Hypocrea rufa/Trichoderma viride: a reassessment, and ... - CBS

Hypocrea rufa/Trichoderma viride: a reassessment, and 

description of five closely related species with and without warted 

conidia 

Walter M. Jaklitsch 1 , Gary J. Samuels 2 *, Sarah L. Dodd 3 , Bing-Sheng Lu 4 and Irina S. Druzhinina 1 

1 Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, Getreidemarkt 

9-166.5, A-1060 Vienna, Austria; 2 United States Department of Agriculture, Agricultural Research Service, Systematic Botany and Mycology 

Laboratory, Rm. 304, B-011A, BARC-W, Beltsville, Maryland 20705, U. S.A.; 3 The Pennsylvania State University, Department of Plant Pathology, 

Buckhout Laboratory, University Park, Pennsylvania 16802, U.S.A. Current address: New Zealand Institute of Crop and Food Research Ltd., 

Private Bag 4704, Christchurch, New Zealand; 4 The Pennsylvania State University, Department of Plant Pathology, Buckhout Laboratory, 

University Park, Pennsylvania 16802, U.S.A. Current address: Agronomy College, Department of Plant Protection, Zhongkai Agrotechnical 

College, Guangzhou 510225, China. 

*Correspondence: Gary J. Samuels, Gary@nt.ars-grin.gov 

Abstract: The type species of the genus Hypocrea (Hypocreaceae, Hypocreales, Ascomycota, Fungi), H. rufa, is re-defined and 

epitypified using a combination of phenotype (morphology of teleomorphs and anamorphs, and characteristics in culture) and 

phylogenetic analyses of the translation-elongation factor 1α gene. Its anamorph, T. viride, the type species of Trichoderma, is 

re-described and epitypified. Eidamia viridescens is combined as Trichoderma viridescens and is recognised as one of the most 

morphologically and phylogenetically similar relatives of T. viride. Its teleomorph is newly described as Hypocrea viridescens. 

Contrary to frequent citations of H. rufa and T. viride in the literature, this species is relatively rare. Although both T. viride and 

T. viridescens have a wide geographic distribution, their greatest genetic diversity appears to be in Europe and North America. 

Hypocrea vinosa is characterised and its anamorph, T. vinosum sp. nov., is described. Conidia of T. vinosum are subglobose 

and warted. The new species T. gamsii is proposed. It shares eidamia-like morphology of conidiophores with T. viridescens, but 

it has smooth, ellipsoidal conidia that have the longest L/W ratio that we have seen in Trichoderma. Trichoderma scalesiae, an 

endophyte of trunks of Scalesia pedunculata in the Galapagos Islands, is described as new. It only produces conidia on a lownutrient 

agar to which filter paper has been added. Additional phylogenetically distinct clades are recognised and provisionally 

delimited from the species here described. Trichoderma neokoningii, a T. koningii-like species, is described from a collection 

made in Peru on a fruit of Theobroma cacao infected with Moniliophthora roreri. 

Taxonomic novelties: Hypocrea viridescens Jaklitsch & Samuels sp.nov., Trichoderma viridescens (A.S. Horne & H.S. Williamson) Jaklitsch 

& Samuels comb.nov., T. gamsii Samuels & Druzhinina sp.nov., T. vinosum Samuels sp.nov., T. neokoningii Samuels & Soberanis sp.nov., T. 

scalesiae Samuels & H.C. Evans sp.nov. 

Key words: Bayesian phylogeny, biogeography, biological control, cacao, endophytes, Hypocrea, Hypocreales, Hypocreaceae, molecular 

identification, morphological key, nomenclature, species identification, systematics, translation elongation factor 1-alpha. 

INTRODUCTION 

Trichoderma viride Pers. (Hypocreales, Hypocreaceae) 

is one of the most commonly reported species of fungi. 

In only the two years 2004 and 2005 T. viride appeared 

in nearly 200 articles that were abstracted by CAB. 

The species is encountered in widely diverse contexts; 

a few examples of activities include organochlorine 

degradation as a soil fungus (Smith 1995), biological 

control in fungus-induced plant disease (Brown & Bruce 

1999; Brown et al. 1999), and as the cause of disease 

in button mushrooms in India (Mishra & Singh 2005). 

It is said to effect seed germination of flowering plants 

(Celar & Valic 2005), and enhance phosphorus uptake 

by plants (Rudresh et al. 2005). It produces enzymes 

(Nobe et al. 2004), degrades cellulosic agricultural 

waste to alcohol (Baig et al. 2004), colonises leaf litter 

(Osono 2005) and is a normal inhabitant of soils (Roiger 

et al. 1991, Hagn et al. 2003). Do all these citations 

refer to only one species, T. viride? Kullnig et al. (2001) 

detected a shockingly high level of misidentification 

of strains that were reported in the literature as T. 

harzianum. If this experience is representative of the 

genus, as it is likely, then not all of these reports actually 

STUDIES IN MYCOLOGY 55: 135–177. 2006. 

refer to T. viride. One example that is representative 

of the degree of inaccuracy in identification is that 

of a biocontrol fungus reported in the literature as T. 

viride (Bastos 1988, 1996 a, b) that was ultimately 

described as the new species T. stromaticum Samuels 

& Pardo-Schultheiss (Samuels et al. 2000); these two 

species are distantly related and morphologically and 

biologically highly dissimilar. Obviously, it is important 

to clarify the identity of T. viride, otherwise the literature 

is meaningless. 

Bisby in 1939 stated that essentially there was 

only one species of Trichoderma, T. viride. In spite of 

some discordant indications, that view held sway until 

1969 when Rifai (1969) monographed the genus and 

characterised T. viride as the only species having globose, 

warted conidia. This immediately raised suspicion about 

all reports of activity by Trichoderma species prior to 

1969. Even with the description of T. saturnisporum 

and T. ghanense, both having warted conidia and both 

being members of T. sect. Longibrachiatum Bissett 

(Samuels et al. 1998), T. viride stood out because 

its conidia were globose as compared to ellipsoidal 

in the other species. Scanning electron microscopy 

(Meyer & Plaskowitz 1989) revealed the existence of 

135

JAKLITSCH ET AL. 

two distinct patterns of conidial ornamentation within 

strains identified as T. viride, viz. more and less strongly 

warted. Strains having the less strongly warted conidia 

were segregated as T. asperellum Samuels et al. 

(Lieckfeldt et al. 1999; Samuels et al. 1999). In a study 

of variation within the morphological species T. viride, 

in addition to recognising T. asperellum and T. viride s. 

str., Lieckfeldt et al. (1999) noted the existence of two 

additional ITS-defined groups that had warted conidia, 

which they referred to as Vd and Ve. The group Vd was 

very closely related to Vb in its ITS1 and 2 sequences 

and its morphology. The group Ve was more distantly 

related and was phenotypically diverse, some of the 

few included strains having smooth conidia and others 

having warted conidia. They (Samuels et al. 1999) 

determined that the group Vb was “true” T. viride by 

comparison with the over two-hundred-year-old type 

specimen of the species that is preserved in Leiden. 

Despite differences in ITS sequences, Samuels et al. 

(1999) could not see consistent phenotypic differences 

between Vb and Vd that would support recognition of 

Vd as a separate taxon. 

Bissett (1991a) proposed to include H. rufa/T. viride 

and its relatives in Trichoderma sect. Trichoderma, 

including also T. koningii Oudem. and T. atroviride 

P. Karst. The monophyly of this group either as 

Trichoderma sect. Trichoderma (e.g. Kullnig-Gradinger 

et al. 2002) or more recently simply as “the viride clade” 

(Samuels 2006), has been affirmed by DNA sequence 

analysis. Since the work of Lieckfeldt et al. (1999) we 

have obtained many additional specimens and cultures 

referable to the viride clade and are able to propose a 

revised taxonomy for this clade. In the present work we 

re-evaluate T. viride groups Vb and Vd and recognise 

group Vd as a distinct species. 

Since the middle of the 19 th century (Tulasne & 

Tulasne 1865), T. viride has been recognised as the 

anamorph of Hypocrea rufa (Pers. : Fr.) Fr., the type 

species of Hypocrea Fr. Like T. viride, H. rufa is possibly 

the most common name used in the identification of 

Hypocrea specimens. Hundreds of specimens in 

herbaria throughout the world are labelled “Hypocrea 

rufa”. However, even a quick glance at specimens 

shows that a plethora of species has been lumped 

under this name. For example, species such as H. 

minutispora B.S. Lu et al./T. minutisporum Bissett and 

H. pachybasioides Yoshim. Doi/T. polysporum (Link : 

Fr.) Rifai have both been incorrectly identified as the 

only distantly related H. rufa. 

Webster (1964) provided the first modern description 

of H. rufa. It is a species that has a stroma that starts 

out semieffused and whitish to tan to reddish brown and 

pruinose and with age becomes darker and cushionshaped; 

the ascospores are hyaline. In our continuing 

work with the viride clade we have found that especially 

the young stroma of most members of the clade is 

distinctive of a number of often sympatric species that 

are best distinguished by their Trichoderma anamorphs 

(Samuels et al. 2006a). We have found indistinguishable 

teleomorphs for both T. viride groups, Vb and Vd. This 

calls for a redefinition and redescription of H. rufa. In 

the present work we refine the description of H. rufa 

136 

and provide an epitype for the species, we describe 

as new a teleomorph for T. viride group Vd, redescribe 

Hypocrea vinosa with its new anamorph T. vinosum, 

and describe the new species T. gamsii, T. neokoningii 

and T scalesiae. 

MATERIALS AND METHODS 

Isolates including NCBI GenBank accession numbers 

of gene sequences investigated in this study are listed 

in Table 1. The locations in European countries are 

indicated with coordinates and map sheets (MTB = 

Messtischblatt). 

Collections and analysis of phenotype 

The isolates originated from three natural sources: 

isolations from ascospores of Hypocrea specimens, 

direct isolations by a variety of means from soil or dead 

herbaceous tissue, and isolations as endophytes from 

sapwood of living stems of Theobroma and related 

tree species, and from Fagus sylvatica. Isolation 

of the stem endophytes was done as reported by 

Evans et al. (2003). A smaller number of isolates was 

obtained from the American Type Culture Collection 

(ATCC), Biologische Bundesanstalt (Berlin), the 

Centraalbureau voor Schimmelcultures (CBS), and 

from individual colleagues. Cultures derived from single 

part-ascospores that were germinated on cornmeal 

agar with 2 % dextrose (CMD, Difco cornmeal agar 

+ 2 % dextrose w/v) and isolated by means of a 

micromanipulator; usually two or more single-spore 

cultures were combined in a single stock culture, and 

such polyspore cultures were used in all subsequent 

analyses. The working set of cultures is maintained on 

cornmeal agar slants at ca. 8 °C, in 20 % glycerine at 

-80 °C, or in liquid nitrogen. 

Representative isolates are deposited at the 

Centraalbureau voor Schimmelcultures, Utrecht, The 

Netherlands (CBS) and the American Type Culture 

Collection, Manassas, VA (ATCC). Isolates listed as 

C.P.K. are those maintained in the collection of Christian 

P. Kubicek, Institute of Chemical Engineering, Research 

Area Gene Technology and Applied Biochemistry, 

Vienna University of Technology, Vienna. Kornerup & 

Wanscher (1978) was used as the colour standard. 

The name of the most commonly cited collectors, G.J. 

Samuels and W.M. Jaklitsch, are abbreviated as G.J.S. 

and W.J. 

Cultures used for study of anamorph micromorphology 

were grown on CMD, PDA or SNA (Nirenberg 

1976), at 20 or 25 ºC for 5–11 d under alternating 12 

h cool white fluorescent light and 12 h darkness; in the 

descriptions that follow, these alternating light conditions 

are referred to when the word “light” is used. 

Morphological analyses of microscopic characters 

were undertaken from material that was first hydrated 

in the case of herbarium material, or wetted in the 

case of living cultures, in 3 % KOH. Autolytic activity, 

which is here defined as usually circular excretions 

at the tips of hyphae, was assessed under direct 

microscopic observation using a 10 × objective.

Table 1. Strains used in phylogenetic analysis, their origin and GenBank numbers. 

HYPOCREA RUFA / TRICHODERMA VIRIDE 

Species Strain 1 Geography Substratum GenBank accession 

number 

ITS1 and 2 tef1 

H./T. viridescens (An) 3 G.J.S. 04-232 Mexico soil under Agave tequillensis DQ841736 DQ841716 

H./T. viridescens (An) CBS 439.95 Northern Ireland mushroom compost DQ315439 AY937413 

H./T. viridescens CBS 333.72 Netherlands unknown DQ315441 DQ307523 

H./T. viridescens (An) CBS 438.95 Northern Ireland mushroom compost DQ315438 DQ307522 

H./T. viridescens G.J.S. 99-175 Australia (VI) Hypoxylon sp. on Nothofagus 

cunninghamii 

H./T. viridescens (An) G.J.S. 97-274 

= BBA 68432 

DQ315437 DQ307521 

Russia cardboard DQ315440 DQ307505 

H./T. viridescens (An) J.B. NZ61 New Zealand, Northland Poukani Forest, soil under fern DQ845419 

H./T. viridescens G.J.S. 99-142 Australia (VI) bark DQ315427 DQ307512 

H./T. viridescens G.J.S. 99-128 Australia (VI) bark DQ315431 DQ307515 

H./T. viridescens (An) G.J.S. 04-81 Italy soil DQ841740 DQ841709 

H./T. viridescens G.J.S. 98-182 = 

W.J. 1223 

= CBS 120067 

Austria Carpinus DQ315425 DQ307511 

H./T. viridescens G.J.S. 89-142 U.S.A. (NC) decorticated wood DQ109532 AY376049 

H./T. viridescens G.J.S. 94-118 

= IMI 374788 

H./T. viridescens G.J.S. 98-129 


France Carpinus bark DQ315424 DQ307510 

France bark AY737773 DQ307542 

H./T. viridescens CBS 119323 Germany Picea abies, wood DQ677648 DQ672607 

H./T. viridescens C.P.K. 2046 U.K. Fagus sylvatica, wood DQ677649 DQ672608 

H./T. viridescens (An) ATCC 32630 Sweden Fagus wood DQ315445 DQ307526 


= N.R. 6969 

Germany soil DQ841743 DQ841717 

H./T. viridescens (An) C.P.K. 2138 Germany Picea abies, wood DQ677647 DQ672606 

H./T. viridescens (An) Tr 6 U.S.A. (OR) Pseudotsuga menziesii, root 

infected with Phellinus weirii 

H./T. viridescens (An) Tr 5 U.S.A. (OR) Pseudotsuga menziesii, root 

infected with Phellinus weirii 


= ICMP 16297 

DQ315444 AY376050 

DQ315443 DQ307525 

New Zealand Pinus radiata, wood DQ315442 DQ307524 

H./T. viridescens CBS 119321 

= C.P.K. 2140 

T4 Austria Fagus sylvatica, wood DQ677651 DQ672610 

of H. viridescens, 

Epitype of T. 

viridescens 


= N.R. 5541 

Japan leaf litter DQ315433 DQ307517 

H./T. viridescens (An) CBS 274.79 Austria wood DQ315428 DQ307513 

H./T. viridescens C.P.K. 947 Austria Picea abies, wood AY665592 DQ672604 

H./T. viridescens (An) C.P.K. 2069 Italy (Sardinia) soil DQ790657 

H./T. viridescens C.P.K. 2043 Austria Fagus sylvatica, wood DQ677646 DQ672605 

H./T. viridescens (An) G.J.S. 05-464 U.K. Fagus sylvatica, trunk 

endophyte 

DQ841714 

H./T. viridescens (An) G.J.S. 04-202 Switzerland soil DQ841729 DQ841710 


= BBA 66069 


= N.R. 5510 

Germany soil DQ315429 DQ307504 

Czech Republic soil DQ315430 DQ307514 

H./T. viridescens (An) ATCC 20898 U.S.A. (NY) soil DQ315434 DQ307518 

H./T. viridescens (An) C.P.K. 2084 Italy (Sardinia) soil DQ790658 

H./T. viridescens (An) J.B. PER43 Peru (Cuzco Dept, La Raya) soil DQ845420 

137


Table 1. (Continued). 


number 

138 


H./T. viridescens (An) J.B. PER52 Peru (Lima Dept, San Luís) soil DQ845421 

H./T. viridescens G.J.S. 05-185 Iran Vitis sylvestris DQ841732 DQ841720 

H./T. viridescens (An) G.J.S. 98-86 Mexico decorticated wood DQ315423 DQ307509 

H./T. viridescens (An) CBS 119322 Great Britain Fagus sylvatica, wood DQ677650 DQ672609 

H./T. viridescens (An) C.P.K. 999 Russia (Central St. For. 

Biosphere Reserve) 

H./T. viridescens (An) C.P.K. 998 Russia (Central St. For. 


H./T. viridescens(An) G.J.S. 05-482 U.K. Fagus sylvatica, stem 

endophyte 

soil AY665699 AY665707 

soil AY665698 AY665706 

DQ841728 

H./T. viridescens (An) G.J.S. 99-18 Japan Pinus radiata, wood DQ315435 DQ307519 

H./T. viridescens Tr 4 U.S.A. (OR) Pseudotsuga menziesii, root DQ315436 DQ307520 

H./T. viridescens (An) CBS 433.34 apple fruit AF456922 AF456905 

T of Eidamia 

viridescens 

Vd 3 (An) G.J.S. 00-67 U.S.A. (WV) decorticated wood DQ315418 DQ307502 

Vd 3 (An) G.J.S. 97-243 U.S.A. (GA) decorticated wood DQ315419 DQ307503 

Vd 3 (An) G.J.S. 94-11 Taiwan bark DQ315422 DQ307508 

Vd 3 (An) G.J.S. 94-10 Taiwan decorticated wood DQ315420 DQ307506 

Vd 3 (An) G.J.S. 94-9 Taiwan bark DQ315421 DQ307507 

H. vinosa/T. vinosum G.J.S. 99-158 

= ICMP 16294 

= CBS 119087, 

T of T. vinosum, 

epitype of H. vinosa 

New Zealand Nothofagus menziesii AY380904 AY376047 


= ICMP 16295 


New Zealand Nothofagus menziesii DQ315447 DQ307528 

H. vinosa/T. vinosum G.J.S. 99-183 Australia bark DQ841744 DQ841719 


= ICMP 16293 

H. vinosa/T. vinosum (An) DAOM 176335 unknown unknown 

Hypocrea sp. RMF 7832 2 unknown unknown 

Vd 1 G.J.S. 03-151 = 

CBS 120068 

Australia bark DQ315446 DQ307527 

Ghana pyrenomycete DQ841738 DQ841711 

Vd 1 G.J.S. 02-87 Sri Lanka bark DQ315461 DQ307544 

Vd 2 (An) C.P.K. 1488 Italy (Sardinia) soil DQ845431 DQ845416 

Vd 2 (An) C.P.K. 2071 Italy (Sardinia) soil DQ845429 

Vd 2 (An) G.J.S. 05-186 Iran Vitis sylvestris DQ841731 DQ841713 

Vd 2 (An) J.B. NZ151 New Zealand (Northland, 

Christchurch) 

clay soil DQ845422 

Vd 2 (An) PER23 2 Peru (Puno Dept, Sillustani) soil DQ845423 

T. gamsii (An) C.P.K. 2070 

= G.J.S. 06-07 


Italy (Sardinia) soil DQ790645 

T. gamsii (An) C.P.K. 2093 Italy (Sardinia) soil DQ790656 



= G.J.S. 06-15 


= G.J.S. 06-14 


Italy (Sardinia) soil DQ790653




number 


= G.J.S. 06-11 






= G.J.S. 06-10 



= G.J.S. 06-09 

= CBS 120075 T 


= G.J.S. 06-08 


T. gamsii (An) J.B. RSA9665 Rep. South Africa (Kwa-Zulu 

Natal) 

T. gamsii (An) J.B. GS33 Guatemala (Zacapa Dept, 

San Lorenzo) 




soil in Eucalyptus plantation DQ845424r 

soil in Pine-oak forest DQ845425 

T. gamsii (An) J.B. R414 Ruanda (Kigali) soil DQ845426 

T. gamsii (An) J.B. RSA9642A Rep. South Africa (Cape 

Province, Capetown) 

soil under Leucodendron DQ845427 

T. gamsii (An) J.B. RO42B Romania (Brasov) flower garden soil DQ845428 

T. gamsii (An) J.B. RSA75 Rep. South Africa (Cape 

Province) 

