA operon spanning the 3' end of the 18S rRNA gene, the first internal transcribed spacer (ITS1), the 5.8S rRNA gene, the second ITS region and the 5' end of the 28S rRNA gene. The PCR reaction mixture and conditions were the same as those used by Crous et al. (2004b).

The ITS nucleotide sequences generated in this study were added to other sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov) and the alignment was assembled using Sequence Alignment Editor v. 2.0a11 (Rambaut 2002) with manual adjustments for visual improvement where necessary. Due to the size and the complexity of the original alignment, the sequences were split over four smaller alignments, each containing genetically similar sequences. The four datasets were each treated identically. Phylogenetic analyses of sequence data were done using PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2002). Phylogenetic analysis of the aligned ITS sequence data consisted of neighbour-joining analysis with the uncorrected (“p”), the Kimura 2-parameter and the HKY85 substitution model in PAUP. Alignment gaps were treated as missing data and all characters were unordered and of equal weight. When they were encountered, ties were broken randomly. Sequence data were deposited in GenBank (Table 1) and the alignments in TreeBASE.

Table 1.

Mycosphaerella and anamorph isolates included in this study for sequence analysis and morphological comparison.

Teleomorph	Anamorph	Strain no.1	Substrate	Country	Collector	ITS GenBank number
Mycosphaerella communis	Dissoconium commune	CPC 11700	Eucalyptus globulus	Spain	P. Mansilla	DQ302948
		CPC 11703	Eucalyptus globulus	Spain	P. Mansilla	DQ302949
		CPC 11792	Eucalyptus sp.	Portugal	A.J.L. Phillips	DQ302950
Mycosphaerella cryptica	Colletogloeopsis nubilosum	CBS 111679; CPC 1576	Eucalyptus nitens	Australia	M.J. Wingfield	DQ302951
Mycosphaerella endophytica	Pseudocercosporella endophytica	CBS 111519; CPC 1191	Eucalyptus sp.	South Africa	P.W. Crous	DQ302952
		CBS 114662; CPC 1193	Eucalyptus sp.	South Africa	P.W. Crous	DQ302953
Mycosphaerella eucalyptorum	—	CBS 118496; CPC 11174	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ302954
Mycosphaerella flexuosa	Stenella sp.	CBS 110743; CPC 673	Eucalyptus globulus	Colombia	M.J. Wingfield	DQ302955
		CBS 111055; CPC 1200	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ302956
		CBS 111163; CPC 1201	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ302957
		CPC 10995	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ302958
Mycosphaerella gamsii	—	CBS 118495; CPC 11138	Eucalyptus sp.	India	W. Gams	DQ302959
Mycosphaerella gracilis	Pseudocercospora gracilis	CBS 111189; CPC 1315	Eucalyptus urophylia	Indonesia	M.J. Wingfield	DQ302960
		CPC 11144	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ302961
		CPC 11181	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ302962
Mycosphaerella heimii	Pseudocercospora heimii	CPC 11441	Eucalyptus sp.	Brazil	A.C. Alfenas	DQ302963
		CPC 11453	Eucalyptus sp.	Brazil	A.C. Alfenas	DQ302964
		CPC 11548	Eucalyptus sp.	Brazil	A.C. Alfenas	DQ302965
		CPC 11716	—	Brazil	A.C. Alfenas	DQ302966
		CPC 11879	Eucalyptus sp.	Portugal	A.J.L. Phillips	DQ302967
Mycosphaerella jonkershoekensis	—	CBS 112224; CPC 3116	Protea lepidocarpodendron	Australia	P.W. Crous	DQ302968
Mycosphaerella lateralis	Dissoconium dekkeri	CPC 11218	Eucalyptus comaldulensis	Bolivia	M.J. Wingfield	DQ302969
		CPC 11293	Eucalyptus tereticornis	Bolivia	M.J. Wingfield	DQ302970
		CPC 11484	Eucalyptus sp.	Spain	P. Mansilla	DQ302971
		CPC 11706	Eucalyptus globulus	Spain	P. Mansilla	DQ302972
		CPC 11729	Eucalyptus globulus	Spain	P. Mansilla	DQ302973
		CPC 11732	Eucalyptus globulus	Spain	P. Mansilla	DQ302974
		CPC 11789	Eucalyptus sp.	Portugal	J.P. Sampaio	DQ302975
Mycosphaerella madeirae	—	CPC 3746	Eucalyptus grandis	Madeira	S. Denman	DQ302976
Mycosphaerella marksii	? Pseudocercospora epispermogoniana	CBS 110981; CPC 1073	Eucalyptus sp.	Tanzania	M.J. Wingfield	DQ302977
		CBS 111670; CPC 1499	Eucalyptus globulus	Uruguay	M.J. Wingfield	DQ302978
		CBS 115501; CPC 5358	Leucadendron tinctum	Madeira	S. Denman	DQ302979
		CBS 116316; CPC 3715	Eucalyptus deglupha	Ecuador	M.J. Wingfield	DQ302980
		CPC 11215	Eucalyptus comaldulensis	Bolivia	M.J. Wingfield	DQ302981
		CPC 11221	Eucalyptus grandis	Bolivia	M.J. Wingfield	DQ302982
		CPC 11222	Eucalyptus grandis	Bolivia	M.J. Wingfield	DQ302983
		CPC 11795	Vepris reflexa	South Africa	P.W. Crous	DQ302984
Mycosphaerella molleriana	Colletogloeopsis molleriana	CPC 11187	Eucalyptus sp.	Spain	M.J. Wingfield	DQ302985
		CPC 11685	Eucalyptus globulus	Spain	P. Mansilla	DQ302986
		CPC 11688	Eucalyptus globulus	Spain	P. Mansilla	DQ302987
		CPC 11709	Eucalyptus globulus	Spain	P. Mansilla	DQ302988
		CPC 11842	Eucalyptus sp.	Portugal	A.J.L. Phillips	DQ302989
		CPC 11845	Eucalyptus sp.	Portugal	A.J.L. Phillips	DQ302990
		CPC 12056	Eucalyptus sp.	Uruguay	M.J. Wingfield	DQ302991
Mycosphaerella nubilosa	? Uwebraunia juvenis	CPC 11246	Eucalyptus globulus	Spain	M.J. Wingfield	DQ302992
		CPC 11249	Eucalyptus globulus	Spain	M.J. Wingfield	DQ302993
		CPC 11487	Eucalyptus sp.	Spain	P. Mansilla	DQ302994
		CPC 11559	Eucalyptus sp.	Spain	P. Mansilla	DQ302995
		CPC 11723	Eucalyptus globulus	Portugal	A.C. Alfenas	DQ302996
		CPC 11761	Eucalyptus globulus	Spain	P. Mansilla	DQ302997
		CPC 11767	Eucalyptus globulus	Portugal	L.P. Phillips	DQ302998
		CPC 11882	Eucalyptus globulus	Portugal	A.J.L. Phillips	DQ302999
		CPC 11885	Eucalyptus sp.	Portugal	A.J.L. Phillips	DQ303000
Mycosphaerella parva	—	CPC 11273	Eucalyptus globulus	Spain	M.J. Wingfield	DQ303001
		CPC 11758	Eucalyptus globulus	Spain	P. Mansilla	DQ303002
		CPC 11759	Eucalyptus globulus	Spain	P. Mansilla	DQ303003
		CPC 11764	Eucalyptus globulus	Spain	P. Mansilla	DQ303004
		CPC 11888	Eucalyptus sp.	Portugal	A.J.L. Phillips	DQ303005
Mycosphaerella perpendicularis	—	CBS 118367; CPC 10983	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ303006
Mycosphaerella pluritubularis	—	CBS 118508; CPC 11697	Eucalyptus globulus	Spain	P. Mansilla	DQ303007
Mycosphaerella pseudafricana	—	CBS 114782; CPC 1230	Eucalyptus globulus	Zambia	T.A. Coutinho	DQ303008
Mycosphaerella pseudocryptica	Colletogloeopsis sp.	CPC 11264	Eucalyptus sp.	New Zealand	J.A. Stalpers	DQ303009
		CBS 118504; CPC 11267	Eucalyptus sp.	New Zealand	J.A. Stalpers	DQ303010
Mycosphaerella pseudosuberosa	Trimmatostroma sp.	CBS 118911; CPC 12085	Eucalyptus sp.	Uruguay	M.J. Wingfield	DQ303011
Mycosphaerella quasicercospora	—	CBS 111161; CPC 1098	Eucalyptus sp.	Tanzania	M.J. Wingfield	DQ303012
Mycosphaerella readeriellophora	Readeriella readeriellophora	CPC 11711	Eucalyptus globulus	Spain	P. Mansilla	DQ303013
Mycosphaerella scytalidii	—	CBS 516.93; CPC 653	Eucalyptus globulus	Brazil	F.A. Ferreira	DQ303014
		CPC 10988	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ303015
		CBS 118493; CPC 10998	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ303016
Mycosphaerella secundaria	—	CBS 111002; CPC 1112	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303017
		CBS 115608; CPC 504	Eucalyptus grandis	Brazil	A.C. Alfenas	DQ303018
		CPC 10989	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ303019
		CBS 118507; CPC 11551	Eucalyptus sp.	Brazil	A.C. Alfenas	DQ303020
Mycosphaerella sp.	Stenella pseudoparkii	CBS 110988; CPC 1090	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303021
		CBS 110992; CPC 1092	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303022
		CBS 110999; CPC 1087	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303023
		CBS 111000; CPC 1088	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303024
		CBS 111049; CPC 1089	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303025
Mycosphaerella sp.	Stenella xenoparkii	CBS 111088; CPC 1299	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303026
		CBS 111089; CPC 1301	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303027
		CBS 111185; CPC 1300	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303028
Mycosphaerella sp.	—	CBS 208.94 / CPC 727	Eucalyptus grandis	Indonesia	A.C. Alfenas	DQ303029
Mycosphaerella sp.	—	CBS 209.94 / CPC 728	Eucalyptus grandis	Indonesia	A.C. Alfenas	DQ303030
Mycosphaerella sp.	—	CBS 110678; CPC 652	Eucalyptus globulus	Brazil	F.A. Ferreira	DQ303031
Mycosphaerella sp.	—	CBS 110679; CPC 653	Eucalyptus globulus	Brazil	F.A. Ferreira	DQ303032
Mycosphaerella sp.	—	CBS 110745; CPC 651	Eucalyptus globulus	Brazil	F.A. Ferreira	DQ303033
Mycosphaerella sp.	—	CBS 110987; CPC 1093	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303034
Mycosphaerella sp.	—	CBS 110991; CPC 1091	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303035
Mycosphaerella sp.	—	CBS 111036; CPC 1101	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303036
Mycosphaerella sp.	—	CPC 10986	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ303037
Mycosphaerella sp.	—	CPC 11002	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ303038
Mycosphaerella sp.	—	CPC 11004	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ303039
Mycosphaerella sp.	—	CPC 12200	Eucalyptus sp.	South Africa	Z.A. Pretorius	DQ303040
Mycosphaerella sp.	—	CPC 12147	Acacia mangium	Thailand	W. Himaman	DQ303041
Mycosphaerella stramenti	—	CBS 118909; CPC 11545	Eucalyptus sp.	Brazil	A.C. Alfenas	DQ303042
Mycosphaerella stramenticola	—	CBS 118506; CPC 11438	Eucalyptus sp.	Brazil	A.C. Alfenas	DQ303043
Mycosphaerella suberosa	—	CPC 11032	Eucalyptus sp.	Colombia	M.J. Wingfield	DQ303044
		CPC 11190	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303045
		CPC 11276	Eucalyptus comaldulensis	Spain	M.J. Wingfield	DQ303046
		CPC 12193	Eucalyptus sp.	—	A.C. Alfenas	DQ303047
Mycosphaerella sumatrensis	—	CBS 118499; CPC 11171	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303048
		CBS 118501; CPC 11175	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303049
		CBS 118502; CPC 11178	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303050
Mycosphaerella suttonii	Kirramyces epicoccoides	CBS 111676; CPC 1550	Eucalyptus grandis	Australia	M.J. Wingfield	DQ303051
		CBS 114768; CPC 1409	Eucalyptus sp.	Brazil	P.W. Crous	DQ303052
		CBS 110907; CPC 63	Eucalyptus grandis	South Africa	P.W. Crous	DQ303053
		CBS 111674; CPC 1581	Eucalyptus grandis	Australia	M.J. Wingfield	DQ303054
		CPC 11279	Eucalyptus tereticornis	Bolivia	M.J. Wingfield	DQ303055
Mycosphaerella verrucosiafricana	—	CBS 118496; CPC 11167	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303056
		CBS 118497; CPC 11169	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303057
		CBS 118498; CPC 11170	Eucalyptus sp.	Indonesia	M.J. Wingfield	DQ303058
Mycosphaerella vespa	Colletogloeopsis sp.	CMW 11558	Eucalyptus sp.	Australia	—	DQ303059
		CMW 11559	Eucalyptus sp.	Australia	—	DQ303060
		CMW 11560	Eucalyptus sp.	Australia	—	DQ303061
		CMW 11563	Eucalyptus sp.	Australia	—	DQ303062
		CMW 11564	Eucalyptus sp.	Australia	—	DQ303063
Mycosphaerella walkeri	Sonderhenia eucalypticola	CPC 11252	Eucalyptus globulus	Spain	M.J. Wingfield	DQ303064
—	Colletogloeopsis zuluensis	CPC 11780	Eucalyptus sp.	South Africa	P.W. Crous	DQ303065
		CPC 11783	Eucalyptus sp.	South Africa	P.W. Crous	DQ303066
		CPC 11962; CMW 17322	Eucalyptus sp.	South Africa	M.J. Wingfield	DQ303067
		CPC 11965; CMW 17326	Eucalyptus sp.	Uruguay	M.J. Wingfield	DQ303068
		CPC 12059	Eucalyptus sp.	Uruguay	M.J. Wingfield	DQ303069
—	Colletogloeopsis sp.	CPC 11786	Eucalyptus sp.	South Africa	P.W. Crous	DQ303070
—	Pseudocercospora basitruncata	CBS 114664; CPC 1202	Eucalyptus grandis	Colombia	M.J. Wingfield	DQ303071
—	Pseudocercospora clematidis	CPC 11657	Clematis sp.	U.S.A.	M.A. Palm	DQ303072
—	Pseudocercospora epispermogoniana	CBS 110693; CPC 823	Eucalyptus grandis	South Africa	G. Kemp	DQ303073
—		CBS 110694; CPC 824	Eucalyptus grandis	South Africa	G. Kemp	DQ303074
		CBS 110750; CPC 822	Eucalyptus grandis	South Africa	G. Kemp	DQ303075
—	Pseudocercospora fatouae	CPC 11648	Fatoua villosa	Korea	H.D. Shin	DQ303076
—	Pseudocercospora natalensis	CBS 111069; CPC 1263	Eucalyptus nitens	South Africa	T.A. Coutinho	DQ303077
—	Pseudocercospora pseudoeucalyptorum	CBS 116359; CPC 3751	Eucalyptus sp.	Madeira	S. Denman	DQ303078
		CPC 10916	Eucalyptus sp.	South Africa	P.W. Crous	DQ303079
		CPC 11713	Eucalyptus globulus	Spain	P. Mansilla	DQ303080
—	Pseudocercospora robusta	CBS 111175; CPC 1269	Eucalyptus robur	Malaysia	M.J. Wingfield	DQ303081
—	Pseudocercospora sp.	CBS 111072; CPC 1266	Eucalyptus pellita	Thailand	M.J. Wingfield	DQ303082
		CBS 111373, CPC 1493	Eucalyptus globulus	Uruguay	M.J. Wingfield	DQ303083
		CPC 11591	Brachybotrys paridiformis	Korea	H.D. Shin	DQ303084
		CPC 11592	Zelkova serrata	Korea	H.D. Shin	DQ303085
		CPC 11654	Morus bombycis	Korea	H.D. Shin	DQ303086
		CPC 11668	Pilea hamaoi	Korea	H.D. Shin	DQ303087
		CPC 11680	Ampelopsis brevipenduncula var. heterophylla	Korea	H.D. Shin	DQ303088
		CPC 11726	Platanus occidentalis	Korea	H.D. Shin	DQ303089
—	Pseudocercospora subulata	CBS 118489; CPC 10849	Eucalyptus botryoides	New Zealand	M. Dick	DQ303090
—	Pseudocercosporella capsellae	CPC 11677	Draba nemorosa var. hebecarpa	Korea	H.D. Shin	DQ303091
—	Readeriella sp.	CPC 11186	Eucalyptus globulus	Spain	M.J. Wingfield	DQ303092
		CPC 11735	Eucalyptus globulus	Spain	P. Mansilla	DQ303093
—	Readeriella mirabilis	CPC 11712	Eucalyptus globulus	Spain	P. Mansilla	DQ303094
—	Septoria eucalyptorum	CBS 118505; CPC 11282	Eucalyptus sp.	India	W. Gams	DQ303095
—	Septoria provencialis	CBS 118910; CPC 12226	Eucalyptus sp.	France	P.W. Crous	DQ303096
—	Stenella sp.	CPC 11671	Lonicera japonica	Korea	H.D. Shin	DQ303097

CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; CMW: Culture collection of Mike Wingfield, housed at FABI, Pretoria, South Africa

Taxonomy

Wherever possible, 30 measurements (× 1000 magnification) were made of structures mounted in lactic acid, with the extremes of spore measurements given in parentheses. Colony colours (surface and reverse) were assessed after 1 mo on MEA, oatmeal agar (OA) and potato-dextrose agar (PDA) (Gams ) 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). Nomenclatural novelties and descriptions were deposited in MycoBank .

RESULTS

For the ITS region, approximately 500 to 560 bases were determined for all isolates (Table 1). The trees resulting from each of the four alignments are depicted in Figs 1, 2, 3, 4. The first alignment contains 102 taxa (including the two outgroups) and 544 characters including alignment gaps. Of these characters, 295 are parsimony-informative, 37 are variable and parsimony-uninformative, and 212 are constant. Neighbour-joining analysis using the three substitution models yielded trees with similar topologies and bootstrap values. Parsimony analysis yielded 243 most parsimonious trees (TL = 1038 steps; CI = 0.620; RI = 0.893; RC = 0.554). The topology of the distance trees differed from the trees obtained using parsimony mainly at the deeper nodes (data not shown). Parts of the distance tree obtained using the HKY85 substitution model are shown in Figs 1, 2, 3, 4. The first alignment and derived tree (Fig. 1) includes M. nubilosa (100 % bootstrap support), species of Colletogloeopsis Crous & M.J. Wingf., the M. molleriana (Thüm.) Lindau complex (95 % bootstrap support), the M. suttonii Crous & M.J. Wingf. complex (100 % bootstrap support) and the M. suberosa Crous, F.A. Ferreira, Alfenas & M.J. Wingf. complex (100 % bootstrap support). One new species of Colletogloeopsis, and four new species of Mycosphaerella are indicated.

Fig. 1.

Distance tree obtained from a neighbour-joining analysis using the HKY85 substitution model on the ITS sequence alignments. The scale bar shows the number of substitutions per site and bootstrap support values from 1000 replicates are shown at the nodes. The tree was rooted to two Botryosphaeria species. New species are indicated in bold, and ex-type strains with a T.

Fig. 2.

Distance tree obtained from a neighbour-joining analysis using the HKY85 substitution model on the ITS sequence alignments. The scale bar shows the number of substitutions per site and bootstrap support values from 1000 replicates are shown at the nodes. The tree was rooted to two Botryosphaeria species. New species are indicated in bold, and ex-type strains with a T.

Fig. 3.

Distance tree obtained from a neighbour-joining analysis using the HKY85 substitution model on the ITS sequence alignments. The scale bar shows the number of substitutions per site and bootstrap support values from 1000 replicates are shown at the nodes. The tree was rooted to two Botryosphaeria species. New species are indicated in bold, and ex-type strains with a T.

Fig. 4.

Distance tree obtained from a neighbour-joining analysis using the HKY85 substitution model on the ITS sequence alignments. The scale bar shows the number of substitutions per site and bootstrap support values from 1000 replicates are shown at the nodes. The tree was rooted to two Botryosphaeria species. New species are indicated in bold, and ex-type strains with a T.

The second alignment (Fig. 2) contains 90 taxa (including the two outgroups) and 535 characters including alignment gaps. Of these characters, 246 are parsimony-informative, 51 are variable and parsimony-uninformative, and 238 are constant. Neighbour-joining analysis using the three substitution models yielded trees with identical topologies and similar bootstrap values. Parsimony analysis yielded 481 most parsimonious trees (TL = 862 steps; CI = 0.613; RI = 0.927; RC = 0.568). The topology of the distance trees differed from the trees obtained using parsimony only in the placement of the Mycosphaerella sp. CPC 11171 clade (data not shown). The second alignment and derived tree mainly includes the M. marksii Carnegie & Keane complex (100 % bootstrap support), the M. heimii Crous complex (60 % bootstrap support), the M. walkeri R.F. Park & Keane (100 % bootstrap support) and the M. parva R.F. Park & Keane complex (100 % bootstrap support). Five new species of Mycosphaerella are indicated in the tree.

The third alignment (Fig. 3) contains 71 taxa (including the two outgroups) and 529 characters including alignment gaps. Of these characters, 237 are parsimony-informative, 71 are variable and parsimony-uninformative, and 221 are constant. Neighbour-joining analysis using the three substitution models yielded trees with identical topologies and similar bootstrap values. Parsimony analysis yielded 4319 most parsimonious trees (TL = 853 steps; CI = 0.626; RI = 0.856; RC = 0.536). The topology of the distance trees differed from the trees obtained using parsimony mainly at the deeper nodes (data not shown). The third alignment and derived tree mainly includes species of Pseudocercospora Speg., Cercospora Fresen., Septoria Sacc. and Stenella Syd. New species indicated in the tree include three in Mycosphaerella, two in Septoria, and two in Stenella.

The fourth alignment (Fig. 4) contains 50 taxa (including the two outgroups) and 570 characters including alignment gaps. Of these characters, 293 are parsimony-informative, 25 are variable and parsimony-uninformative, and 252 are constant. Neighbour-joining analysis using the three substitution models yielded trees with identical topologies and similar bootstrap values. Parsimony analysis yielded eight most parsimonious trees (TL = 627 steps; CI = 0.864; RI = 0.973; RC = 0.841). The topology of the distance trees was similar to that of the topology of the trees obtained using parsimony (data not shown). The fourth alignment and derived tree includes species of Dissoconium de Hoog, Oorschot & Hijwegen, Passalora zambiae Crous & T. Coutinho and Mycosphaerella, with three new species.