T. gamsii (An) C.P.K.2079 

= G.J.S. 06-13 

soil under Protea DQ845430 


T. gamsii (An) G.J.S. 04-09 Texas soil DQ315459 DQ307541 

T. gamsii (An) G.J.S. 92-60 Australia Eucalyptus nitens, stem 

endophyte 

T. gamsii (An) G.J.S. 05-111 


T. gamsii (An) C.P.K. 1010 Russia (Central St.For. 


T. gamsii (An) C.P.K. 1011 Russia (Central St.For. 



= G.J.S. 06-12 

T. neokoningii (An) G.J.S. 04-216 


H. rufa/T. viride G.J.S. 05-104 


DQ315448 DQ307529 

Italy (Pisa) Ricinus communis, stem DQ841730 DQ841722 

soil DQ845432 DQ845417 

soil DQ845433 DQ845418 


Peru Moniliophthora roreri on 

Theobroma cacao 

DQ841734 DQ841718 

Italy peat DQ841741 DQ841727 

H. rufa/T. viride (An) CBS 101526 Netherlands cellulosic tissue X93979 AY376053 

H. rufa/T. viride (An) CBS 119326 Sweden Pinus sylvestris, wood AY665593 DQ672612 

H. rufa/T. viride (An) G.J.S. 99-13 

= NRRL 6955 

H. rufa/T. viride C.P.K. 1995 

= G.J.S. 04-371 

H. rufa/T. viride (An) C.P.K. 1007 Russia (Central St.For. 




Finland soil DQ841733 DQ841712 

France Quercus robur, wood, bark DQ677653 DQ672613 

H. rufa/T. viride (An) G.J.S. 05-463 U.K. Fagus sylvatica, trunk 

endophyte 


= ITB 8212 

= BBA 70239 

soil DQ838533 DQ838539 

soil DQ838532 DQ838538 

DQ841723 

Denmark water-damaged building DQ315456 AF348116 

H. rufa/T. viride (An) ATCC 28020 U.S.A. (VA) soil DQ109535 AY937449 

139




number 

140 


H. rufa/T. viride C.P.K. 965 Czech Republic Picea abies, wood DQ677652 DQ672611 

H. rufa/T. viride (An) CBS 586.95 Estonia Phellinus igniarius 

H. rufa/T. viride C.P.K. 1998 Czech Republic Pinus sylvestris, wood DQ677656 DQ672616 

H. rufa/T. viride (An) Tr 2 U.S.A. (WA) soil DQ315457 AY376052 


= NR 6896 

U.K. leaf litter DQ841737 DQ841715 

H. rufa/T. viride G.J.S. 91-62 U.S.A. (VA) Acer sp., trunk DQ846665 DQ846670 


= ICMP 16298 

New Zealand Pinus radiata, wood DQ313155 DQ288988 

H. rufa/T. viride C.P.K. 2001 Austria Picea abies, wood DQ677659 DQ672619 

H. rufa/T. viride C.P.K. 1996 U.K. Acer pseudoplatanus, wood DQ677654 DQ672614 

H. rufa/T. viride CBS 119327 

= G.J.S. 04-369 

= G.J.S. 04-370 

H. rufa/T. viride CBS 119325 

= G.J.S. 04-372, 

epitype of H. rufa 

and T. viride 

Austria Picea abies, wood DQ677657 DQ672617 

Czech Republic Pinus sylvestris, wood DQ677655 DQ672615 

H. rufa/T. viride C.P.K. 2000 Austria Pinus sylvestris, wood DQ677658 DQ672618 







soil DQ838535 DQ838541 

soil DQ838531 DQ838537 

soil DQ838530 DQ838536 

H. rufa/T. viride (An) G.J.S. 99-16 Japan? Pinus sylvestris DQ315460 DQ307543 

H. rufa/T. viride (An) G.J.S. 92-15 Canada peat DQ315452 DQ307537 

H. rufa/T. viride (An) G.J.S. 04-86 Italy? peat DQ841745 DQ841725 

H. rufa/T. viride (An) J.B. NSW13 Australia (NSW, Wentworth 

Falls near Katoomba) 

Eucalyptus-Banksia forest soil DQ845430 

T.viride Vb 3 (An) Tr 21 U.S.A. (VA) soil AY380909 AY376054 

T.viride Vb 3 (An) G.J.S. 90-95 

= IMI 352470 

T.viride Vb 1 (An) DIS 328g 

= IMI 394148 

U.S.A. (NC) wood DQ315455 DQ307535 

Ecuador Theobroma gileri, trunk 

endophyte 

T.viride Vb 2 (An) G.J.S. 04-40 Brazil Theobroma cacao, trunk 

endophyte 

T. scalesiae (An) G.J.S. 03-74 


Galapagos Islands Scalesia pedunculata, trunk 

endophyte 

H. ochroleuca C.P.K. 1895 U.K. bark 

DQ841739 DQ841724 

DQ315454 DQ307534 

DQ841742 DQ841726 

H. ochroleuca G.J.S. 01-234 Thailand Hypoxylon sp. DQ846666 DQ846668 

T. viride Ve G.J.S. 99-127 Australia (VI) wood DQ315453 DQ307533 

T. viride Ve G.J.S. 99-204 New Zealand Metrosideros sp., bark DQ315450 DQ307531 

T. viride Ve G.J.S. 99-83 Australia (VI) bark AF456921 AF348118 

T. viride Ve G.J.S. 99-86 

= ICMP 16290 

Australia (VI) Eucalptus ? regnans, bark DQ315432 DQ307516 

T. viride Ve G.J.S. 99-191 Australia (VI) wood DQ315451 DQ307532 

T. viride Ve G.J.S. 04-353 U.S.A. (TN) Rhododendron maximum, bark DQ323418 DQ307551 

T. viride Ve (An) G.J.S. 90-97 U.S.A. (NC) bark DQ315449 DQ307530 

T. viride Ve (An) G.J.S. 90-20 U.S.A. (WI) decorticated wood 

T. viride Ve (An) DIS 217i Ecuador Theobroma gileri, trunk 

endophyte 

DQ323420 DQ307549




number 


H. neorufa G.J.S. 96-141 

= ATCC MYA-2680 

U.S.A. (NJ) Acer, bark DQ846667 DQ846669 

H. neorufa G.J.S. 96-132 U.S.A. (NJ) wood AF487653 AF487669 

H. neorufa G.J.S. 96-135, T U.S.A. (NJ) bark AF487655 AF487670 

H. flaviconidia G.J.S. 99-49, T Costa Rica bark AY665696/ 

AY665700 

AY665720 

T. paucisporum (An) G.J.S. 03-69 


= ATCC MYA-3642 

Ecuador Theobroma cacao, rotting pod DQ109527 DQ109541 

T. paucisporum (An) G.J.S. 01-13 


Ecuador Theobroma cacao, rotting pod DQ109526 DQ109540 

= ATCC MYA-3641, 

T 

T. theobromicola (An) DIS 85f, T Peru Theobroma cacao, stem 

endophyte 

DQ109525 DQ109539 

H. pezizoides G.J.S. 01-257 Thailand wood DQ000632 AY937438 

T. hamatum (An) DAOM 167057 

= CBS 102160, 

neotype 

Canada spruce forest soil Z48816 AY750893 

T. pubescens (An) DAOM 166162 U.S.A. (NC) forest soil DQ083027 AY937442 

T. asperellum (An) CBS 433.97, T U.S.A. (MD) sclerotia of Sclerotinia minor X93981 AY376058 

T. asperellum (An) G.J.S. 04-217 Peru Moniliophthora roreri on 

Theobroma cacao 

DQ381957 DQ381958 

1 Abbreviations of culture collections and collectors as follows: ATCC = American Type Culture Collection, Manassas, VA, U.S.A., BBA = 

Biologisches Bundesanstalt, Berlin, Germany; CBS = Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; C.P.K. = Collection of 

C.P. Kubicek, Technische Universität, Abteilung für Mikrobielle Biochemie, Vienna; G.J.S., Tr = Culture collection of the United States Department 

of Agriculture, Systematic Botany and Mycology Lab, Beltsville, MD, U.S.A; DAOM = Canadian Collection of Fungal Cultures, Ottawa, Canada; 

DIS refers to CABI-Bioscience, Ascot, cultures held by G.J.S; ICMP = International Collection of Microorganisms from Plants, Manaaki Whenua 

Landcare Research, Auckland, New Zealand; IMI = CABI-Bioscience, Egham, U.K.; J.B. = John Bissett, Agriculture and Agri-Food, Eastern 

Cereal and Oilseed Research Centre, Ottawa, Canada; W.J. = Culture collection of Walter Jaklitsch, Technische Universität, Abteilung für 

Mikrobielle Biochemie, Vienna; NR = Nippon Roche Corp. Tokyo, Japan 

2 From the culture collection of John Bissett, Agriculture and Agri-Food, Eastern Cereal and Oilseed Research Centre, Ottawa, Canada. 

3 An = isolation made from conidia or directly from substratum, all other cultures derived from ascospores. 

4 T = ex-type culture. 

Coilings, defined as circularly oriented and coiled 

intercalary or terminal parts of hyphae, were detected 

in the same way as autolytic excretions. 

Measurements were made from KOH or water 

mounts and we did not observe any differences when 

the respective reagents were used. Where possible, 

at least 30 units of each parameter were measured 

for each collection. Ninety-five percent confidence 

intervals of the means (CI) are provided; this figure 

represents the interval within which 95 % of the 

individuals of the parameter was found in the analysed 

isolates. The parameters used for analysis are listed in 

Table 2. Chlamydospores were measured by inverting 

a 7–15 d old CMD culture on the stage of a compound 

microscope and observing with a 40 × objective. Data 

were gathered using a Nikon DXM1200 or a Nikon 

Coolpix 4500 digital camera and Nikon ACT 1 software 

and measured either directly on the microscope or by 

using Scion Image (release Beta 4.0.2; Scioncorp, 

Frederick, MD). 

Five types of light microscopy were used, viz. stereo 

microscopy (stereo), bright field (BF), phase contrast 

(PC), Nomarski differential interference contrast (DIC), 

and epifluorescence (FL). The fluorescent brightener 

calcofluor (Sigma Fluorescent Brightener 28 C.I. 40622 

Calcofluor white M2R in 2 molar phosphate buffer at 

pH 8.0) was used for FL. 

Scanning electron microscopy (SEM) was done 

by one of two methods. Material for SEM studies was 

obtained from cultures that were grown on PDA for up 

to 2 weeks at 20–25 °C. Agar blocks with abundant 

conidia were prepared for SEM. For Figs 8 a–h all 

SEM procedures followed the protocols of Meyer & 

Plaskowitz (1989), and for Fig. 10h those of Carta et al. 

(2003) and Erbe et al. (2003). 

141


Sections of Hypocrea stromata were prepared by 

rehydrating small blocks of substratum supporting 

stromata in 3 % KOH. The blocks were supported by 

Tissue Tek O.C.T. embedding medium 4583 (Miles, 

Inc., Elkhart, IN) and sectioned at 12–15 µm on a 

Microtome-Cryostat (International Equipment Co., 

Needham Heights, MA, or Leitz Kryostat 1720, Leica 

Microsystems, Vienna). 

Growth rate trials were performed in darkness on 

potato-dextrose agar (PDA, Difco, Biolab, or Merck) 

and SNA following the procedure described by 

Samuels et al. (2002) with the addition that cultures 

were also grown at 25 ºC under 12 h darkness/12 h 

cool white fluorescent light for 5–7 d. Each growth-rate 

trial was repeated three times and the results of the 

three were averaged. The slope of the growth curve 

was determined using the mean of the colony radius 

(see Samuels et al., 2006a). 

DNA extraction and sequencing methods 

The extraction of genomic DNA was performed as 

reported previously (Dodd et al. 2002). A portion of 

translation elongation factor 1 alpha (tef1) was amplified 

using the primers EF1-728F (Carbone & Kohn 1999) 

and TEF1 rev (Samuels et al. 2002) or TEF1LLErev 

(Jaklitsch et al. 2005). The PCR product of approximately 

600 bp covers the large 4 th and the short 5 th introns of 

the gene. A fragment covering the internal transcribed 

spacers 1 and 2 (ITS1 and 2) of the rRNA gene cluster 

was amplified using ITS1 and ITS4 as the forward and 

reverse primers, respectively (White et al. 1990). DNA 

sequences were obtained using the BigDye Terminator 

cycle sequencing kit (Applied Biosystems, Foster City, 

California). Products were analysed directly on a 3100 

DNA sequencer (Applied Biosystems). Both strands 

were sequenced for each locus. 

Molecular phylogenetic analysis 

Sequences were edited and assembled using 

Sequencher 4.1 (Gene Codes, Wisconsin). Clustal 

X 1.81 (Thompson et al. 1997) was used to align the 

sequences; the alignment of each locus was manually 

edited using MacClade or GeneDoc 2.6 (Nicholas 

& Nicholas 1997). The sequences were deposited in 

GenBank (Table 1). The MSA file for the tef1 locus is 

also available at http://www.isth.info/phylogeny/rufa. 

php. 

The interleaved NEXUS file was formatted using 

PAUP* v. 4.0b10 (Sinauer Associates, Sunderland, 

MA) and manually formatted for the MrBayes v3.0B4 

program. The Bayesian approach to phylogenetic 

reconstructions (Rannala & Yang 2005) was 

implemented using MrBayes 3.0B4 (Huelsenbeck & 

Ronquist 2001). The MODELTEST3-06 package (http:// 

bioag.byu.edu/zoology/crandall_lab/modeltest.htm) 

was used to compare the likelihood of different nested 

models of DNA substitution and select the best-fit model 

for the investigated data set. Both hierarchical LRT and 

AIC output strategies were considered, although the 

preference was given to the latter. The unconstrained 

GTR + I + G substitution model was selected for the 

tef1 locus. 

142 

Metropolis-coupled Markov chain Monte Carlo 

(MCMCMC) sampling was performed with four 

incrementally heated chains (with the default heating 

coefficient λ = 0.2, heats for cold chains 1 and 

heated chains 2, 3 and 4 are 1, 0.83, 0.71 and 0.63, 

respectively) that were simultaneously run for 5 million 

generations for the tef1 alignment, which comprised 

238 sequences. To check for potentially poor mixing 

of MCMCMC, the analysis was repeated at least three 

times. The convergence of MCMCMC was monitored 

by examining the value of the marginal likelihood 

through generations. Convergence of substitution rate 

and rate heterogeneity model parameters were also 

checked. Bayesian posterior probabilities (PP) were 

obtained from the 50 % majority rule consensus of trees 

sampled every 100 generations after removing the 

2000 first trees using the “burn” command. According 

to the protocol of Leache & Reeder (2002), PP values 

lower than 0.95 were not considered significant while 

values below 0.9 are not shown on the resulting 

phylogram. Model parameter summaries after MCMC 

run and burning first samples were collected. For tef1 

mean substitution values were estimated as G↔T = 1, 

C↔T = 3.55, C↔G = 1.28, A↔T = 1, A↔G = 4.68, 

A↔C = 1.5; nucleotide frequencies were estimated as 

0.19 (A), 0.27 (C), 0.2 (G), 0.34 (T); alpha parameter of 

gamma- distribution shape was 0.29. Genetic distance 

was computed in PAUP* v. 4.0b10 under the GTR + I 

model. 

RESULTS 

Phylogeny 

The majority of members of Trichoderma section 

Trichoderma share the same or very similar alleles 

of internal transcribed spacers 1 and 2 (ITS1 and 2), 

rendering this locus inappropriate for recognition of 

some species within the section. Therefore, to infer 

genetic diversity of the H. rufa/T. viride group we used 

intron sequences of the translation elongation factor 1alpha 

(tef1), the most powerful phylogenetic marker as 

yet established in the genus. The resulting Bayesian 

phylogram (Fig. 1), which was obtained from 238 

sequences, corresponds well to the previous analysis 

of related species with T. koningii-like morphology 

(Samuels et al. 2006a). Considering the analysis of 

phenotypes, it is obvious that there are two diverged 

groups named “Large Viride” and “Large Viridescens” 

clades, both of them with significant statistical support. 

Isolates of H. rufa form a compact clade composed of 

mainly European but also North American, Asian and 

Pacific strains showing its cosmopolitan nature. The 

“Large Viride” clade includes additional unresolved 

lineages that apparently represent unnamed species. 

The description of these taxa requires further sampling 

and therefore will be discussed in subsequent 

publications. In this study we have focused on the 

single endophytic strain from the Galapagos Islands, 

T. scalesiae sp. nov., which belongs to the “Large 

Viride” clade but at the same time occupies the most 

distant position from H. rufa. The largest group on

the tree, the “Large Viridescens” clade, splits into 

two independent evolutionary lineages. The terminal 

position of the larger one represents a compact and 

well defined subclade with significant statistical support 

that contains isolates of the former Vd group (Lieckfeldt 

et al. 1999), described as H. viridescens below. 

Similar to H. rufa, this species has mainly European 

origin, also nearly all primary European nodes include 

North American, Central American, Asian and Pacific 

isolates, suggesting the absence of recent allopatric 

speciation in this group of isolates. Another wellsupported 

clade in the vicinity of H. viridescens is 

composed of isolates of H. vinosa. As in the “Large 

Viride” clade this branch contains representatives of 

several well-supported speciation nodes composed of 

strains that are closely related to H. viridescens and 

H. vinosa and undoubtedly represent yet undescribed 

species of Hypocrea/Trichoderma. This diversity will be 

discussed in subsequent publications following further 

investigations and sampling. The material summarised 

in this study is sufficient to prove the existence of another 

phylogenetic species with eidamia-like morphology that 

occupies the second independent lineage within the 

“Large Viridescens” clade. The new species T. gamsii 

forms a homogeneous clade mainly represented by 

isolates from undisturbed soils in Sardinia and Central 

Russia. As in the case of H. viridescens and H. rufa, 

T. gamsii did not evolve as a result of any geographic 

isolation since we also sampled isolates from North 

America and Australia. We describe the most distant 

member of the “Large Viridescens” Clade, once again 

a single strain, as T. neokoningii. The detailed analysis 

of the highly variable intron sequences of the tef1 gene 

has clearly shown that, despite their close relationship, 

H. rufa, H. viridescens, H. vinosa, and a large group of 

isolates that we describe here as T. gamsii represent 

distinct sympatric phylogenetic species. 

Most of the Trichoderma species that have warted 

conidia fall within one of these two large clades. 

Exceptions include T. saturnisporum and T. ghanense, 

both of which are members of the distantly related T. sect. 

Longibrachiatum (Samuels et al. 1998) and clade Ve (Fig. 

1). Clade Ve will be discussed in a future publication. 

All members of the “Large Viridescens” clade are 

characterised by the formation of peculiar, percurrently 

proliferating phialides that are diagnostic of Eidamia 

viridescens, the ex-type of which (CBS 433.34) falls in 

T. viridescens. 

Phenotype: anamorph and cultures 

DNA sequences referred eighty-seven strains to 

the “Large Viridescens” clade and thirty-four to the 

“Large Viride” clade. All of these fungi are typical of 

Trichoderma in producing copious amounts of green 

conidia in pustules or in extensive “lawns” on CMD, 

PDA and SNA. There was a tendency for conidia to 

form more quickly on SNA than on CMD or PDA and 

often conidia did not form on either of the latter media 

while they did form on SNA. Of the three media, SNA 

is overall better for the study of fungi in the viride clade 

in terms of more reliable production of conidia. The 

endophytic T. scalesiae only produced conidia on SNA 


to which a 1 cm 

143 

2 piece of sterile filter paper had been 

added; the conidia only formed at the interface of the 

paper and the agar and on the paper itself. 

There was a tendency for yellow pigment to develop 

in conidia in colonies of the “Large Viridescens” clade 

grown on PDA and SNA at 25 °C for two weeks, and a 

yellow pigment often diffused through CMD. No pigment 

was noted on SNA. Diffusing yellow pigmentation was 

not noted in colonies belonging to the “Large Viride” 

clade. A more or less strong coconut odour was 

detected in PDA and CMD cultures of most members 

of the “Large Viridescens” clade. 

Conidia tended to form in pulvinate to hemispherical 

pustules < 1–3 mm diam. Distinct pustules measuring 

1–5 mm were formed in T. viride/H. rufa on CMD. While 

pustules formed in T. viridescens/H. viridescens reached 

3 mm diam, most often they measured less than 1 mm 

and often no pustules were formed, the conidiophores 

arising in more or less continuous cottony lawns. Often 

conidiophores formed apart from the larger pustules 

in the aerial hyphae and in minute tufts. Pustules in 

both groups tended to be cottony, and individual fertile 

branches could be seen; often conidiophores protruded 

beyond the surface of a pustule, producing a single 

phialide or a few fertile branches near the tip, the rest 

of the conidiophore remaining sterile or nearly so. The 

pustules of T. viridescens were usually less compact 

than in T. viride, and transparent under a 10 × objective. 

In pustules of T. viride produced on CMD, conidia often 

appeared to form at the surface of the pustule. In all 

cases, after one week at 20–25 °C, conidia were deep 

green to dark green 27–28D–F6–8, although lighter 

green conidia were observed in younger cultures. In 

some cultures of T. viridescens grown at 25 °C under 

alternating light on CMD and SNA, conidial masses 

were yellow. Conidia of T. neokoningii on PDA often 

were yellow at first. Often conidia of members of the 

“Large Viridescens” clade became greenish yellow 

when mounted in 3 % KOH. 

Most of the fungi discussed in this work produce 

colonies that are recognizable as typical of Trichoderma 

in producing green conidia in abundance on most media. 

The exception is T. scalesiae, which only produced 

conidia sparingly on SNA to which a 1 cm2 piece of 

sterile filter paper had been added. Conidiophores 

in this species were irregularly branched, similar to 

what was described for T. paucisporum Samuels et 

al. (Samuels et al. 2006b) and for synanamorphs 

of pustulate species of Trichoderma (Chaverri et al. 

2004). Conidia were held in drops of clear liquid, which 

appeared yellow to pale green because of the conidia, 

at the tips of the phialides. 