Several collections represented Mycosphaerella spp. morphologically and phylogenetically distinct from ex-type strains of the morphological species to which they had originally been assigned. These fungi are described as new taxa as follows:

Crous, sp. nov. MycoBank MB500833. Fig. 5.

Fig. 5.

Colletogloeopsis stellenboschiana (CBS 116428). A. Leaf spot. B–E. conidiogenous cells giving rise to conidia. F–I. Conidia. Scale bar = 3.5 μm.

Etymology: Refers to Stellenbosch, where the fungus was collected.

Coniothyrio ovato similis sed conidiis minoribus, (6.5–)7–9(–10) × (33.5(–4) μm, distincta.

Leaf spots amphigenous, circular to subcircular, 0.5–3 mm diam, pale brown, with a raised border and red-purple margin. Conidiomata amphigenous, pycnidial, medium brown, globose, 80–120 μm diam; wall of 3–4 layers of brown textura angularis. Conidiogenous cells discrete, ampulliform to subcylindrical, pale to medium brown, finely verruculose, proliferating 1–3 times percurrently near the apex, 3–6 × 3–4 μm. Conidia holoblastic, solitary, aseptate, ellipsoidal, with subobtuse apex and subtruncate base with minute marginal frill, medium brown, finely verruculose, widest below the middle, (6.5–)7–9(–10) × (3–)3.5(–4) μm.

Holotype: South Africa, Western Cape Province, Stellenbosch Mountain, on leaves of Eucalyptus sp., 4 Dec. 2004, P.W. Crous, CBS H-19688, holotype, culture ex-type CBS 116428 = CPC 10886.

Cultures: Colonies after 3 wk on MEA 15–40 mm diam; on PDA erumpent, spreading, producing copious amounts of slime, olivaceous-black at the centre, aerial mycelium olivaceous-grey, with a vinaceous-grey outer zone and wide olivaceous-black margin that is smooth but uneven; reverse olivaceous-black; on OA surface smoke-grey with a wide, grey-olivaceous border, forming a characteristic yellow pigment; on MEA grey-white on surface, with sectors of smoke-grey; margin thin, submerged, smoke-grey; reverse olivaceous-black; aerial mycelium sparse to moderate, grey-white; colonies fertile.

Host: Eucalyptus sp.

Distribution: South Africa.

Notes: Numerous species of Coniothyrium Corda and several species of Colletogloeopsis cause spots on eucalypt leaves. Colletogloeopsis stellenboschiana is easily distinguished from the taxa occurring on eucalypt leaves (Crous 1998), and from representatives of the “Coniothyrium ovatum” species complex specifically, based on its conidial morphology. Phylogenetically it is closely related to members of the the Colletogloeopsis complex that cause stem cankers on eucalypt trees (Cortinas – this volume)

Crous, sp. nov. MycoBank MB500834. Fig. 6.

Fig. 6.

Mycosphaerella davisoniellae (anamorph Davisoniella eucalypti) (DAR 58999). A. Asci and ascospores. B. Conidiogenous cells and conidia of D. eucalypti. C. conidiogenous cells and conidia of synanamorph. Scale bar = 10 μm.

Anamorph: H.J. Swart, Trans. Brit. Mycol. Soc. 90: 289. 1988.

Asci subcylindrici, subsessiles, 50–70 × 9–12 μm. Ascosporae bi-vel triseriatae, tenuitunicatae, rectae, obovoideae, apicem versus latissimae, in medio uniseptatae, vix vel haud constrictae ad septum, 10–14 × 3–4 μm.

Leaf spots amphigenous, subcircular to irregular, 1–7 mm diam, discrete to confluent, medium brown, surrounded by raised, red-purple margin. Ascomata hypophyllous, embedded in a raised, black, subepidermal stroma, ostiolate, becoming erumpent up to 120 μm diam. Asci subcylindrical, subsessile, straight or slightly incurved, 8-spored, 50–70 × 9–12 μm. Ascospores bi- to triseriate, overlapping, hyaline, thin-walled, straight, obovoid with rounded ends, widest near the apex, medianly 1-septate, not to slightly constricted at the septum, tapering toward both ends, but more prominently toward the base, 10–14 × 3–4 μm. Conidiomata of Davisoniella embedded in the same black subepidermal stroma that contains ascomata, subepidermal, ostiolate, up to 450 μm diam; wall of 2–3 layers of brown textura angularis. Conidiogenous cells subcylindrical to ampulliform or doliiform, 5–15 × 3–4 μm, medium brown, verruculose, proliferating several times percurrently near the apex. Conidia solitary, brown, aseptate, verruculose, thick-walled, oval with an obtuse apex and a truncate to subtruncate base with a prominent basal frill, which can extend up to 2 μm from the brown basal rim of the conidium, (8–)10–12(–14) × 4.5–)5–6(–6.5) μm (av. 11 × 5.5 μm). Synanamorph: Conidiomata intermingled between that of D. eucalypti and ascomata of M. davisoniellae. Conidiogenous cells phialidic, hyaline, subcylindrical to ampulliform, with visible periclinal thickening, 8–15 × 2.5–3.5 μm. Conidia hyaline, curved, subcylindrical, widest in the middle, apex bluntly rounded, obtuse, base truncate, 17–30 × 2–1.5 μm.

In vivo: No cultures available.

Specimen examined: Australia, Darling Ranges W.A., Mundlimup Block, on leaves of Eucalyptus marginata, 24 Nov. 1981, F. Tay, DAR 58999, holotype of D. eucalypti and M. davisoniellae.

Notes: Swart (1988) reported that this fungus is associated with abundant leaf spots on saplings and the foliage of recently felled trees. Conidiomata of D. eucalypti were described as unilocular and subepidermal, occurring in a stroma which could result in some of them appearing as multilocular. Swart (1988) considered the fungus to be the stromatic counterpart of Coniothyrium. Davisoniella eucalypti is clearly related to species in the Colletogloeopsis complex that occurs on eucalypts, having characteristic aseptate, brown, verruculose conidia that arise from percurrently proliferating conidiogenous cells. Davisoniella is unique by virtue of its stroma, that gives rise to the uni- or multilocular conidiomata. Conidia of D. eucalypti exude in slimy masses. In many cases, the exudates included aseptate, hyaline, curved, subcylindrical conidia of a synanamorph. The latter anamorph was produced from unilocular conidiomata that formed in the same stromata that gave rise to D. eucalypti. Surprisingly, many of the stromata investigated also contained ascomata of a Mycosphaerella species, which most likely also belong to the same fungus. The latter state is described here as M. davisoniellae.

Crous & M.J. Wingf., sp. nov. MycoBank MB500835. Fig. 7.

Fig. 7.

Mycosphaerella eucalyptorum (CBS 118496). A. Leaf spot. B. Ostiolar periphysoids. C–D. Ascospores. E–F. Germinating ascospores. Scale bars = 4 μm.

Etymology: Referring to its host, Eucalyptus.

Mycosphaerellae parkii similis, sed ascosporis maioribus, 12–17 × 3.5–4.5 μm, modo B germinantibus, distinguenda.

Leaf spots amphigenous, irregular to sub-circular, 2–20 mm diam, medium brown, with raised, brown borders, and thin, red-purple margins. Ascomata pseudothecial, amphigenous but predominantly epiphyllous, single, black, erumpent, globose, up to 120 μm diam; apical ostiole 10–15 μm diam, with prominent periphyses lining the ostiolar channel; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to ellipsoid, straight or slightly incurved, 8-spored, 35–50 × 8–12 μm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight to slightly curved, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in middle of apical cell, not constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (12–)14–15(–17) × (3.5–)4(–4.5) μm in vivo; some ascospores with slightly asymmetrical apical cells, as commonly observed in M. marksii.

Holotype: Indonesia, on leaves of Eucalyptus sp., Mar. 2004, M.J. Wingfield, CBS H-19689 holotype, culture ex-type CBS 118496 = CPC 11174.

Ascospore germination on MEA after 24 h: Type B. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, not distorting, becoming slightly constricted upon germination, becoming up to 4 μm diam.

Cultures: Colonies on MEA after 3 wk 25–30 mm diam; on MEA flat, spreading, folding, with sparse aerial mycelium, olivaceous-grey, margins smooth, regular, reverse iron-grey; on PDA slightly erumpent, centre olivaceous-grey; outer zone pale olivaceous-grey; reverse iron-grey; on OA with sparse to moderate pale olivaceous-grey aerial mycelium and patches of olivaceous-grey.

Host: Eucalyptus sp.

Distribution: Indonesia.

Notes: Conidia of a Stenella anamorph were found on some lesions. This link is, however, unconfirmed, and isolates did not produce anamorph structures in culture. Mycosphaerella eucalyptorum is phylogenetically closely related to a Mycosphaerella sp. from Colombia that forms Stenella pseudoparkii in culture. Ascospores of M. eucalyptorum (12–17 × 3.5–4.5 μm) germinate with a Type B germination pattern as observed in M. gracilis (10–20 × 2–3 μm) (Fig. 8) and M. marksii (11–22.5 × 2–3.5). It is easily distinguished from these taxa, however, based on its ascospore morphology and growth characteristics in culture (Crous 1998).

Fig. 8.

Mycosphaerella gracilis. A. Ascomata on in leaf tissue. B. Ostiolar region of ascoma. C. Asci. D–E. Germinating ascospores. Scale bar = 3 μmm.

Crous, sp. nov. MycoBank MB500836. Fig. 9.

Fig. 9.

Mycosphaerella gamsii (CBS 118495). A. Leaf spot. B–C. Asci. D–E. Ascospores. F–I. Germinating ascospores. Scale bars: B = 3 μm, F = μm.

Etymology: Named after the collector, well-known mycologist and friend, Prof. dr Walter Gams.

Mycosphaerellae stramenticolae similis, sed ascosporis minoribus, (8–)9–10 × (2–)3 μm, modo C germinantibus, distinguenda.

Leaf spots amphigenous, irregular, 1–20 mm diam, medium brown, with a raised, dark brown border. Ascomata pseudothecial, amphigenous, but predominantly hypophyllous, single, black, subepidermal, becoming erumpent, globose, up to 90 μm diam; apical ostiole 5–10 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to narrowly ellipsoid, straight or slightly incurved, 8-spored, 25–35 × 7–9 μm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid–ellipsoidal, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10 × (2–)3 μm in vivo.

Holotype: India, Palampur, on leaves of Eucalyptus sp., Mar. 2004, W. Gams & M. Arzanlou, CBS H-19690, holotype, culture ex-type CBS 118495 = CPC 11138–11140. 5/6-6

Ascospore germination on MEA after 24 h: Type C. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, but also variable in direction; becoming constricted upon germination, up to 5 μm diam.