The following results pertain to the remaining species 

discussed in this work. It is difficult or impossible to 

define a conidiophore in Trichoderma. Conidiophores 

are mostly formed in pustules. As was noted above, 

pustules tend to be composed of intertwined hyphae 

that terminate in fertile branching systems. For the 

purposes of the present discussion, the conidiophore 

is referred to as the terminal branching system of 

intertwined hyphae that form the pustule. Various 

types of conidiophores were encountered in this study,


and these were largely related to the medium and to 

the clade. In Type 1 (Fig. 3d, e, i), a well-developed 

main axis was not readily visible, or it was short and 

sometimes sinuous. Branching was highly irregular; 

branches were not paired and phialides tended to 

arise singly from the main axis. Phialides were often 

hooked or sinuous (Fig. 3d, e, j, k), cylindrical or 

somewhat swollen at or below the middle. This type 

of conidiophore was only found in the “Large Viride” 

clade, especially in T. viride. The Type 2 conidiophore 

(e.g. Figs 6, 11, 13–14) was formed by all clades. In 

the Type 2 conidiophore there was a more or less 

readily discernable, well-developed main axis, from 

which lateral branches arose at or near 90°; the lateral 

branches were longer with distance from the tip and 

secondary branches were shorter with distance from 

the point of departure of the branch from the main axis. 

Branches often arose in pairs and produced secondary 

branches in pairs. Phialides tended to terminate 

branches in cruciate whorls of 3–4. The phialides were 

straight, cylindrical or somewhat swollen at or below 

the middle. In Type 3, which was common in the “Large 

Viridescens” clade, including T. vinosum, T. gamsii and 

T. neokoningii, the most distinctive characteristic is the 

production of percurrently proliferating phialides (Figs 

5f, g, i, k; 10d–g; 12c, e, f, g; 14h–j), the branching 

system itself is highly variable in extent and form. At 

its simplest, a single phialide percurrently produces 

a second phialide (Fig. 14 h). What appears to be 

continuing percurrent proliferation of phialides results 

in a submoniliform chain of five or more cells (Figs 5g, 

10 e, 12e, f), each cell of the chain being often abruptly 

swollen in the middle and separated by the cell above 

and below by a conspicuous septum. A main axis was 

discernable or not and was often reduced to a few, 

short, verticillately disposed branches or a reticulum 

of branches (e.g. Figs 5i–k; 10d, 12f–h, 14i–j). The 

most extreme form of the third branching type was 

observed in old pustules on CMD and PDA, where 

chains of percurrently proliferating phialides having 

subglobose bases and extremely long, cylindrical 

beaks arose from swollen, subglobose cells (Figs 5k, 

10f, 12f). Percurrently proliferating phialides having 

this morphology were also seen occasionally on more 

typically branched conidiophores (Figs 5d; 12e), on 

conidiophores that produced typical, non-proliferating, 

phialides. Proliferating phialides were rarely seen on 

SNA. Conidiophores of DIS 328g (Vb 1) arose within 

well-developed pustules; they formed a reticulum with 

short fertile branches. The branches tended to be 

sinuous or curved and to be broader than is found in 

other clades that are studied here. The conidiophores 

produced often unicellular lateral branches, each of 

which terminated in 2–4 phialides. The phialides in 

DIS 328g are shorter than in any strain included in this 

study and have a smaller L/W ratio; they were often 

hooked or sinuous. 

The “Large Viridescens” clade includes CBS 433.34, 

which is the ex-type culture of Eidamia viridescens A.S. 

Horne & H.S. Williamson. This species was described 

based on conidiophores produced on PDA; the original 

illustration is highly suggestive of what we have seen in 

the “Large Viridescens” clade, especially the extreme 

144 

form described above as Type 3 and illustrated in 

T. viridescens (Fig. 5k) and T. vinosum (Fig. 10f). 

Conidiophores produced by this culture on SNA (Fig. 

6f, g) were typical of Trichoderma, with a more or less 

uniformly branched conidiophore and typical phialides. 

The culture remained sterile on PDA but produced a 

coconut odour and a diffusing yellow pigment. On CMD 

mononematous conidiophores bearing green conidia 

in appressed phialides developed, but no pustules 

and no proliferating phialides were seen. These 

conidiophores were suggestive of the synanamorph 

conidiophores described by Chaverri et al. (2004) for 

species of Hypocrea/ Trichoderma having conidiophore 

elongations. 

Intercalary phialides were seen in some isolates but 

were neither common nor restricted to any particular 

clade (e.g. Figs 3k, 12d, 13g, 14k). 

Various conidial types were observed in this study. 

These, like conidiophore types, were largely typical of 

clades. Most of the strains produced warted conidia. 

Conidia of T. gamsii (Fig. 12i), T. neokoningii (Fig. 14l) 

and T. scalesiae (Fig. 15h) are smooth. Trichoderma 

viride (Figs 3m, n; 8a–e), DIS 328g (Vb 1), G.J.S. 04- 

40 (Vb 2), and T. vinosum (Fig. 10h–j) have nearly 

globose conidia that have a length/width ratio 1.0– 

1.2. Conidia in G.J.S. 03-151/G.J.S. 02-87 (Vd 1) are 

ellipsoidal, length/width of 1.2–1.4. Conidia of individual 

collections of T. viridescens vary from subglobose to 

ellipsoidal (Figs 6i–m, 8f–h); although the mean L/W of 

all collections in this clade varies from 1.1–1.3, there is 

considerable overlap between this clade and DIS 328g. 

Conidia of T. gamsii and T. neokoningii are unusual 

in being ellipsoidal. Both of these species produce 

T. koningii-like conidiophores and conidia. Conidia of 

T. viride are much more coarsely warted than any of 

the other clades considered here. Warted conidia are 

also produced by members of clade Ve. Conidia in 

this clade are subglobose to ellipsoidal. Ornamented 

conidia were observed for most members of this clade. 

Conidial warts, while often large, are widely spaced 

and thus are not as conspicuous as in members of the 

“Large Viride” and “Large Viridescens” clades that are 

the focus of the present work. 

Chlamydospores were inconsistently produced in 

most clades. Chlamydospores formed in abundance 

in T. gamsii (Fig. 13i) and T. neokoningii (Fig. 14m). 

Chamydospores were especially abundant in T. 

scalesiae (Fig. 15i). Chlamydospores were typical 

of Trichoderma in being globose to subglobose and 

terminal at the ends of hyphae or intercalary within 

hyphal cells. 

Optimal temperature for growth on PDA for all clades 

except T. scalesiae and Vd 1 was 25 °C. The optimum 

for T. scalesiae was 30 °C and the two isolates in Vd 

1 exhibited considerable variation at 30 °C (35–70 mm 

radius after 72 h). Trichoderma vinosum was unusual 

in having a temperature optimum of 20–25 ºC and in 

reaching no more than 5 mm colony radius after 4 days 

at 30 ºC. 

On SNA most isolates reached a radius of no more 

than 40 mm, and usually less, after 72 h at 25–30 °C. On 

SNA only DIS 328g (Vb 1), T. viride and T. viridescens 

demonstrated a clear optimum at 25 °C. On SNA, the

optimum for T. scalesiae was 25–30 °C, as it was for 

T. vinosum. The two isolates of Vd 1 were too variable 

to show a temperature optimum on SNA. G.J.S. 04-40 

(Vb 2) was the fastest growing strain on SNA, reaching 

65 mm after 72 h at 30 °C. This temperature differential 

was not observed on PDA. At 25 °C on PDA DIS 328g 

(Vb 1), G.J.S. 04-40 (Vb 2), T. gamsii, Vd 1 and Vd 2 

reached or exceeded a radius of 45 mm after 72 h. 

Despite their phylogenetic complexity, both T. viride 

and T. viridescens showed very little variation in growth 

rate among their many isolates, both reaching a radius 

of 30–40 mm after 72 h at 25 °C. Significantly, growth 

of isolates in both of these clades, as well as in T. 

vinosum and DIS 328g (Vb 1), was more than 20 mm 

slower at 30 °C than at 20 °C. Trichoderma scalesiae 

was the slowest growing, reaching only 10 mm on SNA 

after 72 h at 25–30 °C and 18 mm on PDA at 30 °C. 

The fastest growing isolate at 30 °C was G.J.S. 04-40 

(Vb 2) reaching 45 mm, although G.J.S. 03-151 (Vd 1) 

reached a radius of 70 mm after 72 h at 30 °C. 

Clade Vd 3, which is a sister to T. viridescens, 

comprises two distinct groups of isolates. The North 

American isolates (G.J.S. 00-67, G.J.S. 97-243) cannot 

be distinguished from T. viridescens in any of their 

morphs and aspects. The Taiwanese isolates (G.J.S. 

94-9 – G.J.S. 94-11) grow significantly more slowly 

than T. viridescens. 

Phenotype: teleomorph 

The stromata of the species included in this study are 

morphologically and anatomically so similar that they 

often cannot be distinguished. The youngest stage, 

when it could be observed, was semieffused, velutinous 

to conspicuously hairy and light tan in colour (Figs 4a, 

d; 2a, c). As perithecial development continued, the 

stroma became pulvinate to tuberculate or turbinate, 

and assumed a brown to rufous colour. Occasionally 

“albino” stromata, off-white to pale yellow, were 

observed in H. rufa (Fig. 2f) and in H. vinosa (Fig. 16i), 

in the latter only in an immature state. Often a velvety 

scurf was also present on mature stromata, the result 

of short hyphal hairs protruding from the stroma surface 

(Figs 2k, l; 4 l; 9b, e). Ostiolar openings were usually 

not visible macroscopically, or were barely visible as 

lighter areas on the stroma surface, sometimes with 

darker margins. The stroma surface, when observed 

in the compound microscope, was composed of small 

pseudoparenchymatous cells. Typically brown pigment 

was unevenly deposited in the walls of these cells 

giving a mottled appearance to the rehydrated stroma 

(Figs 4j, 9a). The stromata typically have a pigmented 

cortical layer underlain by a region of loosely arranged 

hyphae. Asci were cylindrical and had a thin ring in the 

apex; they typically contained 8 uniseriate ascospores. 

Ascospores were hyaline, spinulose and disarticulated 

early to form two halves, or part-ascospores. The 

part-ascospores were dimorphic, the distal part was 

subglobose to broadly conical and the proximal part 

was ellipsoidal or oblong to narrowly wedge-shaped. 

Ascospore sizes were clade-specific. G.J.S. 02-87 (Vd 

1), a teleomorphic member of the “Large Viridescens” 

clade from Sri Lanka, had the smallest ascospores. 


Ascospores of H. vinosa were longer in the distal part 

than in all other species and the width of its proximal and 

distal parts was greater than in all others. Ascospores 

of H. rufa and H. viridescens are nearly identical in 

size. Vb 3 includes two Hypocrea collections from, 

respectively, Virginia and North Carolina. While these 

two collections are sympatric with, but phylogenetically 

distinct from H. rufa, we did not observe any aspect of 

their teleomorph, anamorph or cultural phenotypes that 

would serve to distinguish them from that species. 

Biogeography 

Most of the clades that included more than one strain 

did not show strong biogeographic bias. Hypocrea 

vinosa was originally described from New Zealand 

and, in this work, it is restricted to New Zealand and 

Australia. The “Large Viride” and “Viridescens” clades 

are widely distributed but are more common in North 

America and Europe. These are not tropical fungi. 

Trichoderma viridescens has been found in Peru at high 

elevation. We have seen only one isolate of T. viride 

from a tropical region, i.e. G.J.S. 92-15, from Brazil. 

However, two members of the “Large Viride” clade, DIS 

328g (Ecuador) and G.J.S. 04-40 (Brazil), originated 

in South America. These two endophytic isolates 

apparently represent two distinct species. Trichoderma 

neokoningii was isolated in a tropical region in Peru. 

On the basis of our collecting, T. viridescens is far 

more common and possibly more widespread than 

T. viride. Trichoderma viride and T. viridescens are 

common in Europe as anamorphs, but uncommon as 

teleomorphs if compared to common species like H. 

minutispora. There is a tendency for isolates originating 

in a geographic area (e.g. Taiwan or Europe) to cluster 

together but there was an equally strong tendency for 

clades to comprise strains of mixed origin (e.g. Japan, 

United Kingdom and U.S.A.). Trichoderma gamsii 

includes strains from widely separated locations, viz. 

the Tyrrhenian island of Sardinia (Italy), U.S.A. (Texas), 

Russia and Australia. The clade Vd 3 comprises two 

biogeographically distinct sister clades. Isolates G.J.S. 

00-67 and G.J.S. 97-243 are from eastern U.S.A. 

Isolates G.J.S. 94-9 – G.J.S. 94-11 were collected in 

Taiwan. 

The isolates G.J.S. 04-40 and DIS 328g were 

isolated as endophytes from trunks of Theobroma 

cacao and Th. gileri, respectively, and T. scalesiae was 

isolated as an endophyte from woody, above-ground 

tissue of Scalesia pedunculata. 

Definition of species 

Fig. 1, with T. asperellum as outgroup, demonstrates the 

considerable known and yet to be described taxonomic 

diversity in a large part of T. sect. Trichoderma. 

Despite the existence of several clades that no doubt 

merit taxonomic recognizion, in the current work we 

emphasize the “Large Viride” and “Large Viridescens” 

clades. 

Each of these large clades includes several wellsupported 

internal clades, making it difficult to delimit 

species. In most cases, more or less distinct phenotypic 

apomorphies lead to our decision as to where to draw 

species boundaries. 

145


The greatest phylogenetic diversity is found in 

the “Large Viridescens” clade. At the most distant 

point of this clade, T. gamsii and T. neokoningii can 

be distinguished because they both have smooth, 

ellipsoidal conidia. Trichoderma gamsii is a common 

species in Europe and North America. Trichoderma 

neokoningii is only known from a single culture that was 

collected in Peru as a hyperparasite on a destructive 

pathogen of Theobroma cacao. 

Clade Vd 2 includes European and middle-eastern 

(Iran) isolates that also have smooth, ellipsoidal 

conidia. Clade Vd 1 includes isolates from Sri Lanka 

and Ghana that have ellipsoidal, warted conidia. One 

of these, G.J.S. 02-87 (Sri Lanka), produces a H. rufalike 

stroma but it has smaller ascospores than either H. 

rufa or H. viridescens. We did not observe an eidamialike 

morphology in Vd 1 or Vd 2. 

Hypocrea vinosa is distinguished from H. viridescens 

primarily on the basis of its faster rate of growth and on 

its larger ascospores. It has a conspicuous eidamiamorphology 

when grown on CMD. 

Clade Vd 3 is phenotypically and biogeographically 

diverse. We had originally included all of these 

isolates within T. viridescens. As was noted above, 

the North American isolates (G.J.S. 00-67, G.J.S. 

97-243) cannot be distinguished from H. viridescens, 

whereas the remaining isolates, all from Taiwan, have 

a noticeably slower rate of growth than T. viridescens. 

Their relationship to T. viridescens is indicated by the 

dotted line in Fig. 1. 

Hypocrea/Trichoderma viridescens is a widely 

distributed species that is common in Europe. It is 

phenotypically, phylogenetically and geographically 

diverse, but the phenotypic diversity overlapped to 

such an extent that we were not able to subdivide the 

species. Hypocrea/T. viridescens is characterised by 

north- and south-temperate distribution, relatively slow 

growth, conidiophores that tend to produce paired 

branches on SNA, subglobose to nearly ellipsoidal, 

warted conidia, a coconut odour on PDA and CMD, 

and the conspicuous eidamia-morphology found on 

PDA and CMD. 

The most distant point of the “Large Viride” clade is 

T. scalesiae. This unusual species was isolated as an 

endophyte from the trunk of an endemic daisy tree in 

the Galapagos Islands. It only produced few conidia on 

conidiophores that are atypical in Trichoderma. Even in 

the absence of conidial development, it is recognizable 

146 

as a Trichoderma by its strong odour of coconut and 

also by the production of abundant chlamydospores 

that are typical of Trichoderma. 

A single clade that is sister to H. rufa/T. viride 

includes Vb 1, Vb 2 and Vb 3. The two isolates of Vb3 

were isolated in the mid Atlantic states of the U.S.A. and 

they cannot be distinguished from T. viride (with which 

they are sympatric) morphologically. Apart from the 

phylogenetic difference indicated by sequences of tef1, 

we cannot observe any way to taxonomically separate 

them from H. rufa/T. viride. The single strains that 

comprise Vb 1 and Vb 2 were isolated as endophytes 

from trunks of, respectively, Theobroma gileri and Th. 

cacao in Ecuador and Brazil. Both of them have a faster 

growth rate than H. rufa/T. viride, a difference that is 

especially marked on SNA, and Vb 2 grows faster than 

any of the clades included in the present study. Both 

of these, but especially Vb 2, have somewhat smaller 

conidia than T. viride. Conidiophores of Vb 2 are Type 

1 described on page 144 and typical of T. viride. The 

unusual conidiophores of DIS 328g (Vb 1) and the short 

broad phialides distinguish this clade from its closest 

relatives, Vb 2, Vb 3 or T. viride. The data suggest 

that these two endophytic strains represent distinct 

species; their taxonomy will be discussed in a future 

publication. 

As was the case with H./T. viridescens, H. rufa/T. 

viride is phylogenetically and phenotypically diverse but 

we did not find any hiatus in the characters that would 

enable us to recognise more than a single species. 

The hallmark of T. viride is its remarkably consistent, 

rather slow rate of growth, strongly warted, globose to 

subglobose conidia and this is consistent with the type 

specimen of T. viride (Fig. 8a, b and Samuels et al. 

1999). Moreover, the conidiophores found in T. viride, 

with often solitary, hooked phialides, are consistent 

with what Tulasne & Tulasne (1865) illustrated for their 

concept of H. rufa and T. viride. 

What we have called T. viridescens could have 

perhaps been selected as being typical of T. viride, 

given the overlap in phenotype characters of the 

anamorph, but conidia in this group are not so strongly 

warted and the tendency is for ellipsoidal conidia 

rather than globose. The ex-type culture of Eidamia 

viridescens (CBS 433.34) was included in our analysis. 

Thus we name this clade T. viridescens, with Hypocrea 

viridescens sp. nov. as its teleomorph. 

KEY TO TAXONOMIC AND PHYLOGENETIC SPECIES OF TRICHODERMA SECT. TRICHODERMA 

DISCUSSED IN THIS PAPER 

1. Conidia conspicuously warted, warts usually densely disposed and conspicuous ............................................ 2 

1. Conidia smooth or warted, warts scattered or inconspicuous ............................................................................ 7 

2. Colony radius on PDA after 72 h at 25 °C 50–60 mm ........................................................................................ 3 

2. Colony radius on PDA after 72h at 25 °C < 50 mm ............................................................................................ 4 

3. Conidia (2.7–)3.0–3.7(–4.2) × (2.2–)2.5–3.2(–3.5) μm; Sri Lanka, Ghana ................................................... Vd 1 

3. Conidia (3.0–)3.5–4.0(–4.2) × (3.0–)3.2–3.7(–4.2) μm; endophytic in 

Theobroma cacao, Brazil .............................................................................................................................. Vb 2


4. Conidia (3.0–)3.2–4.0(–4.5) × (2.7–)3.0–3.5(–4.0) μm; conidiophores typically sinuous 

and frequently branched; phialides short and broad, proliferating phialides and/or 

submoniliform hyphae not formed; endophytic in trunk of Theobroma gileri, Ecuador ................................. Vb 1 

4. Conidia larger, (3.0–)3.5–4.5(–8.5) × (2.2–)3.0–4.0(–4.7) μm; not endophytic .................................................. 5 

5. Conidia (3.0–)3.5–4.5(–5.5) × (2.8–)3.4–4.0(–5.0) μm, typically globose, grossly 

warted, L/W (0.8–)1.0–1.2(–1.5); terminal conidiophores often curved, phialides 

often widely spaced and solitary, often hooked or sinuous; proliferating phialides 

usually not formed in pustules ................................................................................................................. T. viride 

5. Conidia (2.7–)3.5–4.5(–8.5) × (2.2–)3.0–4.0(–4.7) μm, globose to ellipsoidal, 

L/W (0.9–)1.0–1.4(–2.0), verruculose; phialides typically forming in whorls, 

straight; proliferating phialides and/or submoniliform conidiophores often formed ............................................ 6 

6. Conidia subglobose, (3.2–)3.5–4.5(–4.7) × (2.7–)3.0–4.0(–4.2) μm, L/W 1.0–1.2(–1.3); 

mean of distal part-ascospores 5.0–6.5 × 4.7–6.2 μm; mean of proximal part-ascospores 

5–7 × 4–5 μm; colony radius on PDA after 72 h at 25 °C typically 25–33 mm; Australia 

and New Zealand .............................................................................................................................. T. vinosum 

6. Conidia subglobose to ellipsoidal, (2.8–)3.5–4.5(–8.5) × (2.3–)3.0–3.7(–4.7) μm, 

L/W (0.9–)1.1–1.4(–2.0); mean of distal part-ascospores 4.2–5.5 × 4.2–4.7 μm; 

mean of proximal part-ascospores 4.5–5.5 × 3.2–4.0 μm; colony radius on PDA after 

72 h at 25 °C typically 35–45 mm; cosmopolitan, more common north-temperate ...................... T. viridescens 

7. Conidia globose to ovoidal, smooth, finely warted or with larger scattered warts .............................................. 8 

7. Conidia smooth, subglobose, ellipsoidal to ellipsoidal, smooth .......................................................................... 9 

8. Conidia subglobose or ovoidal, finely spinulose (often appearing smooth in light microscope), 

(2.8–)3.4–3.6(–7.0) × (2.4–)3–4(–6) µm, L/W 1.0–1.7 ....... T. asperellum Samuels et al. (Samuels et al. 1999) 