Cultures: Colonies on MEA 28–35 mm diam after 3 wk; on MEA spreading, folding, flat, with moderate smoke-grey aerial mycelium in the centre; outer region olivaceous-grey; margins smooth, regular; reverse iron-grey; on PDA with moderate aerial mycelium, pale olivaceous-grey, outer region olivaceous-grey with drops of slime; reverse iron-grey; on OA with moderate aerial mycelium, pale olivaceous-grey, with patches of olivaceous-grey.

Host: Eucalyptus sp.

Distribution: India.

Notes: Mycosphaerella gamsii is phylogenetically closely related to M. stramenticola, but is distinguishable in having a Type C ascospore germination pattern, as is found in species such as M. heimii, M. gregaria, M. molleriana, M. nubilosa and M. walkeri. Mycosphaerella gamsii has ascospores that are 8–10 × 2–3 μm, thus shorter than those of the species listed above, and it also lacks an anamorph in culture.

Crous & M.J. Wingf., sp. nov. MycoBank MB500837. Fig. 10.

Fig. 10.

Mycosphaerella perpendicularis (CBS 118367). A. Leaf spot. B–E. Asci. F–G. Ascospores. H–L. Germinating ascospores. Scale bars: B = 3 μm, H = 5 μm.

Etymology: Referring to ascospores that germinate with germ tubes growing 90° to the long axis of the spore.

Mycosphaerellae heimioide similis, sed ascosporis longioribus, (8–)9–10(–12) × (2.5–)3 μm, modo M germinantibus distinguenda.

Leaf spots amphigenous, irregular to sub-circular, 5–15 mm diam, medium brown, frequently with a orange-red discoloration in the central part; border raised, dark brown. Ascomata pseudothecial, epiphyllous, single, black, subepidermal, globose, up to 90 μm diam; apical ostiole 10–15 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, slightly incurved, 8-spored, 25–35 × 7–8 μm. Ascospores multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–12) × (2.5–)3 μm in vivo.

Holotype: Colombia, Suiza, on leaves of Eucalyptus eurograndis, Jan. 2004, M.J. Wingfield, CBS H-19691, holotype, culture ex-type CBS 118367 = CPC 10983–10985.

Ascospore germination on MEA after 24 h: Type M. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes 90° to the long axis of the spore, and distorting upon germination, becoming up to 5 μm wide.

Cultures: Colonies on MEA reaching 28–37 mm diam after 3 wk; colonies folding, spreading, flat, with sparse aerial mycelium, which is olivaceous-grey on the agar surface, and with smoke-grey aerial mycelium; margins are smooth, regular; reverse iron-grey at the centre, olivaceous-grey in the outer zone; on OA with moderate aerial mycelium, olivaceous-grey at centre, greenish black in outer zone; on PDA olivaceous-grey with some drops of slime, iron-grey in reverse.

Host: Eucalyptus eurograndis.

Distribution: Colombia.

Notes: Germinating ascospores of M. perpendicularis have a characteristic Type M germination pattern, similar to that of M. heimioides. Mycosphaerella perpendicularis can easily be distinguished from M. heimioides, however, by virtue of the fact that the ascospores distort at germination. In addition, the germ tubes of M. heimioides never quite reach 90° to the long axis of the spore, whereas those of M. perpendicularis are at right angles.

Crous & J.P. Mansilla, sp. nov. MycoBank MB500838. Figs 11, 12.

Fig. 11.

Mycosphaerella pluritubularis (CBS 118508). A. Ascus and ascospores. B. Sequence of germinating ascospores, with those after 24 h at the right of the plate. Scale bar = 10 μm.

Fig. 12.

Mycosphaerella pluritubularis (CBS 118508). A. Leaf spot. B–C. Asci. D. ascospore. E–F. Germinating ascospores. Scale bars: B–C = 3 μm, E = 11 μm.

Etymology: Refers to the ascospores that have multiple germ tubes when they germinate.

Mycosphaerellae nubilosae similis, set ascosporis brevioribus, (8–)9–10(–11) × 3(–4) μm, saepe plures quam 2 tubos germinationis proferentibus, distinguenda.

Leaf spots amphigenous, irregular to sub-circular, 5–15 mm diam, pale to medium brown, surrounded by a thin, raised, dark brown border. Ascomata pseudothecial, hypophyllous, single, black, immersed becoming erumpent, globose, up to 100 μm diam; apical ostiole 10–15 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to subcylindrical, straight to slightly incurved, 8-spored, 30–45 × 7–10 μm. Ascospores multiseriate, overlapping, hyaline, prominently guttulate, thin-walled, straight, obovoid with subobtuse ends, medianly 1-septate, widest at the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–11) × 3(–4) μm in vivo.

Holotype: Spain, on leaves of E. globulus, Nov. 2004, J.P. Mansilla, CBS H-19692 holotype, culture ex-type CBS 118508 = CPC 11697).

Ascospore germination on MEA after 24 h: Type F. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, and distorting prominently upon germination, becoming up to 11 μm diam; frequently germinating with more than two germ tubes.

Cultures: Colonies after 3 wk 17–22 mm diam on MEA; on PDA colonies forming copious amounts of slime; surface olivaceous-black with patches of olivaceous-grey and pale olivaceous-grey; aerial mycelium sparse; margins feathery, uneven; reverse iron-grey; on OA surface smoke-grey with patches of olivaceous-grey; on MEA with sparse aerial mycelium, colonies erumpent, iron-grey, margins feathery, irregular; reverse olivaceous-black; colonies sterile.

Host: E. globulus.

Distribution: Spain.

Notes: Mycosphaerella pluritubularis is characterised by its distinct ascospore germination pattern (Type F), but where ascospores form more than two germ tubes, thus distinguishing it from other species like M. nubilosa that have more typical type F germination patterns.

Crous & T. Coutinho, sp. nov. MycoBank MB500839. Fig. 13.

Fig. 13.

Mycosphaerella pseudafricana (CBS 114782). A. Leaf spot. B–E. Asci and ascospores. F–H. Germinating ascospores. Scale bars: B = 3 μm, F = 7 μm.

Etymology: Referring to its morphological similarity to M. africana.

Mycosphaerellae africanae similis, sed ascosporis maioribus, 8–11 × 2.5–3 μm, distinguenda.

Leaf spots amphigenous, irregular to sub-circular, 2–7 mm diam, medium brown, surrounded by a thin, raised, concolorous border. Ascomata pseudothecial, hypophyllous, single, black, immersed becoming erumpent, globose, up to 120 μm diam; apical ostiole 10–15 μm diam; wall of 2–3 cell layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to subcylindrical, slightly incurved, 8-spored, 35–45 × 7–9 μm. Ascospores tri- to multiseriate, overlapping, hyaline to pale brown, guttulate, thin-walled, straight to slightly curved, smooth to finely roughened, fusoid–ellipsoidal with subobtuse ends, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–11) × (2.5–)3 μm in vivo. Spermatogonia similar to the ascomata in morphology. Spermatia hyaline, smooth, rod-shaped with bluntly rounded ends, 3–4 × 1–1.5 μm.

Holotype: Zambia, on leaves of E. globulus, Aug. 1995, T. Coutinho, PREM 54973 holotype, culture ex-type CBS 114782 = CPC 1230; 1229–1231.

Ascospore germination on MEA after 24 h: Type G. Ascospores darkening and becoming verruculose on MEA; germinating from both ends as observed in M. africana, with germ tubes irregular to the long axis of the spore, and distorting prominently upon germination, becoming up to 8 μm wide.

Cultures: Colonies reaching 12–17 mm diam after 3 wk on MEA; colonies erumpent, irregular, surface iron-grey with olivaceous-grey, sparse aerial mycelium in central part; margins catenate, smooth; reverse greenish black; on PDA colonies erumpent, olivaceous-black with sparse olivaceous-grey aerial mycelium in the central part, margins smooth, catenate; reverse greenish black; on OA olivaceous-grey with smooth, catenate margins and green-olivaceous central part.

Host: E. globulus.

Distribution: Zambia.

Notes: Ascospores of M. pseudafricana (8–11 × 2.5–3 μm) germinate with a Type G pattern similar to that observed in M. africana (7–11 × 2–3 μm). Ascospores of M. pseudafricana are more verrucose than those of M. africana, but both taxa have very similar ascospore dimensions and germination patterns. They do differ, in the symptoms with which they are associated. Lesions of M. pseudafricana are generally larger, and they lack the red-purple margin found in M. africana. The easiest means to distinguish these taxa from each other is to compare their growth in culture: colonies of M. africana are black, produce a brown pigment in MEA, and form clusters of chlamydospores, whereas cultures of M. pseudafricana also produce clusters of chlamydospores on MEA, but are iron-grey, and lack the diffuse brown pigment observed in colonies of M. africana.

Crous, sp. nov. MycoBank MB500840. Figs 14, 15.

Fig. 14.

Mycosphaerella pseudocryptica (anamorph Colletogloeopsis sp.) (CBS 118504). A. Ascospores, some with sheath. B. Germinating ascospores. C. Conidiogenous cells and conidia. Scale bar = 10 μm.

Fig. 15.

Mycosphaerella pseudocryptica (anamorph Colletogloeopsis sp.) (CBS 118504). A. Leaf spot. B–C. Asci. D–E. Ascospores. F–H. Germinating ascospores. I–J. Conidia and conidiogenous cells. Scale bars: B–C, I = 4 μm, D = 3.5 μm, F = 7 μm.

Anamorph: sp.

Etymology: Morphologically similar to M. cryptica.

Mycosphaerellae crypticae similis, sed ascosporis minoribus, (11–)12–14(–15) × (3–)3.5(–4) μm, saepe utrinque germinantibus, distinguenda.

Leaf spots amphigenous, irregular to subcircular, 0.5–2 mm diam, pale brown, with a raised, red-brown margin. Ascomata pseudothecial, hypophyllous, arranged in dense clusters in pale brown areas next to the leaf spots associated with conidiomata of the anamorph, black, immersed, globose, up to 70 μm diam; apical ostiole 10–15 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to subcylindrical, straight or slightly incurved, 8-spored, 35–45 × 9–11 μm. Ascospores multiseriate, overlapping, hyaline, granular, thin-walled, straight, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest at the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (11–)12–14(–15) × (3–)3.5(–4) μm, in vivo; frequently encased in an irregular mucous sheath. Mycelium internal, consisting of branched, septate, medium brown, smooth, 3–4 μm wide hyphae. Conidiomata intermixed among ascomata or separate, predominantly on the lower leaf surface, pycnidial, substromatal, up to 120 μm diam; wall of 3–4 layers of brown textura angularis. Conidiophores 0–1-septate, but mostly reduced to conidiogenous cells. Conidiogenous cells discrete, ampulliform to subcylindrical, medium brown, smooth to finely verruculose, proliferating 1–3 times percurrently near apex, but also intercalary and sympodially, 5–15 × 3–5 μm. Conidia holoblastic, solitary, aseptate, fusoid with obtuse to subobtuse apices and truncate bases, medium brown, finely verruculose, (10–)12–14(–17) × (3.5–)4(–6) μm; inconspicuous basal marginal frill present.

Holotype: New Zealand, Wellington Botanical Garden, on leaves of Eucalyptus sp., Mar. 2004, J.A. Stalpers, CBS H-19693, holotype, culture ex-type CBS 118504 = CPC 11267; 11267–11269 (teleomorph), CPC 11264–11266 (anamorph).