8. Conidia subglobose to ellipsoidal, smooth or with large scattered warts; (3.0–)3.2–4.5 

(–5.7) × (2.2–)3.0–3.5(–4.0) μm, L/W = 0.9–1.7 (mean = 1.2) ......................................................................... Ve 

9. Conidia globose to subglobose or broadly ovoidal ........................................................................................... 10 

9. Conidia ellipsoidal to oblong ............................................................................................................................. 13 

10. Conidiophores and conidia typical of Trichoderma, green and typically forming 

in abundance on SNA, PDA and CMD; colonies fast-growing ........................................................................ 11 

10. Conidiophores and conidia verticillium-like, forming in wet heads, sparsely formed 

and very inconspicuous, reliably forming only on SNA .................................................................................... 12 

11. Colonies with a strong coconut-like odour; conidia subglobose to ovoidal, smooth, 

(2.7–)3.0–3.8(–5.0) × (2.3–)2.8–3.5(–4.0) µm, L/W = (0.8–)1.0–1.3(–1.6); often with 

a strong coconut-like odor .................................................................... T. atroviride P. Karst. (Dodd et al. 2002) 

11. Colonies lacking a coconut-like odor; conidia subglobose to ovoidal, finely 

spinulose (often appearing smooth with light microscope), (2.8–)3.4–3.6(–7.0) × 

(2.4–)3–4(–6) µm, L/W 1.0–1.7; rarely with a coconut-like odour .............. T. asperellum (Samuels et al. 1999) 

12. Conidia (3.0–)3.5–4.5(–5.0) × 2.5–3.5 μm; colony radius 24–26 mm after 96 h at 

25 °C on PDA; growing on Moniliophthora roreri on pods of Theobroma cacao, 

Ecuador ........................................................................... T. paucisporum Samuels et al. (Samuels et al. 2006) 

12. Conidia (2.5–)3.0–3.7(–4.0) × 2.2–)2.7–3.2(–3.5) μm; colony radius < 15 mm after 

96 h at 25 °C on PDA; endophyte in stems of Scalesia pedunculata, Galapagos 

Islands ..............� T. scalesiae 

13. Conidia (3.2–)3.5–4.0(–4.5) × 2.5–3.0 μm; Peru .......................................................................... T. neokoningii 

13. Conidia larger, (3.2–)3.5–4.5(–4.7) × (2.2–)2.5–3.5(–3.7) μm; Iran or cosmopolitan temperate ...................... 14 

14. Conidia (3.5–)3.7–4.5(–5.5) × (2.5–)2.7–3.5(–3.7) μm; L/W (1.0–)1.2–1.5(–1.7); 

colony radius on SNA after 72 h at 25 °C typically < 35 mm, north- and south-temperate ................... T. gamsii 

14. Conidia larger, (3.2–)4.0–5.0(–5.8) × (2.2–)2.5–3.0(–3.2) μm; L/W (1.1–)1.4–1.8(–2.1); 

colony radius on SNA after 72 h at 25 °C typically ca. 40–45 mm; Iran ........................................................ Vd 2 

147


Table 2. Continuous characters, geographic distribution and colony phenotype of the Trichoderma species discussed 

148 

Taxa/clades 

Character 

T. gamsii Vd 1 Vd 2 Vb 1 Vb 2 T. scalesiae T. neokoningii 

H. vinosa 

/T. vinosum 

T.viridescens 

/H.viridescens 

T. viride / 

H. rufa 

Peru 

Ecuador Brazil Galapagos 

Islands 

Cosmopolitan 

temperate 

Sri Lanka, 

Ghana 

Europe, North 

America, 

Australia 

New Zealand, 

Australia 

Europe, North 

America, Australia 

and New Zealand 

dominant distribution Europe, North 

America 

Conidia 

ornamentation grossly warted warted warted smooth warted smooth grossly warted grossly warted smooth smooth 

subglobose subglobose subglobose ellipsoidal to 

oblong 

ellipsoidal to 

oblong 

subglobose 

to broadly 

ellipsoidal 

subglobose ellipsoidal to 

oblong 

shape subglobose subglobose to 

ellipsoidal 

(3.2–)3.5–4.0 

(–4.5) 

(2.5–)3.0–3.7(– 

4.0) 

(3.0–)3.5–4.0 

(–4.2) 

(3.0–)3.2–4.0 

(–4.5) 

(3.5–)3.7–4.5 

(–5.5) 

(2.7–)3.0–3.7 

(–4.2) 

(3.2–)4.0–5.0 

(–5.8) 

(3.2–)3.5–4.5 

(–4.7) 

(2.7–)3.5–4.5 

(–8.5) 

length (µm) (3.0–)3.5–4.5 

(–5.5) 

95 % CI 3.99–4.05 3.97–4.02 3.9–4.0 4.3–4.5 3.4–3.5 4.0–4.1 3.6–3.8 3.5–3.8 3.1–3.4 3.7–4.0 

N 720 1233 120 90 60 30 30 30 30 30 

width (µm) (2.8–)3.4–4.0 (2.2–)3.0–3.7 (2.7–)3.0–4.0 (2.2–)2.5–3.0 (2.2–)2.5–3.2 (2.5–)2.7–3.5 (2.7–)3.0–3.5 (3.0–)3.2–3.7 2.2–)2.7–3.2(– 2.5–3.0 

(–5.0) 

(–4.7) 

(–4.2) 

(–3.2) (–3.5) (–3.7) 

(–4.0) (–4.2) 3.5) 

95 % CI 3.75–3.80 3.31–3.35 3.5–3.6 2.7–2.8 2.7–2.9 3.0–3.2 3.2–3.4 3.4–3.6 2.8–3.1 2.6–2.7 

N 720 1233 120 90 60 30 30 30 30 30 

(1.2–)1.4–1.6 

(–1.7) 

(0.9–)1.0–1.1(– 

1.3) 

(0.7–)0.9–1.1 

(–1.3) 

(0.9–)1.0–1.2 

(–1.4) 

(1.0–)1.2–1.5 

(–1.7) 

(1.0–)1.1–1.4 

(–1.6) 

1.0–1.2(–1.3) (1.1–)1.4–1.8 

(–2.1) 

(0.9–)1.1–1.4 

(–2.0) 

L/W (0.8–)1.0–1.2 

(–1.5) 

95 % CI 1.06–1.08 1.20–1.22 1.10–1.13 1.55–1.63 1.0–1.1 1.06–1.14 

N 720 1233 120 90 60 30 30 30 30 30 

Ascospores 

(2.7–)3.2–3.7(– 

4.2) 

(3.7–)5.0–6.5 

(–8.0) 

(3.2–)4.0–5.2 

(–7.5) 

distal length (µm) (3.7–)4.5–5.7 

(–7.7) 

95 % CI 4.91–5.14 4.62–4.72 5.5–5.9 3.4–3.6 

N 120 411 90 30

Table 2. (Continued) 

Taxa/clades 

Character 

T. gamsii Vd 1 Vd 2 Vb 1 Vb 2 T. scalesiae T. neokoningii 

H. vinosa 

/T. vinosum 

T.viridescens 

/H.viridescens 

T. viride / 


(2.5–)2.7–3.5(– 

3.7) 

(3.7–)4.7–6.2(– 

7.7) 

(3.2–)3.7–4.5 

(–5.5) 

(3.2–)4.0–4.7(– 

6.5) 

distal width 

(µm) 

95 % CI 4.25–4.42 4.15–4.23 5.3–5.7 3.0–3.2 

N 120 411 90 30 

(3.5–)4.0–5.0(– 

6.0) 

(4.5–)5.0–7.0(– 

9.0) 

(3.5–)4.5–5.7 

(–8.0) 

proximal length (µm) (3.7–)4.7–6.5(– 

8.0) 

95 % CI 5.4–5.7 5.07–5.21 5.8–6.2 4.3–4.7 

N 120 411 90 30 

(2.2–)2.5–3.2(– 

3.5) 

(3.7–)4.0–5.0(– 

8.0) 

(2.7–)3.2–4.0 

(–5.2) 

proximal width (µm) (3.0–)3.5–4.2(– 

5.2) 

95 % CI 3.77–3.93 3.62–3.70 4.8–5.2 2.8–3.0 

N 120 411 90 30 

Colony radius on PDA 72 h (mm) 

20 °C 26–34 25–38 25–29 30–36 34–40 33 38 39 3 30 

N cultures 18 29 3 4 2 1 1 1 1 1 

25 °C 31–41 32–45 25–33 43–51 53–57 45 54 52 6 42 

N cultures 18 29 3 4 2 1 1 1 1 1 

9–27 9–25 0–10 34–54 25–51 42 12 45 18 36 

30 °C 

N cultures 

17 29 3 3 2 1 1 1 1 1 


Colony radius on SNA 72 h (mm) 

15–27 20–30 14–23 16–24 18–20 27 33 40 3 17 

20 °C 

N cultures 

18 29 3 4 2 1 1 1 1 1 

22–34 24–36 16–27 29–34 29–31 42 42 50 10 30 

25 °C 

N cultures 

18 29 3 4 2 1 1 1 1 1 

7–23 8–22 0–5 25–39 17–33 38 12 63 10 36 

18 29 3 4 2 1 1 1 1 1 

30 °C 

N cultures 

149


150 

tef1 

North 

America 

Central 

America 

South 

America 

0.1 

Africa 

Europe 

Middle 

East 

CBS 433.97 

G.J.S. 04-217 

South-East Asia 

Australia, 

New Zealand 

and Indonesia 

LARGE 

VIRIDESCENS 

CLADE 

LARGE 

VIRIDE 

CLADE 

G.J.S.04-232 




G.J.S.99-175 

G.J.S.97-274 

J.B. NZ61 

G.J.S.99-142 

G.J.S.99-128 

G.J.S.04-81 

G.J.S.98-182 

G.J.S.89-142 

G.J.S.94-118 

G.J.S. 98-129 


C.P.K. 2046 

ATCC 32630 

G.J.S.99-11 

C.P.K. 2138 

Tr 6 

Tr 5 

G.J.S.92-11 


G.J.S. 99-8 


C.P.K. 947 

C.P.K. 2069 

C.P.K. 2043 

G.J.S.05-464 

G.J.S.04-202 

G.J.S.97-272 

G.J.S. 99-10 

ATCC 20898 

C.P.K. 2084 

J.B. PER43 

J.B. PER52 

G.J.S.05-185 

G.J.S.98-86 


C.P.K. 999 

C.P.K. 998 

G.J.S. 05-482 

G.J.S. 99-18 

Tr 4 


G.J.S.00-67 

G.J.S.97-243 

G.J.S.94-11 

G.J.S.94-10 

G.J.S.94-9 

G.J.S.99-158 

G.J.S.02-54 

G.J.S. 99-183 

G.J.S. 99-156 

DAOM 176335* 

RMF7832* 

G.J.S.03-151 Vd 1 

G.J.S.02-87 

C.P.K. 1488 

C.P.K. 2071 

G.J.S. 05-186 

J.B. NZ151 

J.B. PER23 

C.P.K. 2070 

C.P.K.2093 

C.P.K. 2092 

C.P.K. 2091 

C.P.K. 2090 

C.P.K. 2077 

C.P.K. 2076 

C.P.K. 2075 

C.P.K. 2074 

C.P.K. 2073 

J.B. RSA9665 

J.B. GS33 

J.B. R414 

J.B. RSA9642A 

J.B. RO42B 

J.B. RSA75 

C.P.K.2079 

G.J.S.04-09 

G.J.S.92-60 

G.J.S.05-111 

C.P.K. 1010 

C.P.K. 1011 

C.P.K. 2078 

G.J.S. 04-216 

G.J.S. 96-32 


G.J.S.05-104 



G.J.S. 99-13 

C.P.K. 1995 

C.P.K. 1007 

C.P.K. 1006 

G.J.S. 05-463 

G.J.S. 97-271 

ATCC 28020 

C.P.K. 965 


C.P.K. 1998 

Tr 2 

G.J.S. 99-14 

G.J.S. 91-62 

G.J.S. 92-14 

C.P.K. 2001 

C.P.K. 1996 



C.P.K. 2000 

C.P.K. 1009 

C.P.K. 1002 

C.P.K. 1001 

G.J.S. 99-16 

G.J.S. 92-15 

G.J.S. 04-86 

J.B. NSW13 

Tr21 

G.J.S. 90-95 

DIS 328g 

G.J.S. 04-40 

G.J.S. 03-74 

Vd 3 

H. vinosa 

Vd 2 

H. stilbohypoxyli 

G.J.S. 02-134 

G.J.S. 02-96 

G.J.S. 02-139 

G.J.S. 02-135 

G.J.S. 98-8 G.J.S. 97-3 

T. gamsii sp. nov. 

T. neokoningii sp. nov. 


Vb 3 

Vb 1 

Vb 2 

H. viridescens sp. nov. 

T. scalesiae sp. nov. 

H. atroviridis 

H. caribbaea 

G.J.S. 98-43 

DIS 320c T. caribbaeum var. aequatoriale 

G.J.S. 04-355 

DAOM 165782 H. petersenii 

G.J.S. 98-139 

G.J.S. 02-50 

G.J.S. 991-05 H. dingleyae 

G.J.S. 99-194 

G.J.S. 99-202 H. dorotheae 

G.J.S. 02-78 

G.J.S. 97-88 

G.J.S. 96-13 T. intricatum 

C.P.K. 1895 

G.J.S. 01-234 H. ochroleuca 

T. ovalisporum 

DIS 70a 

DIS172i 

DIS172h 

G.J.S. 04-373 

G.J.S. 93-20 

Dis 326h 

G.J.S. 95-28 

G.J.S. 95-175 

DAOM 220105 

G.J.S. 04-318 H. koningiopsis 

DAOM 230813 

G.J.S. 97-273 

G.J.S. 04-376 

G.J.S. 91-6 

G.J.S. 89-122 


G.J.S. 96-120 H. koningii 

ATCC 64262 

G.J.S. 00-168 

G.J.S. 99-127 

G.J.S. 99-204 

G.J.S. 99-83 

G.J.S. 99-86 

G.J.S. 99-191 G.J.S. 04-353 

Ve 

G.J.S. 90-97 

G.J.S. 90-20 

DIS 217i 

DIS 8 

DIS 7 T. erinaceus G.J.S. 99-146 

G.J.S. 99-147 

G.J.S. 85-57 

G.J.S. 00-73 G.J.S. 961-63 


G.J.S. 04-157 

G.J.S. 04-158 H. rogersonii H. muroiana 

G.J.S. 01-327 G.J.S. 96-141 

G.J.S. 96-132 

G.J.S. 96-135 

G.J.S. 99-49 H. flaviconidia 

G.J.S. 03-69 

G.J.S. 01-13 

T. paucisporum 

DIS 85f 

T. theobromicola 

G.J.S. 01-257 H. pezizoides 

G.J.S. 98-170 

DAOM 167057 T. hamatum 

DAOM 166162 

T. asperellum 

T. pubescens 

H. austrokoningii 

H. neorufa 

Fig. 1. Bayesian radial phylogram showing the structure of the Trichoderma section Trichoderma inferred from sequences of two introns of tef1. 

Red colour is used to indicate species described in this study. Arrows show branches leading to species recognised within the section. Dark grey 

filled circles at nodes indicate posterior probability coefficients higher than 0.90 as they were obtained after 5 million generations; black filled 

circles at nodes show support higher than 0.95. Font colours correspond to regions of sampling on the schematic map. The dotted line around 

Vd 3 indicates strong phenotypic similarity despite phylogenetic divergence. 

* - sequences from John Bissett, collection information may be obtained from Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada

DESCRIPTIONS OF THE SPECIES 

Continuous characters not provided in the descriptions 

are given in Table 2. 

1. Hypocrea rufa (Pers. : Fr.) Fr., Summa Veg. Scand., 

Sectio Post. 383. 1849. Figs 2–3, 7a–f, 8 a–e. 

≡ Sphaeria rufa Pers., Obs. Mycol. 1: 20 (1796) : Fr., Syst. 

Mycol. 2: 335. 1822. 

Anamorph: Trichoderma viride Pers., Neues Mag. 

Bot. [Roemer’s] 1: 92. 1794 : Fries, Syst. Mycol. 3: 

215. 1832. 

= Trichoderma lignorum (Tode) Harz, Bull. Soc. Imp. Natur. 

Moscou 44: 116. 1871. 

= Trichoderma glaucum E.V. Abbott, Iowa State Coll. J. Sci. 1: 

27. 1927. 

Stromata when fresh (Fig. 2d, h) 1–4(–6) mm long, 

0.5–1.5 mm high, solitary to gregarious, or aggregated 

in small numbers or crowded in lines along wood 

fibres, at first semi-effused, flat, velutinous, with white 

mycelial margin; becoming pulvinate, more rarely 

turbinate or discoid, circular to irregular in outline, 

surface smooth to slightly uneven to granular, broadly 

attached, margin often becoming free and concolorous 

with stroma surface; at first white, remaining white with 

yellowish ostiolar openings (“albino” form), or more 

commonly becoming variably coloured from the centre: 

first yellowish, then pale ochraceous, light brownish or 

yellow- to orange- to rust-brown (5A4–7, 5B4, 5C6–7, 

6CD5–8), later light to dark reddish brown (7–8CD6–8, 

8E7–8), sometimes with whitish to rust-coloured scurf; 

ostioles invisible or appearing as watery, hyaline, or 

indistinct darker dots, sometimes projecting, convex, 

often irregularly distributed. 

Stromata when dry (Fig. 2a–c, e–g) (0.5–)0.6–3 

(–5.7) mm long, (0.4–)0.6–2(–3.4) mm broad, (0.2–) 

0.3–0.6(–0.9) mm thick (n = 31), KOH – , darker and 

surface more uneven than in fresh stromata, granular 

to finely tuberculate, sometimes extremely uneven with 

perithecial contours visible; ostioles not visible or partly 

convex or semiglobose, appearing as hyaline or brown 

dots, generally hyaline after addition of water (Fig. 2i); 

young stromata velvety to conspicuously hairy (Fig. 

2a, c), with diffuse yellowish orange, yellowish brown 

or (orange-)brown colours, 4B4–5, 5AB5–6, 5CD7–8, 

6B5, 6CD5–8, later light-, 7CD5–8, 7E7–8 to deep 

reddish brown, 8EF5–8 to 7F6–8, 8CD5–6, to dark 

brown, 7EF5–6; albino form white or becoming pale 

yellowish, 4A3–4, with numerous, conspicuous light 

brownish dots (Fig. 2f). 

Stroma anatomy: Cells of stroma surface in face view 

(Fig. 2m) pseudoparenchymatous, (3.5–)5–9(–14.5) 

µm (n = 30) diam, walls to 1 μm thick, reddish brown 

in water, orange-brown in lactic acid, pigment unevenly 

deposited in cell walls, giving a mottled appearance to 

the stroma surface. Ostiolar area in dry stromata (32–) 

40–84(–126) µm (n = 33) diam. Hairs (Fig. 2l) arising 

from the stroma surface, yellowish to pale brown, 

comprising 2–5 cells, apically rounded, rarely branched, 

sometimes consisting of only one inflated cell, (7.5–)10– 


29(–62) μm (n = 79) long, (2–)3.5–5(–6.5) μm wide (n = 

49), walls 0.5–1 µm thick. Cortical region (12–)17–30(– 

35) µm (n = 20) thick, cells forming a textura angularis, 

slightly compressed, reddish brown, in lactic acid 

orange-brown, (2–)3.5–9(–13.5) µm (n = 60) diam in 

vertical section, walls up to 1 µm thick. Cells immediately 

below the cortex comprising a mixture of intertwined 

hyphae, (2.5–)3–6(–6.5) µm (n = 10) wide, vertical and 

parallel between perithecia, and few subglobose to 

angular cells similar to those of the cortex. Perithecia 

(Fig. 2k) (161–)182–245(–307) × (107–)140–210 

(–251) µm (n = 31), flask-shaped, ellipsoidal to globose. 

Ostiolar canal (70–)75–107(–120) µm (n = 21) long, 

(32–)33–55(–69) µm (n = 15) wide at the opening (Fig. 

2j), cells surrounding the ostiolar opening a palisade of 

hyaline, narrowly cylindrical, apically slightly expanded 

cells, plane with the surface or projecting to 14–43 

(–69) µm (n = 15). Peridium colourless, consisting of 

laterally strongly compressed thin hyphae, basally and 

apically pseudoparenchymatous, indistinct, scarcely 

differentiated from and merging with the surrounding 

tissue, apical part flanking the ostioles conspicuously 

thickened. Tissue below the perithecia (Fig. 2n) 

of homogeneous, dense textura epidermoidea, of 

globose to elongate, thin-walled, hyaline cells, (4–)5– 

19(–26) × (3–)4.5–10(–12.5) µm (n = 30), not layered 

but cells gradually smaller and interspersed with few 

narrow hyphae towards the base of the stroma. Asci 

(Fig. 2q) cylindrical, (70–)87–112(–132) (n = 72) µm 

long, including a stipe of (5–)9–17(–22) µm (n = 30), 

(4–)5.5–7(–8.5) µm (n = 72) wide, stipe short, with a 

knob-like base, apical pore minute. Ascospores (Fig. 

2o, p) hyaline, verrucose, verrucae ca. 0.5 µm long 

and diam; dimorphic, distal part subglobose to oval, 

sometimes slightly tapered towards the upper end, 

proximal part oblong to wedge-shaped, the lower end 

broadly rounded. 