Ascospore germination on MEA after 24 h: Type A. Ascospores smooth, becoming olivaceous on MEA, germinating predominantly from both ends, with germ tubes at some angle to the long axis of the spore, and with a constriction at the ascospore septum; ascospores becoming up to 7 μm wide.

Cultures: Colonies slow growing, 3–8 mm diam after 3 wk on MEA; on MEA colonies erumpent, aerial mycelium sparse to absent, margins smooth, surface white-grey to smoke-grey, or with a reddish tinge in patches; reverse fuscous-black; on PDA erumpent, white to smoke-grey with patches of vinaceous-grey; reverse vinaceous-grey, with a diffuse red pigment visible in the agar, up to 2 cm from colony margins; on OA pale grey-olivaceous with a pale vinaceous grey pigment diffusing into the agar.

Host: Eucalyptus sp.

Distribution: New Zealand.

Notes: Ascospores of M. pseudocryptica germinate with a Type A pattern (as observed in M. cryptica), except that they tend to germinate from both ends. It is possible, therefore, that collections of M. pseudocryptica have in the past been confused with those of M. cryptica. Isolates also form a Colletogloeopsis anamorph in culture, which is similar to M. cryptica. Ascospores of M. pseudocryptica are 11–15 × 3–4 μm, and conidia 10–17 × 3.5–6 μm, while ascospores of M. cryptica are 9–17.5 × 2–5.5 μm, and conidia are 8.5–18 × 4–6 μm. Phylogenetically M. pseudocryptica is closely related to the M. molleriana complex (Fig. 1), and distinct from M. cryptica.

Crous & G. Hunter, sp. nov. MycoBank MB500841. Figs 16, 17. Anamorph: sp.

Fig. 16.

Mycosphaerella pseudoendophytica (anamorph Pseudocercosporella sp.) (CBS 113288). A. Ascus and ascospores. B. Germinating ascospores. C. Conidia. Scale bar = 10 μm

Fig. 17.

Leaf spot associated with Mycosphaerella pseudoendophytica (holotype)

Etymology: Named after its morphological similarity to M. endophytica.

Mycosphaerellae endophyticae similis, sed ascosporis modo C germinantibus distinguenda.

Leaf spots amphigenous, irregular to subcircular or angular, 2–5 mm diam, brown, with a raised, dark brown margin. Ascomata pseudothecial, amphigenous, black, subepidermal, erumpent to superficial, globose, up to 120 μm diam; apical ostiole 5–10 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, straight or slightly incurved, 8-spored, 30–40 × 8–10 μm. Ascospores multiseriate, overlapping, hyaline, sparsely guttulate, thin-walled, straight to slightly curved, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in the middle of the apical cell, not to slightly constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–11) × (2–)2.5–3 μm, in vivo. Mycelium internal, consisting of branched, septate, pale to medium brown, smooth, 3–4 μm wide hyphae. Conidiomata in vitro sporodochial, hyaline. Conidiogenous cells aggregated, unbranched or branched, hyaline, smooth, tapering to flat-tipped apical and lateral loci, proliferating sympodially, 8–15 × 2–3.5 μm. Conidia holoblastic, solitary, but frequently undergoing microcyclic conidiation, giving rise to one or several additional conidia, smooth, hyaline, obclavate, apex subobtuse, base long obconically subtruncate to truncate, irregularly curved, 0–3-septate, 12–40 × 1.5–2 μm; hila inconspicuous.

Holotype: South Africa, KwaZulu-Natal, Enon, Richmond, on leaves of E. nitens, 3 May 2000, G. Hunter, CBS H-19694, holotype, culture ex-type CBS 113288 = CMW 9098.

Ascospore germination on MEA after 24 h: Type C. Ascospores smooth, not darkening on MEA, germinating from both ends, with germ tubes parallel to the long axis of the spore, and with a constriction at the ascospore septum; ascospores becoming up to 3.5 μm wide.

Cultures: Similar to those of M. endophytica (Crous 1998).

Host: E. nitens.

Distribution: South Africa.

Notes: Mycosphaerella pseudoendophytica has been known to us for some time, but its formal description required a molecular comparison with ex-type strains of M. endophytica (which it resembles in anamorph morphology), and M. ellipsoidea (which it resembles in ascospore germination pattern). As can be seen here, M. pseudoendophytica (Fig. 4) is clearly a distinct species, sharing features of both of these taxa.

Crous & M.J. Wingf., sp. nov. MycoBank MB500842. Fig. 18. Anamorph: sp.

Fig. 18.

Mycosphaerella pseudosuberosa (anamorph Trimmatostroma sp.) (CBS 118911). A. Colony on MEA. B–C. Broken asci. D–F. Ascospores (note sheath). G–I. Germinating ascospores. J–L. Trimmatostroma conidia produced in culture. Scale bars: B–D = 4 μm, G = 8 μm.

Etymology: Morphologically similar to M. suberosa.

Mycosphaerellae suberosae similis, sed ascosporis minoribus, (11–)12–14(–15) × (3–)3.5(–4) μm, distinguenda.

Leaf spots amphigenous, associated with brown, corky spots on leaf petioles. Ascomata pseudothecial, single to aggregated, black, immersed becoming erumpent, globose, up to 120 μm diam; apical ostiole 10–20 μm diam; wall of 3–6 layers of brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, straight or slightly incurved, 8-spored, 35–45 × 12–16 μm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thick-walled, straight to slightly curved, fusoid-ellipsoidal with obtuse ends, medianly 1-septate, widest at the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (11–)12–14(–15) × (3–)3.5(–4) μm in vivo; frequently surrounded by an irregular mucous sheath.

Holotype: Uruguay, on leaves and petioles of Eucalyptus sp., Apr. 2005, M.J. Wingfield, CBS H-19695, holotype, culture ex-type CBS 118911 = CPC 12085.

Ascospore germination on MEA after 24 h: Type H. Ascospores darkening and becoming verruculose on MEA, germinating from both ends, with germ tubes primarily parallel to the long axis of the spore, and distorting prominently upon germination, becoming up to 11 μm wide.

Cultures: Colonies extremely slow growing, erumpent, uneven, black; aerial mycelium absent; colonies powdery, producing a Trimmatostroma anamorph.

Host: Eucalyptus sp.

Distribution: Uruguay.

Notes: Mycosphaerella pseudosuberosa is morphologically similar, and phylogenetically closely related to M. suberosa. It can be distinguished by its ascospores that are slightly narrower (3–4 μm vs. 3–6 μm), having a mucous sheath, and germinating via two germ tubes (predominantly) that originate from the ends of the spore. Germinating spores exude mucus, and become pale brown and verruculose, which differs from the numerous germ tubes and dark brown ascospores observed in M. suberosa. Furthermore, cultures of M. suberosa are hard and resistant to being cut, while those of M. pseudosuberosa are powdery, producing a Trimmatostroma anamorph in culture. From the phylogenetic data available, it appears that there may be more species within the M. suberosa complex awaiting description (Fig. 1).

Crous & T. Coutinho, sp. nov. MycoBank MB500843. Fig. 19.

Fig. 19.

Mycosphaerella quasicercospora (CBS 111161). A. Ascomata on leaf. B–C. Asci. D. Ascospores. Scale bars = 4 μm.

Etymology: Refers to the fact that this fungus is phylogenetically closely related to species of Cercospora.

Mycosphaerellae nubilosae similis, sed ascosporis brevioribus, 10–14 × 3–4 μm, distinguenda.

Leaf spots amphigenous, irregular to sub-circular, 2–10 mm diam, pale brown, surrounded by a thin, raised, dark brown border; spots becoming confluent with age. Ascomata pseudothecial, hypophyllous, single, black, immersed becoming erumpent, globose, up to 100 μm diam; wall of 2–3 cell layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, straight to slightly incurved, 8-spored, 35–50 × 10–12 μm.

Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, obovoid with subobtuse ends, unequally 1-septate, widest close to the apex of the apical cell, not constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (10–)12–13(–14) × (3–)3.5(–4) μm in vivo; apical cell 4–6 μm long, basal cell 6–8 μm long.

Holotype: Tanzania, on leaves of E. maidenii, May 1995, T. Coutinho, PREM 54971, holotype, culture ex-type CBS 111161 = CPC 1098.

Ascospore germination on MEA after 24 h: Type F. Similar to M. nubilosa.

Cultures: Colonies after 3 wk on MEA reaching 6–15 mm diam; on MEA erumpent with sparse aerial mycelium, pale olivaceous-grey; margins smooth, regular; reverse ochraceous with patches of pale olivaceous-grey; on PDA erumpent, centres white to pale olivaceous-grey, outer zone olivaceous-grey, margins irregular, feathery; reverse smoke-grey in the central part, olivaceous-grey in the outer region; colonies sterile.

Host: E. maidenii.

Distribution: Tanzania.

Notes: Ascospores of M. quasicercospora (10–14 × 3–4 μm) germinate with a Type F germination pattern, similar to that observed in M. nubilosa (11–16 × 3–4.5 μm), but are somewhat shorter, and also cluster phylogenetically apart (Fig. 3). Of particular interest is the fact that it aligns with sequences of Cercospora apii, for which no teleomorph is known. Cultures are sterile, and a re-examination of the original specimen also failed to reveal the presence of a Cercospora state. This is presently the only Mycosphaerella teleomorph clustering with Cercospora apii other than C. acaciae-mangii (Crous ) (Fig. 3).

Crous & M.J. Wingf., sp. nov. MycoBank MB500844. Fig. 20.

Fig. 20.

Mycosphaerella scytalidii (anamorph Stenella sp., synanamorph, Scytalidium-like.) (CBS 118493). A. Leaf spot. B–D. Asci. E–I. Ascospores. J–K. Germinating ascospores. L–M. Conidiophores. N. Conidia. O–R. Mycelium in culture. Scale bars: B, L = 3 μm, J–K = 5 μm.

Anamorph: sp.

Synanamorph: -like.

Etymology: Referring to the Scytalidium-like synanamorph.

Mycosphaerellae parkii similis, sed ascosporis minoribus, 8–10 × (2.5–)3 μm, modi I germinantibus, distinguenda.

Leaf spots amphigenous, irregular to sub-circular, 1–8 mm diam, grey to medium brown, with a raised, dark brown border. Ascomata pseudothecial, amphigenous, single, black, immersed becoming erumpent, globose, up to 90 μm diam; apical ostiole 5–10 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to ellipsoid, straight or slightly incurved, 8-spored, 25–30 × 7–9 μm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid–ellipsoidal with subobtuse ends, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, 8–10 × (2.5–)3 μm in vivo. Mycelium internal and external, consisting of septate, branched, verruculose hyphae, 2–3 μm wide. Caespituli fasciculate, amphigenous on the leaves, brown, up to 50 μm wide and 60 μm high. Conidiophores aggregated in loose fascicles arising from the upper cells of a brown stroma up to 50 μm wide and 30 μm high, or situated on the top of the ascomata; conidiophores medium brown, finely verruculose, 1–4-septate, subcylindrical, straight to geniculate–sinuous, unbranched, 20–40 × 2–4 μm. Conidiogenous cells terminal, unbranched, medium brown, smooth to verruculose, tapering to the flat-tipped apical loci, proliferating sympodially, 7–15 × 2–3 μm, with thickened, darkened, refractive scars. Conidia solitary, or in simple chains, medium brown, verruculose, subcylindrical to ellipsoidal, apex obtuse, base subtruncate, 1–2-septate, frequently constricted at the septa, 7–15 × 3–3.5 μm; hila thickened, darkened, refractive. Aerial mycelium disarticulating into hyaline, smooth arthroconidia that are Scytalidium-like, 12–35 × 3–5 μm.