Colony characteristics: Optimal temperature for growth 

on PDA and SNA 25 °C, colony radius on PDA 30–35 

mm after 72 h in darkness, on SNA 22–31 mm; not 

growing at 35 °C. Colonies grown on PDA 1 wk at 25 

°C with alternating light (Fig. 7a–d) developing conidia 

in several alternating green (28DE5–7 to 27DE3–6 

to 27F7–8) and dull yellow (3A3–4) concentric rings. 

Diffusing pigment not noted. Slight coconut odour 

rarely noted. 

Colonies grown on CMD (Fig. 7e; 25 °C under 

alternating light) >7 cm, filling a Petri plate within 5– 

6 d, thin, hyaline, margin indistinct, diffuse, hyphae 

loosely arranged, no zonation, no pigment formed. 

Autolytic activity absent, coilings and aerial hyphae 

inconspicuous. A weak coconut-like odour formed 

in some but not all strains. Conidiation from 2 d, in 

pustules more or less regularly distributed on the 

plate or forming in a broad band around the margin, 

less frequently in concentric rings, usually starting 

close to the point of inoculation, formed exclusively or 

preceded or accompanied by various amounts of simple 

conspicuously curved conidiophores or minute tufts. 

Pustules (Fig. 3a–b) at first white, becoming green from 

151


152 

k 

a 

b 

g 

j 

c 

m 

n 

l 

d 

h 

o 

p q 

Fig. 2. Hypocrea rufa/Trichoderma viride teleomorph. a–c, e–g. Dry stromata (a, c: immature, downy, b, e–g: mature, f: “albino” stroma). d, 

h. Fresh stromata. i. Stroma reconstituted with water. j. Ostiolar opening in section. k. Section of stroma with perithecia. l. Hairs on surface of 

mature stroma. m. Surface of stroma in face view. n. Subperithecial tissue in section. o–p. Ascospores, o: in cotton blue/lactic acid. q. Asci with 

ascospores. Sources: a– b, p: neotype Scleromyceti Sueciae 303, c: WU 24016; d, g, i–o, q: epitype WU 24013, e: BPI 872089, f: WU 24015, 

h: WU 24011.Scale bars: a, e, g, i = 0.5 mm, b = 0.8 mm, c–d, f = 1 mm, h = 1.5 mm, j, l, q = 20 µm, k = 0.2 mm, m = 25 µm, n = 30 µm, o = 15 

µm, p = 10 µm. 

e 

i 

f


Fig. 3. Hypocrea rufa/Trichoderma viride anamorph. a–b. Conidial pustules formed on CMD. c. Short sinuous elongations arising at the margin 

of pustules. d–l. Type 1 conidiophores from CMD. (d–e, h = “Type 1” conidiophores). Arrows indicate curved, hooked or sinuous phialides. Note 

especially solitary phialides in d and e. m–n. Conidia. Surface view of conidia shown in m, optical section shown in n. a–b from CBS 101526. 

Microscopy: a–b stereo; c bright field; d–h, j–l phase contrast; i, m–n DIC; c, k from CBS 119326; d–e from G.J.S. 89-127; f, m–n from Tr 8. g; l 

from G.J.S. 04-372; h from G.J.S. 05-463; i from G.J.S. 05-104; j from G.J.S. 99-16 9. Scale bars: a–b = 0.5 mm; d–l = 20 µm; m–n = 10 µm. 

153


the 4 th day or later, depending on the isolate, 28D3–5 

or 26E4–6 to 27E4–6, finally 26F5–8 to 27F6–8 after 

1 wk, compact to cottony, pulvinate to hemispherical, 

0.5–2.5(–5.0) mm diam, 0.5–1.6 mm high. 

The structure of typical conidiophores was 

determined after 5–7(–11) d at conditions described 

above: pustules and minute tufts arising on a 8–12 µm 

thick stipe, often with constricted septa, bearing several 

thick primary branches arising at various angles, both 

partly verrucose, further branching dense and complex, 

final long branches thin, bearing short terminal branches 

at various angles, with 1 or 2(–3) terminal phialides. 

Conidiophores (Fig. 3d–l) ill-defined, no main axes 

discernible or at best weakly developed, conspicuously 

and extremely variably curved to sinuous, often seen 

as short elongations on the periphery of pustules; 

branches and phialides generally unpaired. Simple 

conidiophores and microtufts sometimes tending to 

be more regularly paired, with tree-like branching; 

branches sometimes originating on thickened nodes, 

7–11 µm wide with up to 5 branches, often tending to be 

less curved. Phialides (4.0–)6.5–11.2(–18.2) μm long, 

(1.0–)2.5–3.2(–4.0) µm at the widest point, l/w (1.2– 

)2.0–4.5(–13.0), basal width (1.0–)1.7–2.5(–3.0) µm 

(n = 600), originating singly or in groups of 2–3, on rarely 

inflated, 2–3 µm thick cells, usually not paired, very 

variable among isolates, lageniform to long cylindrical, 

typically strongly curved to sinuous, less commonly 

straight, usually with long necks (up to 10 µm), not or 

slightly thickened in various positions, tending to be 

longer and narrower in minute tufts and shorter and 

more swollen when crowded. Conidia (Fig. 3m, n) 

globose to subglobose, infrequently nearly ovoidal, 

(olive-)green, basal scar sometimes visible, coarsely 

tuberculate (Figs 3 m, n; 8 a–e), tubercles up to ca. 

0.7 µm long and 1 µm diam, containing few guttules, 

in aged cultures often in chains. Chlamydospores rare, 

typically subglobose at the tips of hyphal branches, less 

frequently intercalary in hyphal cells, (5–)8–12(–14) μm 

diam (n = 81), hyaline to pale yellowish. 

Colonies grown on SNA (Fig. 7f; 25 °C, alternating 

light) similar to CMD, thin, hyaline, no zonation, no 

pigment, autolytic activity absent, aerial hyphae 

inconspicuous, coilings somewhat more pronounced 

than on CMD. Conidiation similar to CMD, asymmetrical, 

starting in the centre in loosely arranged compact 

pustules of ca. 1–2 mm diam, aggregated to ca. 4 mm 

diam, and on some effuse single conidiophores, green 

(26EF5–7 to 27F6–8) from 3–4 d, dry. Chlamydospores 

(after 15 d at 25 °C) inconspicuous, more frequent than 

on CMD, terminal and intercalary, mainly globose, 

(5.0–) 6.5–10.5(–12.5) µm (n = 21) diam, smooth, 

hyaline to pale yellowish. 

Habitat: Anamorph isolated directly from soil, peat, 

and wood, found also on leaf litter; isolated as an 

endophyte from sapwood of Fagus sylvatica in 

the U.K., isolated from water-damaged building in 

Denmark, not uncommon. Teleomorph uncommon, 

inconspicuous, found on wood, less commonly on bark 

of cut branches, tree tops or 2–120 cm thick logs. In 

Europe found in open coniferous or mixed deciduous 

154 

forests, grassland with single trees or at shady road 

sides, often in piles (to 3 m above the ground) of logs 

at the edge of forests, stored or lying on bare moist 

soil, in leaf litter, in grass, the wood in little to medium 

degree of decomposition and often hard. In Central 

and Northern Europe mainly on coniferous trees 

(Pinus sylvestris, Picea abies), in Western Europe 

more frequent on deciduous trees (e.g. found on 

Quercus robur, Acer pseudoplatanus). Associated with 

Trichaptum abietinum, Stereum sanguinolentum, Sarea 

resinae, Ophiostoma sp., Nectria fuckeliana, Valsa pini, 

Pezicula eucrita, Schizophyllum commune, various 

Corticiaceae; Amphiporthe leiphaemia, Diatrypella sp., 

Bulgaria inquinans. 

Distribution: Teleomorph collected in Europe (confirmed 

in Austria, Czech Republic, U.K., France, Sweden) and 

United States (confirmed in North Carolina, Virginia). 

Anamorph north and south-temperate, including 

Europe (Italy, Sweden, Finland, Switzerland, Denmark), 

Canada, United States, New Zealand, Japan. 

Neotype: Scleromyceti Sueciae No. 303. Epitype, designated here: 

Czech Republic, South Bohemia, Frymburk, 3.4 km north from 

Lipno, MTB 7351/3, 48°38’04’’ N, 14°11’19’’ E, elev. 745 m, on partly 

decorticated logs of Pinus sylvestris, 12–30 cm thick, on the ground 

or elevated in a pile of stored logs at roadside and edge of coniferous 

(Picea / Pinus) forest, soc. Ophiostoma sp., Nectria fuckeliana, 

Valsa pini, Pezicula eucrita, Schizophyllum commune, unidentified 

Corticiaceae, 3 Oct. 2004, W. Jaklitsch, W.J. 2753 (WU 24013; 

culture CBS 119325 = C.P.K. 1997 = G.J.S. 04-372). 

Lectotype of Trichoderma viride: “Prope Parisiis Trichoderma 

viride, Hb. Pers.” Herb. Lugd. Bat. 910 263-877 (L 0018559 = 

“Rijksherbarium No 148-1” cited by Bisby 1939, see Webster 1964; 

lectotype designated by Bisby 1939). Epitype of Trichoderma viride 

designated here: culture C.P.K. 1997, a dry culture deposited with the 

epitype of H. rufa (WU 24013a; living culture CBS 119325 = C.P.K. 

1997 = G.J.S. 04-372). 

Other teleomorphic specimens examined: Austria, Niederösterreich, 

Zwettl, Traunstein, roadside, 1 km after the western end of the 

village, MTB 7556/4, 48°26’10’’ N, 15°05’57’’ E, elev. 830 m, on 

partly decorticated cut logs of Picea abies, up to 45 cm thick, in a 

pile stored at the edge of a Picea/Fagus forest, soc. Ophiostoma 

sp., 5 Oct. 2004, W. Jaklitsch, W.J. 2766 (WU 24015; culture CBS 

119327 = C.P.K. 1999); Steiermark, Liezen, Kleinsölk, close to the 

northeast corner of the Schwarzensee, MTB 8749/1, 47°17’38’’ N, 

13°52’36’’ E, elev. 1170 m, on partly decorticated cut logs of Pinus 

sylvestris, 20–25 cm thick, stored in a pile at roadside and edge of a 

spruce forest, soc. Ophiostoma sp., 7 Oct. 2004, W. Jaklitsch, W.J. 

2773 (WU 24016; culture C.P.K. 2000); Liezen, Weng im Gesäuse, 

Ennstal, Gstatterboden, 0.9 km after the village heading east, MTB 

8453/2, 47°35’ 34’’ N, 14° 39’ 09’’ E, elev. 570 m, on cut log of Picea 

abies, 120 cm thick, 2.5 m above ground, in a pile stored at roadside, 

soc. Trichaptum abietinum, 8 Oct. 2004, W. Jaklitsch, W.J. 2774 (WU 

24017; isolate C.P.K. 2001). Czech Republic, South Bohemia, at 

road side 5.7 km north from Frymburk, MTB 7250/4, 48°42’36’’ N, 

14°08’06’’ E, elev. 750 m, on partly decorticated cut log of Picea abies, 

22 cm thick, on the ground, protected by grass, herbs, soc. Nectria 

fuckeliana, Stereum sanguinolentum, Sarea resinae, immature, 

culture from conidia, 22 Sep. 2003, W. Jaklitsch, W.J. 2408 (WU 

24010; culture C.P.K. 965); 2.7 km before Frymburk approaching 

from Lipno, MTB 7351/3, 48°38’22’’ N, 14°10’52’’ E, elev. 740 m, on 

partly decorticated logs of Pinus sylvestris, 10–43 cm thick, stored 

in a pile between roadside and edge of coniferous (Picea / Pinus) 

forest, mostly immature, 3 Oct. 2004, W. Jaklitsch, W.J. 2758 (WU 

24014; culture C.P.K. 1998). France, La Moselle, Parc Lorraine, 

Héming, between Étang du Stock and Maizières de Vic, 48°43’35’’ 

N, 06°54’07’’ E, elev. 180 m, on cut and mostly corticated branches 

and logs of Quercus robur, 2–40 cm thick, on bare ground, some 

branches squeezed into moist soil, soc. Amphiporthe leiphaemia, 

Diatrypella sp., Bulgaria inquinans, part with white mould, 5 Sep.

2004, W. Jaklitsch & H. Voglmayr, W.J. 2677 (WU 24011; culture 

C.P.K. 1995). Sweden, Uppsala Län, Fredrikslund, pine forest near 

nature reserve Kungshamn-Morga, 1.5 km NE of Fredrikslund, 

59°47’00” N, 17°39’00” E, elev. 50 m, on cut and mostly corticated 

tree tops and branches of Pinus sylvestris, 5–9 cm thick, on the 

ground, 8 Oct. 2003, W. Jaklitsch & S. Ryman, W.J. 2450 (BPI 

872089; cultures CBS 119326 = C.P.K. 984, G.J.S. 04-21 from white 

stroma). United Kingdom, Derbyshire, Baslow, Longshaw Country 

Park, Peak District National Park, 53°18’26” N, 01°36’08” W, elev. 

350 m, on corticated branches and logs of Acer pseudoplatanus, 

2–10 cm thick, on the ground in open grassland, immature, culture 

from conidia, 10 Sep. 2004, W. Jaklitsch & H. Voglmayr, W.J. 2695 

(WU 24012; culture C.P.K. 1996). U.S.A., North Carolina, Macon 

County, northwest of Highlands, Cullasaja Gorge, Dry Falls, on 

bark of recently dead tree, 20 Sep. 1989, G.J.S. et al. (BPI 744477; 

culture G.J.S. 89-127 = CBS 114374); Macon County, Blue Valley 

off Clear Creek Rd., along Overflow Creek, 35°00’ N, 83°15’ E, on 

decorticated wood, 16 Oct. 1990, G.J.S. et al. (BPI 1109386, culture 

G.J.S. 90-95 = IMI 352470 = CBS 120066); Virginia, Giles County, 

Mt. Lake Biological Station, Little Spruce Bog, 37°22’ N, 80°31’ E, 

elev. 1170 m, on branch of Acer sp., 17 Sep. 1991, G.J.S. et al. (BPI 

1112834; culture G.J.S. 91-62). 

Anamorphic cultures studied: see Table 1. 

Typification of H. rufa: This species was first described 

by Persoon in 1796 as Sphaeria rufa. The description, 

comprising just seven words, was enigmatic: 

“periphaerica, irregularis, applanata, mollis, rufa intus 

albida”, and no collecting location or specimen was 

cited. He (Persoon 1801) expanded the description, 

noting that the species was rare in dense forest on 

branches of Fagus and that the rufous colour and 

“scarcely prominent sphaerules [ostiola]” distinguished 

this species from all others. Fries (1822) included S. rufa 

in his Systema mycologicum and he later (Fries 1849) 

placed the species in the new genus Hypocrea. The 

first mention of a specimen was Fries’ (1849) citation 

of Scleromyceti Sueciae 304. Fries created confusion 

in this exsiccate when he misnumbered specimens 

303 and 304. The specimen list gave number 303 

as H. gelatinosa and number 304 as H. rufa but the 

numbers on the actual specimens were reversed, 303 

for H. rufa and 304 for H. gelatinosa (Holm & Nannfeldt 

1962; Hennig Knudsen, in litt. 7 Oct. 2004). Tulasne & 

Tulasne (1865) were the first to establish a link between 

H. rufa and T. viride and their elegant work established 

the taxonomic identity of this species. Scleromyceti 

Sueciae 303 (UPS!) was designated as neotype for S. 

rufa by Rossman et al. (1999). 

Scleromyceti Sueciae 303 (Figs 2 a, b, p) comprises 

a single piece of decorticated wood glued to a piece of 

cardboard on which is written only “Fries, Scler. Suec. 

303. Sphaeria rufa”. Stromata are scattered over the 

surface of the wood. One is young, semi-effused to 

pulvinate, light tan, and fibrous. The mature stromata are 

rufous (ca. 8F8), more or less discoidal, with a deeply 

wrinkled or convoluted, velutinous surface. Ostiolar 

openings are not visible. Hyphal hairs arise from the 

stroma surface. Cells of the stroma surface are angular 

and brown pigment is deposited unevenly in the cell 

walls. No asci remain. Discharged part-ascospores are 

hyaline, spinulose, dimorphic. Subglobose to ovoidal 

parts, which are assumed to be distal part-ascospores, 

measure 3.7–4.7(–5.5) × 3.5–4.5(–5.0) μm (n = 30). 

Ellipsoidal to wedge-shaped parts are assumed to be 

the proximal part-ascospores and measure (4.0–)4.5– 


5.0(–5.7) × (3.0–)3.2–3.7(–4.5) μm (n = 30). Although 

these measurements are somewhat smaller than we 

have found for about 10 collections, the combined 

attributes of anamorph and teleomorph that we describe 

here conform well with Fries’s specimen and with the 

taxonomic identity that was established by Tulasne & 

Tulasne (1865). We have chosen an epitype for H. rufa, 

WU 24013, collected in the Czech Republic, that agrees 

well with the historical concept of H. rufa in teleomorph 

and anamorph. 

Notes: Stromata of H. rufa are usually accompanied 

by the Trichoderma viride anamorph. Conidia found in 

nature are dark green, 26F5–8 to 27F4–8, and often 

citrine- to sulphur-yellow, 4A4–6, hairy patches of 

mycelium are found. Hypocrea rufa cannot be safely 

distinguished from the sympatric H. viridescens by 

the morphology of the teleomorph, and intense yellow 

cottony patches of the accompanying anamorph are 

found in both species. The dry stroma colour reported 

in the description above is difficult to determine due 

to the small size of stromata, which are often solitary 

rather than aggregated. Perithecial measurements are 

only approximated due to indistinct differentiation of 

the peridium from surrounding tissue. Some pulvinate 

stromata approach those of H. pachybasioides or H. 

minutispora in shape and colour when their ostiolar 

openings are clearly visible, but H. rufa differs from 

these species in the presence of hairs, especially in 

young stromata. 

While the anamorph, T. viride, is met frequently on 

wood, but less frequently than T. viridescens, it is difficult 

to find good teleomorph material. Stromata apparently 

develop slowly and in a narrow range of ecological 

conditions, mainly regarding moisture, temperature, 

and age and degree of decay of substrata. Moreover, 

they often develop in open habitats that are susceptible 

to quick desiccation. The frequency of long dry periods 

has increased in recent years, which may contribute to 

the fact that teleomorphs are collected rarely. On the 

other hand, if a habitat is too moist, stromata are soon 

attacked by hyphomycetes, often seen in specimens 

as white mould on the stromata. These are obvious 

reasons why specimens mostly contain immature 

stromata. Anthropogenic influences, particularly cutting 

of logs and branches, strongly facilitate development of 

the teleomorph. 

Webster (1964) refuted Bisby’s (1939) contention 

that H. rufa could not be distinguished from H. gelatinosa 

(Tode : Fr.) Fr. by characterising the anamorphs and 

teleomorphs of the two species. We have examined 

several of the specimens collected in the U.K. and 

cited by Webster (SHU!, now in K). On the basis of the 

stromata alone we could not identify them as H. rufa 

or H. viridescens. However, conidia found on 2540, 

2611, 2618 and 2621 are globose and coarsely warted; 

most likely they are H. rufa. One of those, 2621, has 

conidiophores typical of what we have described for T. 

viride. 

155


2. Hypocrea viridescens Jaklitsch & Samuels, sp. 

nov. MycoBank MB494775. Figs 4–6, 7 g–l, 8 f–h. 

Etymology: viridescens, turning green, in reference to 

the anamorph. 

Hypocreae rufae similis sed anamorphosis differt. Ascosporae: pars 

distalis (3.3–)4.1–5.2(–7.4) × (3.2–)3.8–4.6(–5.4) μm; pars proxima 

(3.4–)4.4–5.8(–7.9) × (2.7–)3.3–4.0(–5.3) μm. Anamorphosis 

Trichoderma viridescens (A.S. Horne & H.S. Williamson) Jaklitsch & 

Samuels. Conidia subglobosa vel ellipsoidea, levia, (2.8–)3.5–4.5(– 

8.5) × (2.3–)3.0–3.7(–4.7) μm, L/W (0.9–)1.1–1.4(–2.0). 

Anamorph: Trichoderma viridescens (A.S. Horne 

& H.S. Williamson) Jaklitsch & Samuels, comb. nov. 

MycoBank MB501049. 

≡ Eidamia viridescens A.S. Horne & H.S. Williamson, Ann. Bot. 

37: 396. 1923. 

Stromata when fresh (Fig. 4a–g) ca. 0.5–3.5 mm diam, 

ca. 0.5–1.5 mm thick, single, loosely gregarious or 

densely aggregated in lines, young downy, covered 

with yellowish to rust-coloured hairs, first with white 

inconspicuous margin; later glabrous, pulvinate, 

centrally attached, margins free, outline circular, 

angular to irregular, ostioles invisible to inconspicuous 

or light to dark dots, surface smooth to finely granular 

by perithecial contours, young light brown 5B5–6 to 

6CD5–6, yellow-brown to nearly orange-brown 6CD7- 

8, intensely orange-rust-reddish brown 8(B)C7–8; 

mature mostly dark reddish brown, 7DE7–8, 8EF6–8 

(to 9F7–8, 10CD7–8, 10F7–8), margin usually dark; 

rarely rosy-brownish with dark spots to reddish brown 

8CD5-6, more rarely 6A6–7 to 7A5–6. 

Stromata when dry (Fig. 4h, i) (0.2–)0.6–1.6(–3.6) 

mm (n = 277) diam, 0.2–0.5(–0.8) mm (n = 33) thick, 

unchanged or slightly darkened in 3 % KOH; young thin, 

semi-effuse to effuse, hairy, white to yellowish brown to 

rust-brown, 4A4, 5BD4–7 to 6–7CD5–8, with white to 

rust margin; later effluent, discrete and pulvinate with 

circular, angular to irregular outline; surface uneven, 

tubercular to wrinkled; ostioles invisible or appearing 

as light dots with darker marginal rings, not or slightly 

projecting, convex; colour in the great majority of 

stromata deep and dark reddish brown 7–9EF5–8. 


(Fig. 4 l) glassy, isodiametric to slightly elongated, (2–) 

3.5–8.5(–19) × (1.5–)2.5–6(–10) µm (n = 313, 210), 

walls thickened and pigment not uniformly deposited. 

Hairs arising from cells of the stroma surface, usually 

abundant when young, scant on mature stromata, 1–4celled, 

thick-walled, (5–)10–24(–47) μm (n = 240) long, 

(2–)3.2–5.0(–6.5) µm (n = 83) wide, base to 7 µm wide, 

apically rounded, pale brownish, smooth to slightly 

verruculose. Ostiolar area of dry stromata (24–)34–64 

(–79) µm (n = 33) diam. Cortical region of stroma (Fig. 

4k) (15–)18–36(–60) µm (n = 63) thick, around the entire 

stroma except the point of attachment, reddish brown 

to yellow-brown, cells in section of textura angularis, 

(2.0–)3.5–7.5(–17) × (1.7–)2.8–5.0(–8.5) μm (n = 279, 

150), with walls thickened in the outer part, thinner 

towards the interior. Cells immediately below the cortex 

comprising a mixture of intertwined, (3–)4–6(–6.5) µm 

wide hyphae (n = 15), vertical and parallel between 

perithecia, and hyaline, subglobose to angular, mainly 

156 

isodiametric cells, (3–)5–9.5(–13) µm (n = 30) diam, 

flanking the ostioles. Perithecia flask-shaped, often 

somewhat narrowed towards the base, sometimes 

ellipsoidal to globose, (136–)220–287(–337) μm high, 

(72–)150–224(–280) μm diam (n = 149); peridium at 

the base (17–)19–26(–30) µm, laterally (12–)13–20 

(–22) µm (n = 15) thick, hyaline. Ostiolar canal (53–) 

70–98(–130) μm (n = 138) long, (30–)33–49(–57) µm 

(n = 15) wide at the opening, the opening formed by a 

palisade of hyaline, apically elongate narrowly clavate 

cells, even with the stroma surface or projecting up to 

15 µm (n = 15). Subperithecial tissue homogeneous, 

dense textura angularis to t. epidermoidea, cells 

(sub-)globose to elongate to subangular or curved, 

(3.5–) 4.5–15(–39) µm (n = 337) long, (2–)4.5–10 

(–17) µm (n = 240) wide, hyaline to pale brownish, 

with ca. 0.5 µm thick walls, tissue not layered but cells 

gradually smaller and interspersed with few narrow, ca. 

3–4 µm wide hyphae towards the base of the stroma; 

in the central part of the stroma (point of attachment) 

stratified by a palisade (Fig. 4n) of oblong refractive 

glassy cells, ca. 14–31 × 4–9 µm, above the smallercelled 

pseudoparenchyma, sometimes irregular 

brownish pigment in patches incorporated through the 

whole tissue; basally delimited by the reddish brown 

cortex. Asci (Fig. 4o) cylindrical, (56–)82–101(–118) × 

(3–)5–7(–9) μm (n = 314), including a stipe of (4–)6– 

22(–33) μm (n = 31); apical pore minute. Ascospores 

hyaline, verruculose to verrucose with verrucae ca. 0.5 

µm long and diam, dimorphic; distal part subglobose, 

oval to wedge-shaped, (3.3–)4.1–5.2(–7.4) × (3.2–) 

3.8–4.6(–5.4) μm (n = 411); proximal part oblong to 

wedge-shaped, lower end broadly rounded, (3.4–)4.4 

–5.8(–7.9) × (2.7–)3.3–4.0(–5.3) μm (n = 411). 


on PDA and SNA 25 °C; after 72 h in darkness colony 

radius on PDA 32–45 mm, on SNA 24–37 mm; typically 

slower at 30 °C than at 20 °C; not growing at 35 °C. 

Colonies grown on PDA for 1 wk at 25 °C with alternating 

light and darkness (Fig. 7g–j) developing conidia in 3 or 

4 distinct concentric rings, but often remaining sterile 

or producing very few conidia. Conidial masses green 

(28D–E5–6). Diffusing pigment not noted or reverse 

only slightly yellowish (3A3–4 to 3B4). Coconut odour 

typically present. Autolytic activity more pronounced at 

15 °C, coilings more frequent at 30 °C. 

Colonies grown on CMD (Fig. 7k; 25 °C) filling 

the Petri plate within 5–6 d, thin, hyaline, regularly 

circular, hyphae loosely arranged radially, no zonation. 

Autolytic activity inconspicuous, coilings abundant 

in some isolates. Aerial hyphae often scarce during 

fast growth, becoming abundant, particularly towards 

the margin, broad zone at the margin becoming 

downy. A diffuse greenish yellow pigment (1B2–6, 

2A3–3B4 to 29A2–3) often diffusing through the 

whole culture. A strong coconut-like odour usually 

formed. At 15 and 30 °C slower development with 

less conidiation and less strong coconut-like odour 

formed, coilings more frequent at 30 °C. Conidia 

forming within 2–10 d at 25 °C under alternating light, 

typically effuse, conidial pustules uniformly dispersed 

throughout the colony, developing in a broad marginal

a 

d e f 

g h i 

b 

k l 

m 

n 

c 


Fig. 4. Hypocrea/Trichoderma viridescens teleomorph. a–g. Fresh stromata (a, d–e: immature, b–c, f–g: mature). h–i. Dry mature stromata. 

j. Surface of stroma reconstituted with water, showing ostioles and unevenly distributed pigment. k. Surface of stroma and ostiolar opening 

in section. l. Surface of stroma in face view, including unicellular hair. m. Subperithecial tissue in section. n. Palisade of cells above point of 

attachment of stroma in section. o. Asci with ascospores in cotton blue/lactic acid. Sources: a, l, o: WU 24025. b–c: WU 24027. d, f: holotype WU 

24029. e: WU 24024. g, j, m–n: WU 24019. h: WU 24018. i: WU 24028. k: G.J.S. 98-182. Scale bars: a = 2.7 mm, b–c, e = 2 mm, d, g = 1 mm, 

f = 2.5 mm, h = 0.4 mm, i = 0.5 mm, j = 0.2 mm, k = 30 µm, l = 10 µm, m, o = 15 µm, n = 35 µm. 

j 

o 

157


Fig. 5. Hypocrea/Trichoderma viridescens anamorph from CMD. a. pustule. b–k. Conidiophores; arrows show examples of percurrently proliferated 

phialides. b–c. “Type 1” conidiophores more or less typical of Trichoderma. d. Conidiophore with “Type 1” branching and typical Trichoderma 

phialides and one branch with elongated and percurrently proliferated phialides. g. showing long submoniliform chains of proliferated phialides. 

i–k. showing branching typical of the original concept of Eidamia viridescens; vesicles and proliferated phialides in k very similar to the original 

illustration of E. viridescens. l. Intercalary phialide shown at i. a from G.J.S. 92-11; b, k from ATCC 32630; c from G.J.S. 99-3; d from G.J.S. 98- 

129; e–f, h from CBS 333.72; g, j from G.J.S. 05-115; i from G.J.S. 98-192; l from G.J.S. 99-142. Microscopy: a. stereo. b, e–g, i–j phase contrast. 

d, h, k–l DIC. c fluorescence. Scale bars: a = 0.5 mm; b–g, j, k = 20 µm; h–i, l = 10 µm. 

158


Fig. 6. Hypocrea/Trichoderma viridescens anamorph. a–h. from SNA. a–b. Pustules. c. A long terminally fertile conidiophore extending beyond 

the surface of the pustule. d–g. Conidiophores typical of “Type 2,” Trichoderma branching and phialides. h. inercalary phialide. i–m. Conidia, 

showing variation in shape; in i surface view in optical section. i–k from SNA; l–m from CMD. n. Chlamydospores on CMD. Microscopy: a–d 

stereo. e–g phase contrast. h–m DIC. a–b from G.J.S. 05-115; c–d, g–i, l–m from CBS 433.34 (ex-type of Eidamia viridescens); e–f from G.J.S. 

04-232; j from G.J.S. 05-115; k from G.J.S. 99-142. Scale bars: a = 1 mm; b–d = 0.5 mm; e–g, n = 20 µm; h –m = 10 µm. 

159


band, or developing in indistinct concentric rings; 

after ca. 6–7 d (not always) followed by the development 

of loose, polymorphic tufts (Fig. 5a) of ca. 0.2–1.5 

mm diam on thick, thick-walled and verrucose stipes, 

confluent to 3 × 2 mm, sometimes in up to three 

concentric rings or more irregularly disposed, never 

dense or compact; conidial masses light to dark green 

from 5 d onwards, 28A4–5 to 27F5–8, development 

slow, final colour often after 14 d; sometimes conidia 

yellow at first, then becoming green. Inoculation in 

the middle of the plate often resulting in more regular 

distribution of tufts. Conidiophores typically visible at 

the surface of the pustules. 

Conidiophores (Fig. 5b–k) dimorphic, representing 

Types 2 and 3 (described on page 144), the two types 

typically not forming in the same culture (but see e.g. CBS 

333.72 or CBS 119322 with both); conidiophores of both 

types found on CMD and PDA, Type 2 conidiophores 

alone found on SNA. Conidiophores of Type 2 (Fig. 6) 

more or less typical of Trichoderma, conidiophores at 

the surface of the pustule projecting from the pustule, 

often with a sterile stipe and a few terminal branches or 

only few phialides produced at the tip, or conidiophores 

branched along the length with fertile branches widely 

spaced; conidiophores forming toward the interior 

of the pustule tending to have a discernable straight 

to sinuous main axis with progressively longer fertile 

branches tending to be paired and arising at right 

angles to the main axis, branches terminating in 1–3 

phialides in a cruciate whorl. Phialides from Type 2 

conidiophores lageniform, slightly swollen in the middle 

when more crowded, straight (Fig. 6d–g), less frequently 

sinuous or hooked, phialides sometimes appearing to 

be proliferated percurrently. Conidiophores of Type 3 

(Fig. 5) developing in many PDA and CMD cultures, 

main axis barely or not discernable, branching at acute 

angles with respect to the parent hypha, branches 

rebranching or not, each branch terminating in 1 or 2 

phialides; phialides appearing to proliferate percurrently 

(5f–i, k), often resulting in a submoniliform chain of 2–6 

cells (Fig. 5f, g), the terminal cell of which is a phialide; 

phialides and subtending cells distinctive in being 

swollen in the middle and more or less conspicuously 

constricted above and below the middle, phialides 

sometimes with a long, cylindrical neck. Conidiophores 

often arising from variously distorted and swollen 

cells in pustules (Fig. 5 j, k); submoniliform chains of 

proliferated phialides are often conspicuous in older 

pustules. Phialides measured from both types of 

conidiophores, the longer phialides found on Type 2 

conidiophores, (4.8–)7.2–11.5(–20.5) μm long, (1.5– 

)2.5–3.5(–5.0) μm at the widest point, (1.0–)1.5–2.5(– 

4.0) μm wide at the base, L/W (1.4–)2.2–4.5(–10.4) (n 

= 1125); arising from a (1.1–)2.1–3.3(–5.0) μm wide 

cell (n = 1064). Conidia (Fig. 6h–m, 8f–h) subglobose 

to ovoidal to ellipsoidal, warted. Intercalary phialides 

present but not common on all media (Fig. 5i, 6h). 

Chlamydospores (Fig. 6n) present or absent, terminal 

or intercalary and subglobose, (2.5–)8–12(–18) μm 

diam (n = 390). 

On SNA (Fig. 7 l; 25 °C) colonies similar to CMD, thin, 

hyaline, hyphae loosely arranged radially, no zonation, 

160 

aerial hyphae and coilings often more pronounced, 

chlamydospores (mostly intercalary and angular to 

ellipsoidal) more frequent than on CMD, no odour and 

no pigment formed. Conidiation (Fig. 6a–g) first effuse 

on long aerial hyphae in proximal and central parts 

of the colony, spreading across the whole plate, then 

condensed into small loose tufts or pustules up to ca. 1 

mm diam, confluent to 6 × 4 mm at the proximal margin, 

becoming green from 5 d onwards, later also in a ring 

at the margin, more effuse, pustules becoming dark 

green (27F4–8) after ca. 2 wk. At 30 °C autolytic activity 

conspicuous and submoniliform terminal branches of 

conidiophores more frequent. 

Habitat: Anamorph isolated from bark, decorticated 

wood, mushroom compost, paper board, apple core, 

root of Pseudotsuga menziesii infected with Phellinus 

weirii, leaf litter; isolated as an endophyte from trunks 

of Fagus sylvatica. Stromata forming on wood, more 

rarely on bark of Picea abies and Fagus sylvatica, 

less commonly on Corylus avellana, Salix caprea, 

or overgrowing fungi such as resupinate polypores, 

mostly on cut and usually at least partly decorticated 

branches and logs 2–100 cm thick, stored in piles, 

lying on bare ground, in grass, in leaf litter, on acidic 

or calcareous soils, mostly in open forests, at shady 

roadsides, associated with Ophiostoma spp., Nectria 

fuckeliana, Diatrype stigma s.l., Chlorociboria (green 

wood), Capronia cf. pilosella, Hypocrea lixii, Ascocoryne 

sarcoides, Corticium roseum, Hypoxylon sp.; usually 

on little to medium-decomposed, often hard wood. 

Distribution: Common, especially as anamorph, in 

north- and south-temperate regions: Europe (Austria, 

Czech Republic, France, Germany, England, Northern 

Ireland, Russia, Sweden) and North America (U.S.A.: 

Oregon, North Carolina, Georgia; Mexico); Australia, 

New Zealand; found also in Taiwan and Japan. 

Holotype of teleomorph: Austria, Kärnten, Völkermarkt, 

Eisenkappel-Vellach, Vellacher Kotschna, MTB 9653/1, 46°24‘02‘‘ N, 

14°34‘06‘‘ E, elev. 970 m, on split and partly decorticated branch of 

Fagus sylvatica, 7–8 cm thick, on the ground, soc. Corticium roseum, 

31 Oct. 2005, H. Voglmayr & W. Jaklitsch, W.J. 2881 (WU 24029; 

culture CBS 119321 = C.P.K. 2140). 

Epitype designated here: C.P.K. 2140 deposited as a dry culture 

together with the holotype of H. viridescens as WU 24029a. 

Neotype of Eidamia viridescens, dried culture of original strain CBS 

433.34 (herb. CBS 7868), isolated from rotten apples, U.K. 

Additional teleomorphic specimens examined: Australia, Victoria, 

vic. Healesville, Toolangi State Forest, Myer’s Creek Rd., Wirrawalla 

Rainforest Walk and Myrtle Walking Track in Myrtle Gully, elev. 575 

m, with Nothofagus cunninghamii, Eucalyptus regnans and tree 

ferns, 23 Aug. 1999, on bark of recently dead tree, G.J.S. 8600 (BPI 

746813; culture G.J.S. 99-142); between Yarram and Traralgon, 

Tarra Valley, Tarra-Bulga National Park, Tarra Rainforest Walk, 

elev. 250 m, with Eucalyptus spp., Nothofagus cunninghamii and 

tree ferns, 22 Aug. 1999, on Nothofagus cunninghamii infected with 

Hypoxylon sp., G.J.S. 8568 (BPI 746781; culture G.J.S. 99-175); 

between Yarram and Traralgon, Tarras Bulga National Park visitor 

center, Forest Trail, in Nothofagus cunninghamii and Eucalyptus 

regnans forest, on bark, G.J.S. 8582 (BPI 746793; culture G.J.S. 

99-128). Austria, Oberösterreich, Grieskirchen, Natternbach, forest 

close to Gaisbuchen, MTB 7548/3, 48°24’39” N, 13°41’40” E, elev. 

580 m, on branch of Fagus sylvatica on leaf litter in spruce forest, 1 

Aug. 2004, H. Voglmayr, W.J. 2553 (WU 24022; culture C.P.K. 2043); 

Steiermark, Liezen, Kleinsölk, walking path between Schwarzensee


Fig. 7. Cultures of H. rufa/T. viride and H./T. viridescens after one wk at 25 °C. a–f. H. rufa/T. viride. a. CBS 101526. b. W.J. 2450. c. G.J.S. 05-104. 

d. Tr 2. e–f. W.J. 2450; all from PDA except e (CMD) and f (SNA). g–l. H./T. viridescens. g. G.J.S. 98-86. h. G.J.S. 99-175. i. G.J.S. 05-185. j. G.J.S. 

98-182. k–l. W.J. 2306; all from PDA except k (CMD) and l (SNA). 

161


Fig. 8. Scanning electron micrographs of conidia of T. viride and T. viridescens. a–e. T. viride. f–h. T. viridescens. a–b. from type specimen (L); 

c–d: G.J.S. 92-15; e: G.J.S. 90-95; f: G.J.S. 92-11; g: G.J.S. 94-11; h: G.J.S. 89-142. Scale bars = 5 µm except b = 10 μm 

162

and Putzentalalm, MTB 8749/1, 47°17’12’’ N, 13°52’13’’ E, elev. 

1170 m, on log segment of Picea abies, 100 cm thick, in grass, soc. 

Nectria fuckeliana, 6. Aug. 2003, H. Voglmayr & W. Jaklitsch, W.J. 

2306 (WU 24018; culture CBS 119324 = C.P.K. 942); (Ost-)Tirol: 

Lienz, Defereggental, Hopfgarten in Defereggen, in Dölsach, at 

roadside between the current transformer and the beverage depot, 

MTB 9041/3, 46°55’23’’ N, 12°32’41’’ E, elev. 990 m, on stored log 

of Picea abies, 16 cm thick, in grass, 4. Sep. 2003, W. Jaklitsch, 

W.J. 2374 (WU 24019; culture C.P.K. 947); Vienna, 23 rd district, 

Maurer Wald, MTB 7863/1, 48°08’57” N 16 14’50” E, elev. 360 m, 

on decorticated branch of Carpinus betulus on the ground, in mixed 

deciduous forest, soc. Tubeufia cerea, 3 Oct. 1998, W. Jaklitsch, 

W.J. 1223 (WU 24009, BPI 747557; culture G.J.S. 98-182 = CBS 

120067). France, Haute Garonne, Bord de la Garonne, 31000 

Toulouse, on bark of Carpinus sp., 11 Dec. 1994, J.F. Magni, comm. 

F. Candoussau (BPI 737860; culture G.J.S. 94-118 = IMI 374788); 

Pyrénées Atlantiques, Isle de Sauveterre de Bearn, elev. 100 m, on 

bark of recently dead tree, 25 Oct. 1998, G.J.S. & F. Candoussau 

(BPI 748308; culture G.J.S. 98-129 = CBS 101928). Germany, 

Baden-Württemberg, Freiburg, Landkreis Schwarzwald-Baar-Kreis, 

Furtwangen, shortly before Kaltenherberg coming from Gasthof 

Thurner, MTB 8015/1, 47°59‘36‘‘ N, 08°10‘50‘‘ E, elev. 1000 m, on 

mostly decorticated cut logs of Picea abies, 20–40 cm thick, in a 

pile in grass at road side, part with white mould, 2 Sep. 2004, W. 

Jaklitsch & H. Voglmayr, W.J. 2664 (WU 24023; culture C.P.K. 2138); 

Freiburg, Landkreis Breisgau-Hochschwarzwald, St. Märgen, shortly 

after Glashütte, coming from Hexenloch, on the right side of the road 

close to a bridge, MTB 8014/2, 47°59’37’’ N, 08° 07’32’’ E, elev. 750 

m, on mostly decorticated cut branch of Picea abies, 4 cm thick, on 

very moist ground, 2 Sep. 2004, H. Voglmayr & W. Jaklitsch, W.J. 

2665 (WU 24024; culture C.P.K. 2044); Freiburg, Landkreis Lörrach, 

Todtnau, at the crossing to St. Blasien, MTB 8113/4, 47°48’11’’ N, 

07°56’01’’ E, elev. 490 m, on mostly decorticated cut logs of Picea 

abies, up to 35 cm thick, in pile, wet, in grass, soc. old Ophiostoma 

sp., white mould, 3 Sep. 2004, W. Jaklitsch & H. Voglmayr, W.J. 2670 

(WU 24025; culture CBS 119323 = C.P.K. 2045); Bavaria, Starnberg, 

Tutzing, Erling, at the Hartschimmel terrain, 47°56’41’’ N, 11°10’37’’ E, 

elev. 700 m, on partly decorticated branch of Fagus sylvatica, 13–15 

cm thick, on the ground deep in leaf litter, 3 Sep. 2005, W. Jaklitsch, 

W.J. 2838 (WU 24028; culture C.P.K. 2139). Sweden, Stockholms 

Län, Nothamn, mixed forest at the coast, MTB 4179/3, 60°01’42’’ N, 

18°50’46’’ E, elev. 10 m, on corticated branch of Corylus avellana, 2– 

2.5 cm thick, in moss in mixed forest of Picea abies, Pinus sylvestris, 

Betula pendula, Corylus avellana, Sorbus aucuparia, soc. Diatrype 

stigma s.l., 7 Oct. 2003, W. Jaklitsch, W.J. 2447 (WU 24020; culture 

C.P.K. 983); Uppsala Län, Sunnersta, forest opposite the virgin forest 

Vardsätra Naturpark across the main road, MTB 3871/2, 59°47’24’’ N, 

17°37’51’’ E, elev. 15 m, on cut branch of Salix caprea, 7.5 cm thick, 

on bare, very moist soil in mixed forest, soc. Capronia cf. pilosella, 8 

Oct. 2003, W. Jaklitsch, W.J. 2453 (WU 24021; culture C.P.K. 985). 