Holotype: Colombia, Angela Maria, on leaves of Eucalyptus urophylla, Jan. 2004, M.J. Wingfield, CBS H-19696 holotype, culture ex-type CBS 118493 = CPC 10998.

Ascospore germination on MEA after 24 h: Type I. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, lateral branches present, and spore distorting upon germination, becoming up to 5 μm wide.

Cultures: Colonies on MEA reaching 18–30 mm diam after 3 wk; colonies erumpent, folding, margin smooth, irregular, aerial mycelium moderate, pale olivaceous-grey; reverse iron-grey; on PDA with moderate aerial mycelium, olivaceous-grey with patches of pale olivaceous-grey; reverse olivaceous-black; on OA pale olivaceous-grey with patches of olivaceous-grey and iron-grey.

Host: Eucalyptus urophylla.

Distribution: Colombia.

Notes: Several other as yet undescribed species occur on Eucalyptus leaves in Colombia, and some, such as M. longibasalis Crous & M.J. Wingf. (Crous 1998) (Fig. 21), is still not known from culture. Isolate CPC 10986 clusters with CPC 11002, and in culture they are distinct from CPC 11004. We were, however, unable to trace these isolates back to ascomata due to several species being present on the same leaf spots. Thus, further collections will be required before these taxa can be named.

Fig. 21.

Mycosphaerella longibasalis. A–B. Broken asci. C–F. Ascospores. Scale bars = 4 μm.

Mycosphaerella scytalidii is phylogenetically closely related to the Mycosphaerella sp. represented by CPC 11002 and CPC 10986 (Fig. 4). For reasons explained above, however, we presently cannot name the latter species. Mycosphaerella scytalidii is also noteworthy based on the fact that it forms a Stenella anamorph, as well as a Scytalidium-like synanamorph in culture. Numerous species of Mycosphaerella form clusters of chlamydospores on their hyphal tips in culture (M. bellula, M. jonkershoekensis) (Crous ), leading to the impression that they could develop into Trimmatostroma-like anamorphs. None, however, have been reported to form Scytalidium anamorphs. Many species of Mycosphaerella form aerial mycelium that remain hyaline, with wide, disarticulating cells, suggesting that this anamorph morphology may be more prevalent in species of Mycosphaerella than previously realised. Ascospores of M. scytalidii germinate with a Type I pattern, but none of the species on Eucalyptus with this germination pattern form a Stenella anamorph in culture.

Crous & A.C. Alfenas, sp. nov. MycoBank MB500845. Fig. 22.

Fig. 22.

Mycosphaerella secundaria (CBS 118507). A. Leaf spot. B–C. Broken asci. D–H. Ascospores. Scale bars: B, H = 3 μm.

Etymology: Referring to the ecology of this fungus as a secondary coloniser on lesions of M. suberosa.

Mycosphaerellae parkii similis, sed ascosporis minoribus, 8–10 × 2.5–3 μm, distinguenda.

Occurring as a secondary colonist on leaf spots caused by M. suberosa, or M. perpendicularis. Ascomata pseudothecial, amphigenous, single, inconspicuous, sparsely distributed, black, subepidermal, rarely erumpent, globose, up to 90 μm diam. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to narrowly ellipsoid, straight or slightly incurved, 8-spored, 20–30 × 7–9 μm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, ellipsoidal with subobtuse ends, medianly 1-septate, widest close to the apex of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, 8–10 × 2.5–3 μm in vivo.

Holotype: Brazil, Bahia, Teixeira de Freitas, on leaves of Eucalyptus sp., 8 Jun. 2004, A.C. Alfenas, CBS H-19697, holotype, culture ex-type CBS 118507 = CPC 11551–11553.

Ascospore germination on MEA after 24 h: Type D. Similar to M. parkii.

Cultures: Colonies on MEA after 3 wk reaching 25–35 mm diam; on MEA olivaceous-grey, flat, spreading, folding, with sparse aerial mycelium and smooth, even margins; reverse iron-grey; on PDA iron-grey with olivaceous-grey aerial mycelium in central part, and drops of slime throughout; reverse iron-grey; on OA flat, spreading, olivaceous-grey.

Host: Eucalyptus spp.

Distribution: Brazil, Colombia.

Notes: When this species was initially collected in 1992 (CPC 504), it was noted that it occurred in lesions ascribed to M. suberosa, presumably as a secondary pathogen. We have now been able to recollect this fungus where it had colonised lesions caused by M. suberosa, as well as those of Cryptosporiopsis eucalypti Sankaran & B. Sutton on eucalypts in Brazil. In the same phylogenetic clade accommodating M. secundaria from Brazil, isolates collected in Colombia were also found which were apparently associated with lesions caused by M. perpendicularis (Fig. 2). Mycosphaerella secundaria has thus far only been collected in association with other species of Mycosphaerella that we believe are the primary pathogens. Mycosphaerella perpendicularis (ascospores 8–10 × 2.5–3 μm) was originally treated as M. parkii (ascospores 8–15 × 2–3.5 μm) (Crous 1998).

Additional culture examined: Brazil, Picadao, Conceicao da Barra, on leaves of E. grandis, 27 Apr. 1992, A.C. Alfenas, CBS 115608 = CPC 504.

Crous & A.C. Alfenas, sp. nov. MycoBank MB500846. Fig. 23.

Fig. 23.

Mycosphaerella stramenti (CBS 118909). A–B. Asci. C–F. Ascospores. G. Germinating ascospore. Scale bar = 3 μm.

Etymology: Refers to the occurrence of this fungus on leaf litter.

Mycosphaerellae parkii similis, sed ascosporis minoribus, (8–)10–12(–13) × 3(–3.5) μm, modo I germinantibus, distinguenda.

Leaf spots absent, ascomata associated with leaf litter. Ascomata pseudothecial, amphigenous, but predominantly hypophyllous, single, black, immersed becoming erumpent, globose, up to 120 μm diam. Asci aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to subcylindrical, straight or slightly incurved, 8-spored, 25–40 × 7–8 μm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight to slightly curved, fusoid–ellipsoidal with subobtuse ends, medianly 1-septate, widest in middle of apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)10–12(–13) × 3(–3.5) μm, in vivo.

Holotype: Brazil, Minas Gerais, Belo Oriente, on leaf litter of Eucalyptus sp., 24 Jan. 2004, A.C. Alfenas, CBS H-19698, holotype, culture ex-type CBS 118909 = CPC 11545–11547.

Ascospore germination on MEA after 24 h: Type I. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, and lateral branches also present; ascospore constricting at the septum, becoming up to 5 μm wide.

Cultures: Colonies on MEA reaching 20–27 mm diam after 3 wk; on MEA colonies erumpent, spreading, aerial mycelium sparse, surface folding, pale olivaceous-grey, with central part having patches of smoke-grey; margin feathery, irregular, reverse greenish black; on PDA surface olivaceous-black with patches of smoke-grey aerial mycelium in central part; margins feathery, irregular, reverse greenish black; on OA olivaceous-black with smoke-grey aerial mycelium; margins irregular, feathery.

Host: Eucalyptus sp.

Distribution: Brazil.

Notes: Ascospores of M. stramenti germinate with a Type I pattern. Several taxa are known to have this pattern of ascospore germination (Crous 1998), from which M. stramenti can be distinguished by its ascospore dimensions and cultural characteristics. Phylogenetically it is closely related to M. endophytica (Fig. 4).

Crous & A.C. Alfenas, sp. nov. MycoBank MB500847. Fig. 24.

Fig. 24.

Mycosphaerella stramenticola (CBS 118506). A. Ascomata on leaf. B. Asci. C–D. Ascospores. E–F. Germinating ascospores. Scale bars: B = 8 μm, C = 3 μm, E = 6 μm.

Etymology: Latin stramentum = leaf litter, the substrate from which this fungus was collected.

Mycosphaerellae crystallinae similis, sed ascosporis minoribus, 8–11 × 3–3.5 μm, distinguenda.

Leaf spots absent, associated with leaf litter. Ascomata pseudothecial, amphigenous, single, black, immersed becoming erumpent, globose, up to 90 μm diam; apical ostiole 5–10 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to subcylindrical, straight or slightly incurved, 8-spored, 30–35 × 7–9 μm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid-ellipsoidal with subobtuse ends, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–11) × 3(–3.5) μm, in vivo.

Holotype: Brazil, Bahia, Eunapolis, on leaf litter of Eucalyptus sp., 23 May 2004, A.C. Alfenas, CBS H-19699 holotype, culture ex-type CBS 118506 = CPC 11438–11440.

Ascospore germination on MEA after 24 h: Type I. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, and distorting prominently upon germination, becoming up to 6 μm wide; lateral branches also present.

Cultures: Colonies on MEA reaching 22–38 mm diam after 3 wk; colonies flat, spreading; aerial mycelium sparse; margins smooth, regular, surface olivaceous-grey with drops of slime; reverse iron-grey; on OA pale olivaceous-grey in the centre due to moderate aerial mycelium; olivaceous-grey in the outer region; on PDA olivaceous-grey with drops of slime, margin thin, iron-grey on surface and reverse.

Host: Eucalyptus sp.

Distribution: Brazil.

Notes: Mycosphaerella stramenticola is phylogenetically closely related to isolates CPC 727–728 (Fig. 2), which represent an undescribed taxon from Indonesia. Mycosphaerella stramenticola has ascospores that germinate with a Type I pattern, thus being similar to those of M. crystallina (11–15 × 3–4 μm), M. ellipsoidea (8–11 ×2–3 μm), M. endophytica (8–11 × 2–3 μm), M. lateralis (7–16 × 2–3 μm), M. irregulariramosa (7–10 × 1.5–2.5 μm) and M. tasmaniensis (10–13 × 2.5–4 μm). Ascospores of M. stramenticola are 8–11 × 3–3.5 μm, and thus being wider than those of M. ellipsoidea, M. endophytica, and M. irregulariramosa. Furthermore, cultures of M. stramenticola are sterile, while all the other species listed here produce anamorphs in culture.

Crous & M.J. Wingf., sp. nov. MycoBank MB500848. Fig. 25.

Fig. 25.

Mycosphaerella sumatrensis (CBS 118499). A. Leaf spot. B–C. Asci. D–G. Ascospores. H–I. Germinating ascospores. J–K. Hyphae with exudate droplets. Scale bar = 4 μm.

Etymology: Refers to Sumatra, where this fungus was collected.

Mycosphaerellae keniensi similis, sed ascosporis maioribus, 12–16 × 3–4 μm, distinguenda.

Leaf spots amphigenous, irregular to subcircular, 2–10 mm diam, pale brown with a dark brown, raised border, and thin, red-purple margin. Ascomata pseudothecial, amphigenous but predominantly epiphyllous, single, black, subepidermal to erumpent, globose, up to 80 μm diam; apical ostiole 15–20 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid, straight or slightly incurved, 8-spored, 30–40 × 9–11 μm. Ascospores multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in middle of apical cell, not constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (12–)13–15(–16) × (3–)4 μm, in vivo.