United Kingdom, Devon, Exeter, Stoke Woods, close to the parking 

place Forest Walks, SX919959, 50°45’10” N, 03°31’54” W, elev. 30 

m, on mainly corticated branch of Fagus sylvatica, 4 cm thick, on 

the ground in leaf litter, 8 Sep. 2004, H. Voglmayr, W. Jaklitsch & J. 

Webster, W.J. 2686 (WU 24026; culture C.P.K. 2046); North East 

London, Epping Forest, between Robin Hood Roundabout and Hill 

Wood, 43–34/1, 51°39’15” N, 00°02’13” E, elev. 40 m, on branch 

of Fagus sylvatica on the ground in leaf litter, soc. and partly on a 

resupinate polypore, soc. Hypocrea lixii, Ascocoryne sarcoides, 

Diatrype decorticata, 16 Sep. 2004, H. Voglmayr & W. Jaklitsch, 

W.J. 2723 (WU 24027; culture CBS 119322 = C.P.K. 2047). United 

States, North Carolina, Macon Co., Nantahala National Forest, W of 

Franklin, Wayah Bald, elev. 5340 ft., 26 Sep. 1989, on decorticated 

wood, G.J.S., C.T. Rogerson, W.R. Buck, R.C. Harris (BPI 744478, 

culture G.J.S. 89-142 = CBS 120065). 

Anamorphic isolates studied: See Table 1. 

Notes: The teleomorph of H. viridescens is usually 

associated with its anamorph, with dark green conidia 

and sometimes showing citrine- to sulphur-yellow hairy 

patches as in H. rufa. Culture CBS 433.34 is the extype 

culture of E. viridescens and a dried specimen of 

this culture thus becomes neotype (herb. CBS 7868) 

in the absence of an original specimen. Figs 5 and 6 in 

the original publication represent the species well. 

Separation of H. viridescens and H. rufa can be 


difficult. These species are sympatric in teleomorph 

and anamorph, although we have not seen stromata 

of H. rufa from Australia or New Zealand, where 

stromata of H. viridescens are common in Nothofagus 

forests. Many collections of H. viridescens produce 

distinctly ellipsoidal conidia, a form not found in H. rufa. 

Phialides of H. rufa are often solitary and hooked to 

sinuous, and conidiophores lack a discernable main 

axis, and are also usually distinctly curved to sinuous, 

whereas conidiophores of T. viridescens observed from 

SNA, and often also on CMD, tend to be more typical 

of Trichoderma, with paired branches that increase in 

length with distance from the tip. Phialides in pustules 

of H. rufa do not proliferate percurrently, a common and 

distinctive feature of H. viridescens. A coconut odour is 

typical of T. viridescens but unusual in T. viride. Conidia 

of T. viridescens are lighter green and less coarsely 

tuberculate than those of T. viride. Downy immature 

stromata are more rusty, fresh colour is less variable 

and usually darker reddish brown than in H. rufa. 

3. Hypocrea vinosa Cooke, Grevillea 8: 65 (Dec 1879) 

[non Pat., 1881]. MycoBank MB176917. Figs 9–11, 16. 

Anamorph: Trichoderma vinosum Samuels, sp. nov. 

MycoBank MB445737. 

Trichodermati viridescenti simile, sed crescens tardius in agaro PDA, 

conidia uniformiora et ascosporae minores. 

Holotypus Hypocrea vinosa: Waitaki 307 (K, herb. Cooke), epitypus 

designated here: PDD 88476. Holotypus Trichoderma vinosum: 

Cultus in agaro siccus G.J.S. 8702 (PDD 88476). 

Stromata when dry (Fig. 15f-i) 1–2 mm diam, 0.5–1.5 

mm thick, unchanged or slightly darkened in 3 % 

KOH; solitary or in groups of 3 or fewer; tuberculate 

to pulvinate, broadly attached, often erumpent through 

bark cankers or lenticels; at first almost white to 

yellowish brown or light brown, becoming darker brown 

to reddish brown (7–8D–E8); stroma surface scurfy 

due to short, rust-coloured hairs, plane to wrinkled or 

tubercular from perithecial contours; ostiolar openings 

not visible or less frequently appearing as few viscid 

dots. 


(Fig. 9a) pseudoparenchymatous, (2–)3–8(–10) × 2–6 

(–8) μm (n = 90), walls slightly thickened, often with 

uneven deposit of pigment and surface appearing 

mottled, cells of stroma surface often obscured by 

hairs. Hairs (Fig. 9b, e) often abundant, especially on 

young stromata, cylindrical, unbranched, comprising 

2–4 cells, 10–25(–35) μm long, 3–5 μm wide. Cortical 

region (Fig. 9d-g) of stroma 20–40(–55) μm (n = 10) 

thick, reddish brown to yellow-brown, around the entire 

stroma except the point of attachment, cells in section 

of textura angularis, (2.0–)2.5–6.5(–10.2) × (1.0–)1.7– 

5.2(–8.5) μm (n = 90), walls thickened. Cells immediately 

below the cortex (Fig. 9e–g) comprising intertwined 

hyphae and textura epidermoidea to t. angularis toward 

the ostiolar canal. Ostiolar canal (Fig. 9d, g) ca. 90 

μm long; the ostiolar opening formed by a palisade of 

narrowly clavate, hyaline filaments even with stroma 

surface. Perithecia (Fig. 9c–d) elliptical in section, 150– 

190 μm high, 90–100 μm diam. Subperithecial tissue 

163


Fig. 9. Hypocrea vinosa/T. vinosum teleomorph. a. Cells at surface of stroma in face view. b. Short hairs arising from the surface of the stroma. 

c. Longitudinal section through a stroma. d. Median, longitudinal section through a perithecium. e. Section through the upper part of a stroma 

showing short hairs arising from the surface, a pigmented cortical area and intertwined hyphae below. f–g. Ostiolar region of a perithecium. h. 

cells of the interior of a stroma below a perithecium. i– j. Asci and ascospores; a thin ring can b seen in j. All from G.J.S. 02-54. Scale bars: a–b, 

e–i = 20 μm; c = 200 μm, d = 100 μm, j = 10 μm. 

164

homogeneous, of textura angularis to t. epidermoidea 

with some longer hyphal elements, cells (2.7–)4.5–14 

(–23.5) μm long, (1.5–)3.7–7.7(–10) μm wide (n = 90), 

walls slightly thickened. Asci (Fig. 9i) cylindrical, 63– 

103(–123) × (3.5–)4–6(–8) μm, sessile, apex with an 

obscure ring. Ascospores (Fig. 9j) hyaline, verruculose; 

distal part subglobose, (3.7–)5.0–6.5(–8.0) × (3.7–)4.7– 

6.2(–7.7) μm (n = 90); proximal part wedge-shaped 

to almost oblong or ellipsoidal, (4.5–)5.0–7.0(–9.0) × 

(3.7–)4.0–5.0(–8.0) μm (n = 90). 


on PDA and SNA 20–25 °C, after 72 h in darkness 

colony radius on PDA 20–35 mm, on SNA 16–23 mm; 

at 30 °C < 5 mm; not growing at 35 °C. Colonies grown 

on PDA for 1 wk at 25 °C under alternating light and 

darkness producing white mycelium; sterile, conidia 

forming within 3 wk in compact pustules at the periphery 

of the colony; no diffusing pigment or distinctive odour 

noted. 

Colonies grown on CMD reaching > 7 cm radius 

within 10 d at 20–25 °C under alternating light and 

darkness, no pigment or a pale yellow diffusing pigment 

formed; typically producing a coconut-like odour; 

conidia forming in poorly developed to compact, 1–3 

mm diam pustules, at the periphery of the colony or in 

2 or 3 concentric rings, grey-green (27D4–5) or darker 

green. 

Colonies grown on SNA reaching > 7 cm radius 

within 10 d at 20–25 °C under alternating light and 

darkness, no diffusing pigment formed, no distinctive 

odour detected; conidial production variable, abundant 

to scant, in 1–2 mm diam, hemispherical to pulvinate 

pustules formed in a broad band around the margin 

and adjacent to filter paper; conidia dark green; fertile 

conidiophores protruding terminally from pustules or 

not. 

Conidiophores dimorphic, representing Types 2 

and 3 (described on page 144), the two types typically 

not forming in the same culture; conidiophores of both 

types found on CMD (Fig. 10a–g), Type 2 conidiophores 

alone found on SNA. Conidiophores on SNA (Fig. 11c–i) 

lacking a well-developed main axis, or main axis short; 

main axis often nodose, branches tending to be paired 

and separated by short internodes; branches tending 

to produce a profusion of phialides directly or at the tip 

of short secondary branches; branches and phialides 

tending to arise at right angles with respect to their 

subtending cells. Protruding conidiophores unbranched 

or sparingly branched along the length, producing one 

or few cylindrical phialides at the tip. Phialides narrowly 

lageniform, more swollen in the middle when crowded 

than when widely spaced, straight, (6.0–)7.7–11(–14.5) 

μm long, (2.0–)2.2–3.0(–3.7) μm at the widest point, 

(1.2–)1.5–2.2(–2.7) μm wide at base, L/W = (0.4–)2.4– 

4.6(–6.5) (n = 90). Intercalary phialides not common. 

On CMD Type 3 conidiophores forming in compact 

pustules, comprising vesiculose cells branching in an 

irregular fashion, each branch terminating in one or a 

few phialides; phialides lageniform to greatly swollen, 

often proliferating percurrently to form a phialide, often 

submoniliform chains of proliferated phialides (Fig. 


10c–g) found in pustules. Conidia subglobose, warted 

(Fig. 10h–i). Chlamy-dospores not observed. 

Habitat: On corticated hardwood trees including 

Nothofagus menziesii. 

Distribution: Australia and New Zealand. 

Holotype of teleomorph: New Zealand, Waitaki, [?Berggren] 307 

(K, herb. Cooke). Epitype, designated here, PDD 88476. Holotype 

of anamorph (Trichoderma vinosum): a dried culture of G.J.S. 8702 

deposited as PDD 88476. 

Additional specimens examined: Australia, New South Wales, Blue 

Mountains, Morton National Park, vic. Bundnadoon, Fairy Bower 

Track, on bark of recently dead tree, 19 Aug. 1999, G.J. Samuels 

8742, K. Põldmaa, E. Lieckfeldt (BPI 746681; culture G.J.S. 99-156 

= ICMP 16293); Blue Mountains National Park, Grand Canyon from 

Evans Lookout, elev. 850 m, on bark of recently dead tree, 16 Aug. 

1999, G.J.S. 8739 & K. Põldmaa (BPI 746677; culture G.J.S. 99-183 

= ICMP 16289). New Zealand, South Westland, Jackson River Rd., 

Martyr River, roadside facing mixed Nothofagus/broad-leaf forest, 

44°06’ S, 168°32’ E, 7 May 2002, S.R. Pennycook (BPI 842436; 

culture G.J.S. 02-54 = CBS 119086 = ICMP 16295); Westland, Lower 

Buller River Gorge, “Sinclair’s Castle”, at point where Ohikanui 

River joins the Buller River, along floodplain of Ohikanui River, in 

Nothofagus and tree ferns, elev. 50 m, 41°51’ S, 171°43’ E, 6 Sep. 

1999, G.J.S. 8702 & S. Dodd (PDD 88476, epitype designated here, 

isoepitype BPI 746637; culture G.J.S. 99-158 = CBS 119087 = ICMP 

16294). 

Notes: As was noted by Cooke (1879), H. vinosa differs 

from H. rufa in having slightly larger ascospores. The 

type specimen of this species is in poor condition. It 

consists of three pieces of decorticated wood with 

stromata on one of them. The stromata are dark reddish 

brown, discrete, discoidal, 1–2 mm diam, broadly 

attached to the substratum, with a slight tendency to 

have free margins; the surface is plane, perithecial 

contours or ostiolar openings are not visible. The stroma 

becomes light orange-brown in KOH. Ascospores are 

hyaline, conspicuously spinulose, dimorphic. Distal 

part-ascospores (n = 10) are globose to subglobose, 

5.0–6.7 × 5.0–5.5 μm; proximal part-ascospores are 

oblong, 5.7–7.2 × 4.5–5.2 μm. 

Webster (1964) attributed a Trichoderma anamorph 

to a New Zealand collection (PDD 10466) and Rifai 

(1969) assigned that anamorph to the T. aureoviride 

aggregate. According to Webster, the specimen that 

he examined fits the type of H. vinosa well. However 

our study of PDD 10466 reveals it to be distinct from 

the type specimen of H. vinosa. PDD 10466 is a poor 

specimen, with only one old stroma. That stroma is 

light brown (ca. 6D8). Ascospores are hyaline and 

dimorphic. Thirty ascospores in asci were measured; 

they are smaller than in the type collection. The distal 

parts are (3.7–)4.0–5.0(–6.0) × (3.2–)3.5–4.2(–5.0) 

μm; the proximal parts are (3.5–)4.0–5.5(–6.3) × 

(2.7–)3.0–4.0(–4.5) μm. The specimen includes a dry 

PDA culture. Conidia are green, ellipsoidal to narrowly 

ovoidal, (3.0–)3.2–3.5(–3.7) × 2.2–2.7 μm, smooth. 

Conidiophores in the dry culture have collapsed but 

we did not observe an eidamia-like development. The 

specimen PDD 10466 was collected in the Auckland 

region of the North Island of New Zealand, which is 

biologically distinct from the type locality of H. vinosa. 

Thus we are convinced that PDD 10466 is not H. vinosa, 

although we cannot identify it to any known species. 

165


Fig. 10. Hypocrea vinosa/T. vinosum anamorph on CMD. a–g. Conidiophores; note vesiculose development suggestive of Eidamia viridescens 

in a, d, f–g; phialides proliferating percurrently to form submoniliform chains in d–f. More or less typical Trichoderma conidiophores in b. h–j. 

Conidia. h. SEM, i. in optical section, j. in surface view. a–b, h from G.J.S. 99-158; c, e, i–j from G.J.S. 99-183; d, f from G.J.S. 99-156; g 

from G.J.S. 02-54. Microscopy: a, i–j. DIC; b–c, e phase contrast; d, f–g fluorescence. Scale bars: a–g = 20 μm; h = 6 μm; i–j = 10 μm. 

166


Fig. 11. Hypocrea vinosa/T. vinosum anamorph on SNA. a–b. Pustules. c–i. Conidiophores. All from G.J.S. 02-54. Scale bars: a = 1 mm, b = 

0.5 mm; c–i = 20 um. 

167


The ellipsoidal conidia and collapsed conidiophores 

are similar to T. koningii, and in New Zealand three 

species having this morphology are known, viz. H./T. 

austrokoningii, H./T. dingleyae and H./T. dorotheae. 

However, conidia of PDD 10466 are smaller than in 

these species. As far as we are aware, the culture 

that Webster obtained from that specimen is no 

longer available. The illustrations provided by Rifai are 

consistent with many species of Trichoderma in sect. 

Trichoderma. Smooth conidia were illustrated. 

The specimen that we have designated as epitype 

also agrees well with the type of H. vinosa. This is 

perhaps not surprising given the relative non-specificity 

of Hypocrea specimens. The type of H. vinosa was 

collected in Waitaki, which is located on the east coast 

of Otago, on the South Island of New Zealand, where 

Nothofagus is common. It is at least possible that the 

type specimen was collected on Nothofagus. The 

epitype and one additional collection were made from 

N. menziesii in Westland, a region on the east coast of 

New Zealand that is slightly north of Otago. Thus, the 

epitype was collected close to the type locality. 

4. Trichoderma gamsii Samuels & Druzhinina, sp. 

nov. MycoBank MB501050. Figs 12–13, 16 a–d. 

Teleomorph: None known 

Etymology: Named to honour K. Walter Gams, peripatetic 

mycologist (fan of Sardinia) and trichodermist. 

Trichodermati koningii simile, sed conidia majora, (3.2–)4.0–5.0(–6.2) 

× (2.0–)2.5–3.0(–3.2) μm. Conidiophora dimorphica, conidiophora 

Trichodermati similia in agaro SNA evoluta; conidiophora ad 

Eidamiam spectantia in agaro CMD evoluta. 

Holotypus: C.P.K. 2074 (dried in BPI 872183; ex-type culture C.P.K. 

2074 = G.J.S. 06-09 = CBS120075). 

Characteristics in culture: Optimum temperature for 

growth on PDA and SNA 25–30 °C in intermittent light; 

after 72 h on PDA colony radius 50–60 mm, on SNA 

colony radius 40–48 mm; at 35 °C < 7.5 mm on PDA 

and SNA. Colonies grown on PDA in intermittent light 

forming conidia sporadically within 1 wk 25 °C. On PDA 

after 1 wk in light typically a dense white mycelium 

covering the 9-cm-diam Petri plate; conidial production 

variable, some colonies remaining sterile and more or 

less conidia forming in other colonies; conidia tending 

to form in 3 broad, often obscure, concentric rings; 

conidia form in a dense, continuous lawn between the 

inoculum plug and the edge of the Petri plate, typically 

a shade of yellow (2–3B–D 8). 

On SNA at 25 °C in light reaching > 7 cm radius 

after 1 wk in light; conidia forming abundantly in (2–) 

3–4 conspicuous concentric rings, 27E5–8, large, 

produced in rather broad sheets or in flat pulvinate, often 

confluent pustules 1–2 mm diam. No pigment diffusing 

through the agar, no distinctive odour. Conidiophores 

(Fig. 13a-g) more or less uniformly branched for a 

short distance, solitary phialides common, internodes 

between branches typically long, occasionally short and 

branches crowded; often branches arising from swollen 

nodes on the conidiophore, occasionally conidiophores 

terminating in a long extension; extension sterile for a 

168 

long distance below the tip, a single phialide forming at 

the tip. Phialides lageniform, at most slightly swollen in 

the middle, straight; solitary or terminating branches in 

pairs or in appressed heads of several, (5.2–)8.5–10 

(–18.5) μm long, (1.5–)2.2–3.0(–4) μm at the widest 

point, (1.0–)1.5–2.2(–2.7) μm at the base, L/W = (1.4–) 

2.4–4.2(–8.4) (n = 300); arising from a (1.0–)1.8–3.0 

(–5.0) μm wide cell. Intercalary phialides common. 

On CMD at 25 °C in light reaching > 7 cm radius 

within 10 d; conidia forming in a broad band of discrete 

to confluent pustules around the colony margin; 

pustules densely cottony, individual conidiophores 

not apparent, 0.5–1.0(–3) mm diam, green (27–28E– 

F5–8) or yellow in part, conidiophores also forming in 

the scantily produced aerial mycelium; less frequently 

colonies remaining sterile; sometimes with a pale yellow 

diffusing pigment; typically with a more or less strong 

coconut-like odour. Colonies grown on SNA with filter 

paper at 25 °C in light > 7 cm radius within 10 d; conidia 

forming as in darkness, conidial production often heavy 

and in larger pustules over the filter paper, at first yellow 

(1A7), then green (27C–F8), pustules cottony to dense 

and individual conidiophores not seen. Conidiophores 

(Fig. 12a–h) comprising convoluted and intertwined, 

2–3 μm wide hyphae from which phialides arise. 

Phialides produced laterally from hyphae and solitary, 

or terminating branches and solitary or held in pairs, 

typically swollen in the middle and the tip elongated 

and cylindrical; percurrently proliferated phialides 

conspicuous, often forming submoniliform chains. 

Chlamydospores typically abundant, subglobose, 

terminal on hyphae, (3.0–)8.5–12(–15.7) × (3.0–)8– 

11.5(–15.5) μm. 

Conidia (Figs 12i, 13h) ellipsoidal, oblong, or narrowly 

ovoidal, smooth. 

Habitat: Soil, isolated once as an endophyte of a tree 

fern and once from the stem of Ricinus communis. 

Known distribution: Italy, common in Sardinia, also 

present in the U.S.A. (Texas), Australia, and Central 

Russia. 

Holotype: Italy, Sardinia, north-east side of the Tyrrhenian island, 

close to Aglientu, isolated by Q. Migheli from soil under Pistacia 

lentiscus and Myrtus communis, C.P.K. 2074 (BPI 872183; culture 

C.P.K. 2074 = G.J.S. 06-09 = CBS 120075). 