Holotype: Indonesia, Northern Sumatra, on leaves of Eucalyptus sp., Feb. 2004, M.J. Wingfield, CBS H-19704, holotype, culture ex-type CBS 118499 = CPC 11171, CBS 118501 = CPC 11175, CBS 118502 = CPC 11178.

Ascospore germination on MEA after 24 h: Type J. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, but also with one or two lateral branches forming at the spore ends; ascospores becoming slightly constricted and up to 4 μm wide.

Cultures: Colonies 8–19 mm diam on MEA after 3 wk; erumpent, with sparse aerial mycelium, smoke-grey; margin smooth, but irregular; reverse olivaceous-black; on PDA erumpent, olivaceous-grey with a thin whitish border; iron-grey in reverse; on OA smoke-grey, appearing olivaceous-black in the centre due to collapse of the aerial in copious amounts of slime.

Host: Eucalyptus sp.

Distribution: Indonesia.

Notes: Mycosphaerella sumatrensis is phylogenetically distinct from other species occurring on Eucalyptus (Fig. 2). Ascospores (12–16 × 3–4 μm) germinate with Type J germination patterns, as do M. colombiensis (11–15 × 3–4 μm) and M. keniensis (7–11 × 2.5–3 μm). However, ascospores of M. sumatrensis are larger than those of M. keniensis, and it has no anamorph, while M. colombiensis occurs in close association with its Pseudocercospora anamorph (Crous 1998).

Crous & M.J. Wingf., sp. nov. MycoBank MB500849. Fig. 26.

Fig. 26.

Mycosphaerella verrucosiafricana (CBS 118496). A. Leaf spot. B–C. Asci. D–E. Ascospores. F–G. Germinating ascospores. Scale bars: B = 3 μm, F = 8 μm.

Etymology: Refers to M. africana, to which it is morphologically similar.

Mycosphaerellae africanae similis, sed ascosporis latioribus, verruculosis, (7–)8–9(–10) × 3(–3.5) μm, distinguenda.

Leaf spots amphigenous, irregular to sub-circular, 5–15 mm diam, pale brown to grey, surrounded by a raised, dark brown border, and a thin, red-purple margin. Ascomata pseudothecial, amphigenous but chiefly hypophyllous, single, black, immersed becoming erumpent, globose, up to 60 μm diam; apical ostiole 10–15 μm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to narrowly ellipsoid, straight or slightly incurved, 8-spored, 18–27 × 7–8 μm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, ellipsoid with obtuse ends, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (7–)8–9(–10) × 3(–3.5) μm in vivo.

Holotype: Indonesia, Northern Sumatra, on leaves of Eucalyptus sp., Feb. 2004, M.J. Wingfield, CBS H-19705 holotype, culture ex-type CBS 118496 = CPC 11167, CBS 118497 = CPC 11169, CBS 118498 = CPC 11170).

Ascospore germination on MEA after 24 h: Type E. Ascospores becoming dark brown and verruculose on MEA, and germinating from both ends, with germ tubes irregular to the long axis of the spore; frequently with more than two germ tubes, and distorting prominently upon germination, becoming up to 9 μm diam.

Cultures: Colonies on MEA 12–22 mm diam after 3 wk; erumpent, spreading, with smooth, uneven margins; upper surface cracking open; aerial mycelium sparse to absent; colonies sectoring, olivaceous-grey; margin thin, iron-grey; reverse greenish-black; on PDA with moderate aerial mycelium, and spots of slime appearing spread over the iron-grey surface; reverse greenish black; on OA colonies submerged; aerial mycelium almost completely absent, greenish black; forming chains of dark brown, thick-walled chlamydospores that aggregate into small microsclerotia (on all media); colonies sterile.

Host: Eucalyptus sp.

Distribution: Indonesia.

Notes: Mycosphaerella verrucosiafricana is distinguished from other taxa currently known from Eucalyptus in that it has a characteristic ascospore germination pattern. Germinating ascospores turn brown and verruculose, but germinate with more than two germ tubes, which grow irregular to the long axis of the spore (Type G, becoming type E with age). Young ascospores just beginning to germinate can be confused with those of M. africana, as they initially also have only two germ tubes, though the ascospores are more distinctly verruculose than those of M. africana. Within a few hours of germination, additional germ tubes appear, and the pattern is more similar to that of Type E, which is seen in M. suberosa. Mycosphaerella verrucosiafricana is distinguished from M. suberosa in that the germ tubes remain hyaline, and ascospores and leaf spots are quite distinct from those of M. suberosa.

Z.Q. Yuan, de Little & Mohammed, Nova Hedwigia 71: 416. 2000. Fig. 27.

Fig. 27.

Pseudocercospora subulata (CBS 118489). A. Leaf spot. B. Conidiophores. C–E. Conidia. Scale bars = 5 μm.

= Pseudocercospora pseudobasitruncata U. Braun & M. Dick, New Zealand J. For. Res. 32: 228. 2002.

Specimen examined: New Zealand, North Island, KeriKeri, on leaves of E. botryoides, 17 Oct. 2003, M.A. Dick, CBS 118489 = CPC 10849.

Cultures: Colonies reaching 25–35 mm diam after 3 wk on MEA; pale olivaceous-grey, erumpent, with moderate to extensive aerial mycelium; margin regular, smooth, reverse iron-grey; on PDA pale olivaceous-grey, margin thin, olivaceous-grey, reverse iron-grey; on OA central part erumpent, pale olivaceous-grey, outer zone olivaceous-grey, flat and spreading.

Host: E. botryoides.

Distribution: New Zealand.

Notes: Pseudocercospora subulata is morphologically similar to P. pseudobasitruncata, and hence they are listed here as synonyms. The culture used in this study was obtained from lesions colonised by both P. crousii U. Braun & M. Dick and P. subulata. Although the culture was obtained from a single germinating conidium, it is sterile, and we were unable to rule out the possibility that it may represent P. crousii and not P. subulata. Further collections and cultures are required to undertake DNA sequence comparisons with the Pseudocercospora Speg. species recently described from eucalypts by Braun & Dick (2002).

Crous, sp. nov. MycoBank MB500850. Figs 28, 29.

Fig. 28.

Conidiophores and conidia of Septoria eucalyptorum (CBS 118505). Scale bar = 10 μm.

Fig. 29.

Septoria eucalyptorum (CBS 118505). A. Conidiomata forming on CLA, with conidial masses. B. Conidial mass. C–D. Conidia. Scale bars = 4 μm.

Etymology: Refers to its host, Eucalyptus.

Septoriae linicolae similis, sed conidiis brevioribus, 8–22 × 2–2.5 μm, distinguenda.

Leaf spots absent, conidiomata associated with leaf litter. Mycelium internal, consisting of smooth, branched, septate, pale brown, 2–2.5 μm wide hyphae. Conidiomata pycnidial, immersed, brown, globose on leaves, up to 160 μm diam; wall consisting of 3–6 cell layers of textura angularis. Conidiophores lining the inner layer of the conidioma, dense aggregated, subcylindrical, straight to curved, 0–1-septate, mostly reduced to conidiogenous cells. Conidiogenous cells terminal, unbranched, hyaline, smooth, subcylindrical, proliferating sympodially near the apex, 5–10 × 2–2.5 μm. Conidia solitary in vivo, but undergoing microcyclic conidiation in vitro, finely guttulate, subcylindrical to narrowly obclavate, with obtuse to subobtuse apex, and long subtruncate base, straight to curved, 1(–3)-septate, (8–)12–16(–22) × 2(–2.5) μm; hila inconspicuous, 0.5–1 μm diam.

Holotype: India, Palampur, on Eucalyptus leaf litter, Feb. 2004, W. Gams & M. Arzanlou, CBS H-19700, holotype, cultures ex-type CBS 118505 = CPC 11282, CPC 11283.

Cultures: Colonies after 3 wk on MEA 30–40 mm diam; on MEA pale white to smoke-grey; aerial mycelium sparse; colonies spreading, margins even, smooth; reverse fuscous-black with patches of vinaceous-grey; on PDA producing large amounts of slime, with thread-like tufts of aerial mycelium; surface pale purplish grey (centre) with a zone of vinaceous-grey, and a pale vinaceous-grey, flat, spreading marginal region; reverse vinaceous-grey with patches of pale vinaceous-grey; on OA pale vinaceous-grey (centre) with a zone of purplish grey, a wide, flat margin concolorous with the medium; conidiomata frequently formed along circardian growth lines.

Host: Eucalyptus sp.

Distribution: India.

Notes: Sankaran et al. (1995) listed several species of Septoria on Eucalyptus, most of which have been redisposed to other genera. The exceptions are S. eucalypti G. winter & Roum. (conidia filiform–acicular, 1-septate, 14–18 × 1.5 μm) and S. mortolensis Penz. & Sacc. (conidia 0–2-septate, 50–55 × 3–3.5 μm). Gadgil & Dick (1999) recently described S. typica Gadgil & M. Dick, which is characterised by having filiform, sigmoid or falcate, 1-septate conidia, 65–70 × 2–3 μm. Septoria eucalyptorum is distinct from this species in having conidia that are subcylindrical to narrowly obclavate, 1(–3)-septate, 8–22 × 2–2.5 μm. DNA sequence data in the present study (Fig. 3), show that Septoria eucalyptorum is closely allied to S. linicola (on Linum, conidia filiform, 1–3-septate, 17–40 × 1.5–3 μm) and S. protearum (on Protea, conidia subcylindrical to narrowly obclavate, (0–)1–3(–4)-septate, 6–30 × 1.5–2 μm). To fully resolve this relationship, however, other loci will need to be sequenced, as the ITS domain is insufficient to distinguish species complexes in Septoria.

Crous, sp. nov. MycoBank MB500851. Fig. 30.

Fig. 30.

Septoria provencialis (CBS 118910). A. Leaf spot. B–C. Conidiogenous cells. D–E. Conidia. Scale bars = 3 μm.

Etymology: Refers to the Provence in France where the fungus was collected.

Septoriae mortolensi similis, sed conidiis brevioribus, 12–45 × 2.5–4 μm, distinguenda.

Leaf spots amphigenous, dark brown, angular, confined by leaf veins, 1–6 mm diam, becoming confluent with age. Mycelium internal, consisting of smooth, branched, septate, hyaline, 3–4 μm wide hyphae. Conidiomata amphigenous on leaves, pycnidial, immersed, brown, globose, up to 200 μm diam; wall consisting of 2–4 cell layers of textura angularis. Conidiophores lining the inner surface of the conidioma, densely aggregated, subcylindrical to ampulliform, straight to slightly curved, 0–2-septate, 6–25 × 3–5 μm. Conidiogenous cells terminal, unbranched, hyaline, smooth, subcylindrical to ampulliform, proliferating sympodially or several times percurrently near the apex, 6–10 × 3–5 μm. Conidia solitary in vivo, finely guttulate, subcylindrical to narrowly obclavate, with subobtuse apex, and obconically subtruncate base, variously curved to irregular, mostly widest in the middle of the basal cell, tapering towards the apex, (1–)2(–3)-septate, (12–)30–40(–45) × 2.5–3(–4) μm.

Holotype: France, Provence, Cheval Blanc camping site, on juvenile Eucalyptus leaves, 29 Jul. 2005, P.W. Crous, CBS H-19701, holotype, cultures ex-type CBS 118910 = CPC 12226, CPC 12227–12228.

Cultures: Colonies 10–15 mm diam after 3 wk on MEA; colonies erumpent, surface irregular, catenate, olivaceous-grey with cream to pale rosy-buff spore masses; aerial mycelium absent; margins smooth, regular, with a thin outer zone that is pale olivaceous-grey to slightly rosy-buff; colonies olivaceous-black in reverse.

Host: Eucalyptus sp.

Distribution: France.

Note: Conidia of S. provencialis (12–45 × 2.5–4 μm) are most similar to S. mortolensis (50–55 × 3–3.5 μm), although on average, they are much shorter.

Crous & M.J. Wingf., sp. nov. MycoBank MB500852. Fig. 31.

Fig. 31.

Stenella pseudoparkii (teleomorph Mycosphaerella sp.) (CBS 19702). A–C. Germinating ascospores. D–E. Conidiophores. F–H. Conidia. Scale bars: A = 5 μm, D = 3 μm.

Teleomorph: sp.

Etymology: Morphologically similar to M. parkii and its anamorph, S. parkii.

Stenellae parkii similis, sed conidiis brevioribus, 20–50 × 2.5–3 μm, distinguenda.

Leaf spots amphigenous, irregular to subcircular, 3–7 mm diam, pale brown, with a raised border. Conidiophores arising singly from superficial mycelium, brown, smooth to finely verruculose, 1–4-septate, subcylindrical, straight to variously curved, unbranched, 15–60 × 3–4 μm. Conidiogenous cells terminal, unbranched, medium brown, smooth, tapering to flat-tipped apical loci that are darkened and refractive, proliferating sympodially, 15–25 × 2–3 μm. Conidia solitary to catenulate in simple chains, medium brown, verruculose, cylindrical or narrowly obclavate, with subobtuse apex, and long obconically subtruncate base, straight to curved, 1–5-septate, 20–50 × 2.5–3 μm; hila thickened, darkened and refractive.

Holotype: Colombia, on leaves of Eucalyptus sp., 1995, M.J. Wingfield, CBS H-19702 holotype, culture ex-type CBS 110999 = CPC 1087; 1088–1092.

Ascospore germination on MEA after 24 h: Type D. Ascospores smooth, not darkening on MEA, germinating from both ends, with germ tubes parallel to the long axis of the spore, and some lateral branches; ascospores distorting, becoming up to 5 μm wide.

Cultures: Colonies after 3 wk on MEA 23–30 mm diam, pale olivaceous-grey, spreading, with moderate aerial mycelium, and smooth, irregular margins; colonies folding, erumpent; reverse olivaceous-black; on PDA pale olivaceous-grey with moderate aerial mycelium and copious amounts of slime; margins submerged in the agar; reverse olivaceous-grey; on OA pale olivaceous-grey, colonies folding with moderate aerial mycelium, and a thin olivaceous-grey margin.

Host: Eucalyptus sp.

Distribution: Colombia.

Notes: Several species of Mycosphaerella were present on the lesions from which S. pseudoparkii was isolated, and it was not possible to trace the ascospores back to the specific ascomata. The description of the Mycosphaerella teleomorph thus has to await further collections. The ascospores that shot out onto MEA germinated with a Type D pattern, which together with its Stenella anamorph, resulted in it being identified as M. parkii (Crous 1998). Phylogenetically, S. pseudoparkii is distinct from M. parkii, and most closely related to M. scytalidii, which has a Type I germination pattern. Stenella pseudoparkii has shorter conidia (20–50 × 2.5–3 μm) than Stenella parkii (25–200 × 2–2.5 μm) (Crous & Alfenas 1995, Crous 1998).

Crous & M.J. Wingf., sp. nov. MycoBank MB500853. Fig. 32.

Fig. 32.

Stenella xenoparkii (teleomorph Mycosphaerella sp.) (CBS 111185). A–B. Conidiophores. C–G. Conidia. Scale bar = 4 μm.

Teleomorph: sp.

Etymology: refers to the morphological similarity with M. parkii and its anamorph, S. parkii.

Stenellae parkii similis, sed conidiis brevioribus, 12–50 × 3–5 μm, distinguenda.

Leaf spots amphigenous, irregular to subcircular, 2–10 mm diam, pale brown, with a raised border and thin, red-purple margin. Conidiophores arising singly from superficial mycelium, medium brown, finely verruculose, 1–2-septate, subcylindrical, straight to variously curved, unbranched, 30–60 × 3–4 μm. Conidiogenous cells terminal, unbranched, medium brown, verruculose, tapering to flat-tipped apical loci that are darkened and refractive, proliferating sympodially, 10–25 × 3–4 μm. Conidia catenulate in branched chains, medium brown, verruculose, cylindrical or narrowly obclavate, with subobtuse apex, and subtruncate base, straight to curved, 0–2-septate, 12–50 × 3–5 μm; hila thickened, darkened and refractive.

Holotype: Indonesia, on leaves of E. grandis, Mar. 1996, M.J. Wingfield, holotype PREM 54968, isotype in CBS H-19703, cultures ex-type CBS 111185 = CPC 1300; 1299–1301.

Ascospore germination on MEA after 24 h: Type D. Similar to M. parkii.

Cultures: Colonies after 3 wk on MEA 25–35 mm diam; on MEA spreading, slightly erumpent, margins smooth but irregular; aerial mycelium sparse to moderate; surface olivaceous-black, but central part grey due to aerial mycelium; reverse olivaceous-black; on PDA olivaceous-black with mucous droplets and aerial mycelium that is olivaceous-grey in the central part, but has a reddish tinge in the outer region; reverse greenish black; on OA iron-grey with sparse to moderate olivaceous-grey aerial mycelium.

Host: Eucalyptus grandis.

Distribution: Indonesia.

Notes: The specimen on which this species is based was originally identified as representing M. parkii. The original identification was based on its characteristic leaf spots, ascospore germination patterns and dimensions, and the presence of a Stenella anamorph. Teleomorph material was not retained, and hence only the anamorph, which forms in culture, can be named. Conidia of S. xenoparkii (12–50 × 3–5 μm) are shorter and wider than those of S. pseudoparkii (20–50 × 2.5–3 μm) and S. parkii (25–200 × 2–2.5 μm) (Crous & Alfenas 1995, Crous 1998).

DISCUSSION

In this study we have described 21 new species of Mycosphaerella or its anamorphs from Eucalyptus leaves. Some of these new species arise from a re-examination of specimens and cultures treated previously (Crous 1998). The species in this earlier study had been described primarily on the basis of morphology and without the support of DNA sequence comparisons.

Results of this study are similar to those of Crous et al. (2004b) showing that there are several species of Mycosphaerella on eucalypts that have distinct cultural characteristics and can be separated based on phylogenetic analyses, but that share the same symptoms, morphological characterisitics and ascospore germination patterns. It is clearly very difficult to accurately identify Mycosphaerella species on eucalypts in the absence of DNA sequence analyses. Identifications or species described based solely on morphological characteristics must consequently be viewed with some circumspection.

A good example of the confusion arising from identifications based solely on phenotypic characters is found in the case of M. parkii. In the present study, we reconsidered several collections originally identified as M. parkii based on symptoms, ascospore dimensions, germination patterns, and the presence of a Stenella anamorph in culture. The “M. parkii”-like isolates were consequently shown to represent several species. Because of insufficient material being available, only two anamorph species S. xenoparkii and S. pseudoparkii, could be named.

Cryptic species were also found among isolates originally identified as M. africana. These identifications were based on the presence of fusoid–ellipsoidal ascospores that are constricted at the septum, that darken upon germination, and that produce colonies that are relatively slow-growing. These isolates are described here as M. verrucosiafricana and M. pseudafricana. Other examples of cryptic species were found in the case of M. pseudoendophytica, which is morphologically similar to M. endophytica, M. pseudosuberosa, which is similar to M. suberosa, and M. pseudocryptica, which is similar to M. cryptica.

In this study we have applied only DNA sequences of the ITS region. Although this locus has been very useful in delimiting species of Mycosphaerella from Eucalyptus, it is not always sufficient to derive conclusions for all species complexes (Crous , Hunter – this volume). For example, it is not suitable for distinguishing species in anamorph genera such as Cercospora and Septoria. In contrast, sequences of the ITS region appear to be useful for distinguishing species with Pseudocercospora, Ramularia and most other Mycosphaerella anamorph genera that we have considered (Crous & Groenewald, unpubl. data). It appears, therefore, that the ITS region has evolved at different rates in different anamorph genera associated with Mycosphaerella, and that it is more conserved in Cercospora and Septoria, two genera that always cluster together.

In this study, we have described several new Mycosphaerella species from leaf litter. This suggests that there are numerous endophytic Mycosphaerella species that sporulate once leaves have died. The biology of Mycosphaerella species suggests that these fungi are probably not saprobes but rather that they infect living leaf tissue and only sporulate after leaf fall. Virtually nothing is known of this life-habit of Mycosphaerella species, and it would be intriguing to follow the infection patterns of species that are not primary pathogens.

Mycosphaerella secundaria was one of the more intriguing fungi arising from this study. This fungus has been collected on several occasions, but its unique nature was not confirmed previously. Mycosphaerella secudaria is always found on leaf spots caused by M. suberosa. This is an unusual habit for a species of Mycosphaerella, and its ecological role deserves further study.

Dissoconium dekkeri (teleomorph: M. lateralis) was originally described as a potential hyperparasite of powdery mildew (De Hoog et al. 1983, 1991), and has since been isolated from many different hosts (Crous ). Jackson et al. (2004) showed that M. lateralis is not a hyperparasite of M. nubilosa and M. cryptica, the two species with which M. lateralis frequently co-occurs. Jackson et al. (2004) also showed that D. dekkeri can infect Eucalyptus leaves. Mycosphaerella lateralis and M. communis occur on leaves of numerous Eucalyptus spp., and they are frequently found on leaf spots caused by other Mycosphaerella species, as well as unrelated fungi (Crous unpubl. data). The ecological role of M. lateralis, however, remains to be determined.

The linking of yet another anamorph genus to Mycosphaerella, namely Davisoniella, draws an interesting parallel to morphologically similar coelomycete genera. Of particular interest, are the taxa currently accommodated in Colletogloeopsis, particularly those that are Coniothyrium-like and to which D. eucalypti is definitely closely related if not congeneric. Presently no cultures are available, the relationship between these taxa remains to be proven, and hence the anamorph is best retained in Davisoniella.

In this study, we have added 21 species to the number of Mycosphaerella spp. presently known to occur on Eucalyptus leaves and stems. We suggested that there could easily be at least as many Mycosphaerella spp. on eucalypts as there are species of that genus. This would imply that only 14 % of the species of Mycosphaerella from eucalypts have presently been described. This means that significant challenges face the taxonomists who wish to distinguish Mycosphaerella spp. form eucalypts in future. Most likely, in future studies, DNA sequence comparisons based on multiple genes will be required to accurately identify these fungi. Given the enormity of this task, focus will clearly need to be directed to those species that are primary pathogens. However, the primary pathogens are so easily confused with other less important species, that all material will ultimately have to be thoroughly studied and understood.