Additional cultures examined: Australia, A.C.T., Canberra, Australian 

National Botanic Garden, isolated from xylem of Eucalyptus nitens, 

Aug. 1992, P.J. Fisher D-12-153 (culture G.J.S. 92-60). Italy, Pisa, 

isolated from stem of Ricinus communis, Q. Migheli, G. Vanacci 

899 (BPI 872184; culture G.J.S. 05-111 = CBS 120072); Sardinia, 

data as for holotype (cultures C.P.K. 2070 = G.J.S. 06-07 = CBS 

120073, C.P.K. 2073 = G.J.S. 06-08 = CBS 120074, C.P.K. 2075, 

C.P.K. 2076, C.P.K. 2077, C.P.K. 2078, C.P.K. 2079 = G.J.S. 06-13, 

C.P.K. 2090 = G.J.S. 06-14, C.P.K. 2091, C.P.K. 2092, C.P.K. 2093). 

Russia, Central Forest State Biosphere Reserve, Tverskaya district 

(cultures C.P.K. 1010 and C.P.K. 1011) isolated from terric histosol 

soil type under Piceetum composite forest with Alnus glutinosa. 

United States, Texas, Gaines County, farm soil, C. Howell TK 77 

(culture G.J.S. 04-09). 

Notes: The closest morphological comparison of 

T. gamsii is with T. koningii. The most conspicuous 

difference between the two species is the production of


Fig. 12. Trichoderma gamsii on CMD. a–h. Conidiophores. More or less typical Trichoderma conidiophores in a, e; examples of percurrently 

proliferating phialides shown at arrows. Note submoniliform chains of percurrently proliferating phialides in b, f; thin arrow in d pointing to 

intercalary phialide. i. Conidia. a, d, i from C.P.K. 2077; b from C.P.K. 2078; c, e from G.J.S. 04-09; f from G.J.S. 05-111; g–h from C.P.K. 2073. 

Microscopy: a–h PC, i DIC. Scale bars a–h = 20 μm, i = 10 μm. 

169


Fig. 13. Trichoderma gamsii on SNA. a–g. “Type 2” conidiophores. Intercalary phialide shown in g (arrow). h. Conidia. I. Chlamydospores. a 

from C.P.K. 2073; b–d from G.J.S. 05-111; e, g from C.P.K. 2079; h from C.P.K. 2075; f, i from C.P.K. 2091. Microscopy: a–c, e–g = PC, d, h = 

DIC; i = Bright field. Scale bars: a–g, i = 20 μm, h = 10 μm. 

170


Fig. 14. Trichoderma neokoningii. a–b. Colonies on PDA (a) and SNA (b) after 1 wk at 25 °C, alternating light. c–d. Pustules from SNA. 

e–j. Conidiophores. e. Conidiophore protruding from the surface of a pustule with widely spaced branches and nearly cylindrical phialides. 

f–g. Conidiophore sparingly branched above and densely branched below. h. Percurrently proliferating phialides (arrow). i–j. Eidamia-like 

conidiophores with conspicuous proliferating phialides. k. Intercalary phialide. l. Conidia. m. Chlamydospore. e–h, k–m from SNA; i–j from PDA. 

All from G.J.S. 04-216. Microscopy: c–d = stereo; e, i, k = PC; f–h, j–l = DIC; m = bright-field. Scale bars: a–b in 9-cm-diam Petri dishes, c = 1 

mm, d = 0.5 mm, e–j, m = 20 μm; k–l = 10 μm. 

171


proliferating phialides in T. gamsii on CMD and PDA. 

Trichoderma gamsii has the largest conidium length/ 

width ratio that we have yet seen in Trichoderma. 

Conidiophores of T. gamsii are neither as extensive nor 

as uniformly branched as they are in T. koningii. 

Fisher et al. (1993) recovered T. gamsii (as T. 

koningii) as an endophyte from xylem of Eucalyptus 

nitens in Australia in low frequency. 

The isolate G.J.S. 04-09 (Howell TK 77) is resistant 

to the fungicide Bayton (C. Howell, pers. comm.). 

5. Trichoderma neokoningii Samuels & Soberanis, 

sp. nov. MycoBank MB501051. Fig. 14. 

Etymology: with reference to a similarity to T. koningii 

Oudem. 

Trichodermati koningii Oudem. simile sed conidia in pustulis compactis 

in agaro SNA formata, inconspicue crescens, et conidiophora 

Eidamiae similia in agaro PDA; conidia ellipsoidea, glabra, (3.2–) 

3.5–4.0(–4.5) × (2.7–)3.0–3.5(–4.0) μm. Habitat in America australi. 

Teleomorph: none known. 

Colony characteristics: Optimum temperature for 

growth on PDA 25 °C, on SNA 30 °C; after 72 h in 

darkness colony radius on PDA at 25 °C ca. 40 mm and 

at 30 °C 35 mm, on SNA at 25 °C 30 mm, at 30 °C ca. 

35 mm; not growing at 35 °C. Radius of colonies grown 

on PDA and SNA for 1 wk at 25 °C with alternating 

light ca. 70 mm. Colonies on PDA cottony, white, on 

CMD and SNA aerial mycelium scant, colonies nearly 

invisible; no diffusing pigment or distinctive odour 

noted. Colonies on PDA (Fig. 14a) producing conidia 

in 2–3 mm diam hemispherical pustules in the aerial 

mycelium, especially around the original inoculum, or 

in continuous lawns around the original inoculum and 

at the periphery of the colony. Conidiophores on PDA 

at first of Type 2, but with age becoming Eidamia-like, 

conidiophores short and irregularly branched, swollen 

cells developing in pustules (Fig. 14j), phialides 

abruptly swollen in the middle and the cylindrical neck 

proliferating conspicuously to form chains of 2 or 3 (Fig. 

14i, j). On SNA (Fig. 14b) and CMD large, 2-4 mm diam, 

pulvinate pustules scattered throughout the colony. 

Conidia on PDA at first yellow, becoming greyish green 

(27D–E5), on SNA and CMD deep green to dark green 

(28E–F8). Pustules formed on SNA (Fig. 14c) and 

CMD, densely woolly, conidiophores poorly visible at 

the surface of the pustule. Conidiophores on CMD and 

SNA exclusively of Type 2 (Fig. 14e), often sterile over 

a more or less long distance and producing a single 

terminal phialide or whorl of phialides (Fig. 14g), or 

tending to be regularly branched, with branches tending 

to be paired and increasing in length with distance from 

the tip; branches tending to be widely spaced, each 

branch terminating in a single phialide or in a whorl of 

2 or 3 phialides (Fig. 14e–f). Conidiophores produced 

toward the interior of the pustule more densely branched 

with shorter internodes between branches, branches 

tending to be paired and closely-spaced, terminating in 

1 to several phialides. Phialides produced near the tips 

of conidiophores tending to be solitary, cylindrical or 

slightly swollen in the middle, often constricted above 

172 

the middle to form a long cylindrical neck; phialides 

produced toward the interior of the pustule or on more 

densely disposed branches (Fig. 14i) shorter than 

those produced at the tip and conspicuously swollen in 

the middle, (4.5–)5–10(–13.5) μm long, (2.0–)2.2–3.0 

(–3.2) μm at the widest point, (1.2–)1.5–2.0(–2.2) μm 

at the base, L/W = (1.4–)1.8–4.6(–6.8) (n = 30); arising 

from a (1.5–)2.0–3.0(–4.0) μm wide cell. Percurrently 

proliferating phialides common on SNA (Fig. 14h). 

Intercalary phialides uncommon (Fig. 14f) on SNA or 

CMD. Conidia (Fig. 14k) oblong to ellipsoidal, smooth, 

green. Chlamydospores (Fig. 14 l) abundant on SNA, 

terminal, subglobose, (6.2–)7.5–10.7(–13.7) × (6.0–) 

6.7–9.2(–10.7) μm (n = 30). 

Habitat: Isolated from the pseudostroma of 

Moniliophthora roreri infecting a pod of Theobroma 

cacao. 

Known distribution: Peru, known only from the type 

collection. 

Holotype: Peru, Cuzco, Crialo, isolated from Moniliophthora roreri 

sporulating on cacao pod, date unknown, W. Soberanis ‘D’ (BPI 

872182; ex-type culture G.J.S. 04-216 = CBS 120070). 

Notes. There is little morphological distinction between 

T. neokoningii and T. koningii and T. koningiopsis. The 

latter two species (Samuels et al. 2006a) produce conidia 

abundantly on most media in the aerial mycelium or in 

poorly developed pustules, not in conspicuous pustules 

as they occur in T. neokoningii. Conidiophores of the 

former two species are also more regularly branched and 

do not produce conspicuously percurrently proliferating 

phialides. Trichoderma koningii is a species of northtemperate 

regions whereas T. koningiopsis is commonly 

found in tropical soils. Trichoderma koningiopsis grows 

faster than T. neokoningii on PDA. Although T. koningii 

and T. koningiopsis are members of Trichoderma sect. 

Trichoderma, they are phylogenetically distinct from 

T. neokoningii. Trichoderma gamsii is closely related 

to, and morphologically very similar to T. neokoningii; 

conidia of the common temperate T. gamsii are 

significantly longer than those of T. neokoningii. 

6. Trichoderma scalesiae Samuels & H.C. Evans, sp. 

nov. MycoBank MB501052. Figs 15, 16 e. 

Teleomorph: none known. 

Etymology: With reference to Scalesia pedunculata, 

the host plant from which T. scalesiae was isolated. 

Trichodermati paucisporo simile sed conidia (2.5–)3.0–3.7(–4.0) × 

(2.2–)2.7–3.2(–3.5) μm et inconspicue crescens; coloniae radius 

< 15 mm post 96 horas 25 °C in agaro PDA. 

Holotypus: BPI 872181 


on PDA 30 °C, for growth on SNA 15–30 °C; after 72 h 

in darkness colony radius on PDA ca. 18 mm, on SNA 

ca. 10 mm; not growing at 35 °C. Radius of colonies 

grown on PDA, CMD and SNA for 1 wk at 25 °C with 

alternating light and darkness 30–40 mm. On PDA 

aerial mycelium cottony, white to yellow (ca. 2B7); on 

CMD and SNA aerial mycelium scant and colonies 

nearly invisible; no diffusing pigment or distinctive odour

detected on PDA; no diffusing pigment, a faint coconutlike 

odour detected on CMD; a faint yellow diffusing 

pigment but no distinctive odour detected on SNA. 

Colonies on PDA and CMD sterile, on SNA conidia 

sparingly produced in cottony aerial mycelium over 


the filter paper, none elsewhere. Conidiophores (Fig. 

15a–g) formed in the aerial mycelium, 25–45 μm long, 

monophialidic or sparingly to profusely branched, 

branches arising at or near 90° with respect to the 

main axis, typically not paired, each branch producing 

Fig. 15. Trichoderma scalesiae. a–g. Conidiophores formed in the aerial mycelium at the interface of filter paper and agar. h. Conidia. i. 

Chlamydospores. All from G.J.S. 03-74. Scale bars: a = 0,5 mm, b, f–g = 20 μm, c-e, h, i = 10 μm, d = 0.5 mm; e–j, m = 20 μm; k–l = 10 μm. 

173


Fig. 16. Hypocrea/Trichoderma species. a–d. Trichoderma gamsii. a–b. conidial pustules on SNA (G.J.S. 05-111). c–d. Colonies grown 1 wk at 

25 °C alternating light (G.J.S. 05-111). c. PDA. d. SNA. e. T. scalesiae (G.J.S. 03-74), 1 wk at 25 °C alternating light. f–i. H. vinosa stromata. f. 

From the type collection. g. G.J.S. 99-158; h. G.J.S. 02-54, immature with hairs at stroma surface. i. G.J.S. 99-156, immature. Scale bars: a–b 

= 0.5 mm; f = 1 mm, g–i = 0.5 mm. 

174

one or a few phialides along the length or rebranching, 

each secondary and terminal branch terminating in 1–4 

phialides, a single watery drop of conidia held at the tip 

of each phialide. Phialides slightly swollen in the middle 

or tapering uniformly from base to tip, straight, curved 

or sinuous, (8.7–)10–15(–17) μm long, 2.0–3.0(–4) 

μm at the widest point, (1.5–)2.0–2.2(–2.5) μm at the 

base, L/W = (0.7–)0.8–1.0(–1.3) (n = 30), arising from 

a (1.7–)2.0(–2.5(–3.0) μm wide cell. Conidia (Fig. 15h) 

subglobose, (2.5–)3.0–3.7(–4.0) × (2.2–)2.7–3.2(–3.5) 

μm (n = 30), smooth, pale green. Chlamydospores 

(Fig. 15i) abundant on SNA, terminal and intercalary, 

(7.0–)8.0–8.7(–10.0) × (6.5–)7.7–9.5(–10.5) μm (n = 

30). 

Habitat: Endophytic within woody tissues of Scalesia 

pedunculata (Asteraceae), the “Daisy Tree” of the 

Galapagos islands. 

Holotype: Ecuador, Galapagos Islands, Santa Cruz, Mt. Cocker, 

isolated from stem of Scalesia pedunculata (Asteraceae), 19 May 

2000, H.C. Evans W2022d (BPI 872181; ex-type culture G.J.S. 03- 

74 = CBS 120069). 

Notes: Trichoderma scalesiae produced conidia only 

sparingly and then only on SNA to which sterile filter 

paper squares had been added. In Trichoderma the 

coconut-like odour is typical of sect. Trichoderma; 

thus even in the absence of conidia this species was 

predicted to be a member of that section. Normal 

Trichoderma species produce conidia abundantly on 

common mycological media. In the current work we 

have found that conidial production on CMD and PDA 

can be unreliable, whereas the carbohydrate-poor SNA 

can be relied on to stimulate conidial production. In the 

case of T. scalesiae presumably the cellulose content 

of filter paper was an absolute requirement for conidium 

production. We did not attempt to stimulate conidium 

production on other media by adding filter paper squares 

to them. Most likely, if this species had been seen in the 

past it would have been recorded as being sterile. The 

recently described T. paucisporum Samuels, Suarez & 

Solis, also a member of sect. Trichoderma, is similar to 

T. scalesiae in that it produces conidia sparingly on SNA, 

although it does produce conidia in very low numbers 

and sporadically on CMD and SNA. Filter paper was 

not necessary for conidium production on SNA by T. 

paucisporum. The conidiophores of T. paucisporum are 

morphologically very similar to those of T. scalesiae 

(Samuels et al., 2006). Conidiophores in both species 

are similar to what were described as synanamorphs 

of some Trichoderma species by (Chaverri et al. 2004). 

Trichoderma paucisporum was originally isolated from 

Ecuador, from cacao pods infected with Moniliophthora 

roreri and lying on the ground. 

Scalesia species, which are endemic to the 

Galapagos Islands, are considered to be the plant 

equivalents of Darwin’s finches. Scalesia pedunculata, 

a tree, is the largest of the daisy trees. For information 

concerning S. pedunculata see references found at 

http://www.arkive.org/species/GES/plants_and_algae/ 

Scalesia_penduculata/more_info.html. In addition to T. 

scalesiae, we have also identified T. harzianum Rifai 

as an endophyte in trunks of S. pedunculata (Samuels, 

unpubl.). 

DISCUSSION 


This is the eighth article in our series dealing with 

members of the phylogenetically and taxonomically 

complex Trichoderma sect. Trichoderma (Dodd et al. 

2002, 2003, Druzhinina et al. 2004, Holmes et al. 2004, 

Lieckfeldt et al. 1999, Lu & Samuels 2003, Samuels et 

al. 1999, 2006a, b). 

In the introduction to the current work we suggested 

that reports of T. viride refer to more than one species. 

We have shown that even the classical morphological 

concept of T. viride, i.e. a green-conidial species with 

globose, warted conidia, is paraphyletic. In the present 

paper we distinguish the two most common species 

of Trichoderma that have warted conidia and describe 

a new species that has warted conidia, T. vinosum. 

We augment the known diversity of morphological 

expression in Trichoderma to include the peculiar 

Eidamia morphology. During the course of the study, 

additional species having warted conidia or that were 

closely related to T. viride or T. viridescens were revealed 

to us. In addition we describe a second species, T. 

scalesiae, that but for DNA sequence analysis and 

careful examination of cultures on SNA, would be 

reported as a sterile unknown fungus. Although we 

present results from sequencing of only a single 

gene, tef1, the resulting clades conform to phenotypic 

apomorphies in defining species. Clearly, from Fig. 

1 it can be seen that additional species remain to be 

characterized. These will be discussed in forthcoming 

publications. 

In the present work, as in the accompanying article 

in this issue that concerns T. koningii (Samuels et al. 

2006a), the question that we have had to answer was 

how to delimit taxonomic species. As we continue 

to collect new specimens and submit them to DNA 

sequencing and phylogenetic analysis, we find 

within clades considerable homoplasy in the few 

morphological features that are available for analysis. 

Despite the formation of a teleomorph by several 

species, this morph is so highly conserved within the 

viride clade as to be virtually useless in species-level 

taxonomy while at the same time being diagnostic 

of the clade. Similarly, while the basic conidiophore 

morphology in the viride clade, defined here as “Type 

2”, signals membership in the clade, it varies only little 

among the species. For this reason, one cannot fault 

earlier mycologists for having recognized few species 

of Trichoderma, or even later mycologists for having 

failed to recognize the differences between T. viride 

and T. viridescens. In both the T. viride and T. koningii 

works our strategy for recognition of taxonomic species 

was to closely integrate phenetic and phylogenetic 

characters. These two analyses were undertaken 

independently in two laboratories, and therefore 

were unbiased. Species were recognized when the 

concordance between both approaches were found. 

Although we clearly understand that the phylogeny of 

one single locus, in this case tef1, is useful to formulate 

a hypothesis concerning the phylogeny of the fungus, 

we also understand that use of this single gene region 

is not powerful enough to falsify a species hypothesis. 

175


Thus, we rely on the concordance between phenotype 

and genotype. It is also important to note that for 

some groups the high observed phylogenetic diversity 

enabled us to recognize a species (e.g. T. gamsii), 

while in other cases, when the diversity was low and 

both sets of characters were unclear, we refrained from 

proposing a new taxonomy (Vd 1–3). 

The formation of warted conidia in the viride 

clade seems to suggest that this character is derived 

within Trichoderma and that smooth conidia are the 

primitive state. However, we are not yet in a position 

to discuss evolution of most phenotypic characters 

in Trichoderma, and conidium ornamentation is not 

an exception. Tuberculate conidia are produced by 

completely unrelated species of Trichoderma, including 

T. saturnisporum and T. ghanense (both sect. 

Longibrachiatum, Samuels et al. 1994), and Bissett 

(1991b) illustrated warted conidia in T. virens (sect. 

Pachybasium) using scanning electron microscopy. 

On the other hand the eidamia-like morphology of 

conidiophores produced on rich media is apomorphic 

for the large viridescens clade defined here, and the 

coconut-like odour produced by several members of 

the entire viride clade. 

The level of knowledge of Trichoderma species is 

perhaps greater than for any other genus of fungi. We 

acknowledge the difficulty of identifying Trichoderma 

species on the basis of their phenotype, despite the 

fact that morphology-based keys to species, and 

illustrations of at least the most common species 

is available at www.nt.ars-grin.gov. However, every 

species is represented in GenBank by sequences of 

two or more genes and by a multiplicity of correctly 

identified strains (Samuels 2006). Moreover, all 

known species of Trichoderma may be identified using 

molecular markers at www.isth.info. The majority of 

species can be safely identified by the DNA barcoding 

based on ITS1 and 2 loci (Kopchinskiy et al. 2005). 

However, since species from Trichoderma section 

Trichoderma share the same or very similar alleles of 

ITS1 and 2, they should be identified by an integration 

of the barcode method (TrichOKEY) and Trichoderma 

similarity search TrichoBLAST (Kopchinskiy et al. 

2005) as described in Druzhinina et al. (2006). If a 

researcher has access to a sequencing facility, there 

is no need to misidentify a species of Trichoderma. We 

strongly recommend that individuals check the identity 

of strains that are of interest and we urge editors of 

journals reporting properties of Trichoderma species 

to require that the identity of the strains be verified 

by members of the International Subcommittee on 

Taxonomy of Hypocrea (ISTH) which can be accessed 

at www.isth.info. 

ACKNOWLEDGEMENTS 

We thank Hermann Voglmayr for the collection of Hypocrea 

teleomorphs, Svengunnar Ryman and Roland Moberg for support 

of excursions in Sweden. Drs Moberg and Hennig Knudsen were 

instrumental in identifying type material of Sphaeria rufa. Cultures 

were provided by Drs John Bissett (DAOM), Charles Howell (USDA), 

Toru Okuda (Nippon Roche), Walter Gams (CBS), Willies Soberanis 

176 

(Tarpoto, Peru), Helgard Nirenberg (BBA, Berlin), and Doustmorad 

Zafari (Bu Ali SIna University, Hamadan, Iran). Dr. Adnan Ismaiel 

(BPI) and Mag. Monika Komon-Zelazovska obtained many 

sequences from Trichoderma cultures. Ms Ellen Bloch (NY) patiently 

located and expedited the loan specimens of Hypocrea. Mr James 

Plaskowitz (BPI) and Dr Eric Erbe (USDA, Beltsville), respectively, 

performed the electron microscopy. A.T. Gräfenhan kindly reviewed 

an earlier version of this paper. The financial support by the Austrian 

Science Fund (FWF Project P16465-B03) to W.M.J. is gratefully 

acknowledged. This study was supported in part by the United States 

National Science Foundation (PEET) grant 9712308, “Monographic 

Studies of Hypocrealean Fungi: Hypocrea and Hypomyces” to the 

Pennsylvania State University, Department of Plant Pathology. 

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