Hypocrea lixii, the teleomorph of Trichoderma harzianum

Abstract

Cultures derived from ascospores of Hypocrea lixii (= H. nigricans, H. lentiformis) produced the morphological species Trichoderma harzianum in pure culture. Trichoderma harzianum, the most commonly found species of the genus, is also one of the most species frequently used in biocontrol of plant pathogens.
It has not been connected previously to a teleomorph. The connection was confirmed by DNA sequence analysis. Similar to the
anamorph, the teleomorph collections have a wide geographic distribution. Described in the 19th century, Hypocrea lixii is epitypified by a collection from Thailand.

Full text

richoderma harzianum Rifai (Ascomycota, Hy-
pocreales, Hypocreaceae) is a cosmopolitan species
that is found in a variety of substrata. This species
is known for its effectiveness in biocontrol of plant-pathoge-
nic fungi, specially soil-borne diseases (WELLS et al. 1972,
AL-HEETI & SINCLAIR 1988, YANG et al. 1995, BAILEY &
LUMSDEN 1998, MATHEW & GUPTA 1998). In addition, T. har-
zianum has been reported to have potential in the enhancement
of plant growth and resistance to plant pathogens (BAILEY &
LUMSDEN 1998, GROMOVICH et al. 1998).
Cultures derived from ascospores of fresh collections of
Hypocrea lixii Pat. produced the morphological species T.
harzianum in pure culture. In an unpublished study, H. lixii
isolates were shown to group with isolates of T. harzianum
based on phylogenies of four genes, translation elongation fac-
tor-1α, calmodulin, actin and ITS rDNA, and morphological
and cultural data (CHAVERRI et al. 2002). Despite the fact that
T. harzianum is genetically diverse, several phylogenetic li-
neages can be found that include cultures from specimens of
H. lixii. Thus, there is no doubt of the genetic link between the
two morphs.
Hypocrea lixii was originally described on the basis of a
single collection from a rotting Ganoderma basidioma in Pa-
pua New Guinea (PATOUILLARD 1891). It is morphologically
indistinguishable from the type collection of H. lentiformis
Rehm, described from leaves of a palm (Euterpe sp.) in sou-
thern Brazil (REHM 1898) and from a paratype collection of
H. nigricans f. octospora Doi (1972), which was collected
in the western Pacific island of New Britain, also on decaying
palm leaves. Although we have not been able to study type
material of H. nigricans f. nigricans (Imai) Doi, the descrip-
tion of the anamorph and teleomorph of this species provided
by DOI (1972) and DOI & DOI (1980) is morphologically con-
sistent with T. harzianum and H. lixii. We have collected many
specimens from New World and Asian tropical regions as well
as from North America and Europe that agree with type ma-
terial of H. lixii.
The purpose of the present paper is to redescribe and epi-
typify H. lixii.
Material and methods
The majority of the specimens examined in this study were
collected and deposited in the BPI culture collection. In addi-
tion, herbarium specimens were studied from BPI, FH, HBG,
NY. Cultures were obtained from single-ascospore isolations
from fresh specimens of Hypocrea lixii. The herbarium spe-
cimens of Hypocrea were rehydrated briefly in 3% KOH. Re-
hydrated stromata were supported by Tissue-Tek O.C.T.
Compound 4583 (Miles Inc., Elkhart, Indiana) and sectioned
at a thickness of approx. 15 µm with a freezing microtome.
Thirty-four teleomorph characteristics were evaluated, inclu-
ding: substratum; diameter, height, color and shape of the stro-
ma; texture of surface of the stroma; perithecium shape, length
and width; reaction to 3% KOH, color and width of perithe-
cium wall; ostiolar canal length; thickness, color and 3% KOH
reaction of stroma outer region; shape, length and wall thick-
ness of cells of the outer middle (immediately below the outer
region) and inner region (below perithecia) of the stroma; as-
cus length and width; distal and proximal part-ascospore
length and width. Measurements of continuous characters
Hypocrea lixii, the teleomorph of Trichoderma harzianum
Priscila CHAVERRI1and Gary J. SAMUELS2
Cultures derived from ascospores of Hypocrea lixii (= H. nigricans, H. lentiformis) produced the morphological species
Trichoderma harzianum in pure culture. Trichoderma harzianum, the most commonly found species of the genus, is also
one of the most species frequently used in biocontrol of plant pathogens. It has not been connected previously to a teleo-
morph. The connection was confirmed by DNA sequence analysis. Similar to the anamorph, the teleomorph collections
have a wide geographic distribution. Described in the 19th century, Hypocrea lixii is epitypified by a collection from
Thailand.
Mycological Progress 1(3): 283–286, August 2002 283
1Corresponding author. The Pennsylvania State University, De-
partment of Plant Pathology, 301 Buckhout Lab., University Park,
Pennsylvania 16802, USA. Current address: United States Depart-
ment of Agriculture, Agricultural Research Service, Systematic Bo-
tany and Mycology Lab., Rm. 304, B-011A, BARC-W, Beltsville,
Maryland 20705-2350, USA. Fax: 1 (301) 504-5810.
Email: priscila@nt.ars-grin.gov
2United States Department of Agriculture, Agricultural Research Ser-
vice, Systematic Botany and Mycology Lab., Rm. 304, B-011A,
BARC-W, Beltsville, Maryland 20705-2350, USA
©DGfM 2002
T

were gathered using the image-capturing software Scion Ima-
ge beta 4.0.2. Confidence intervals (a = 0.05), minimum and
maximum values for the morphological characters measured
were calculated using Systat 8.0 (SPSS, Inc., Illinois).
Results
Analysis of morphological data of type specimens of H. lenti-
formis, H. lixii, and H. nigricans f. octospora, and additional
specimens of H. lixii show that these species are indistinguish-
able from each other and therefore represent one species. Be-
cause Hypocrea lixii Pat. is the oldest name, it is the correct
name for this species.
Several cultures derived from specimens of Hypocrea
identified by Y. Doi as H. nigricans (IFO 31294, 31289,
30611, 31285, 31286) were examined and were morpholo-
gically identical to T. harzianum. Unfortunately we were not
able to study the Hypocrea specimens from which those cul-
tures were derived. The type specimen of H. nigricans f. ni-
gricans was not available from TNS. Specimens deposited by
Doi in NY as H. nigricans are indistinguishable from the type
collection of H. lixii.
We were not able to obtain the holotype specimen of H.
nigricans f. octospora for study, but a paratype of this form,
deposited in NY, was studied. Hypocrea nigricans f. octo-
spora was distinguished from f. nigricans because it has eight
green part-ascospores and eight smaller colorless part-asco-
spores. Within a perithecium of H. nigricans f. octospora,
ascospore pigmentation and size segregated in various patterns,
viz. 8:0, 4:4, 2:4:2, 2:2:2:2, or 1:6:1 suggesting that the loss
of pigment and diminution in size of ascospores is the result
of a mutation or a rare allele. The illustration provided by DOI
(1972, Fig 41) suggests that the smaller, colorless part-asco-
spores are in fact malformed and probably not viable, further
suggesting a ‘spore killer’ mutant (VAN DER GAAGA et al.
2000, RAJU & PERKINS 1991). DOI (1972) noted that collec-
tions of f. octospora often only had 8 normal part-ascospores
(as opposed to the expected 16), which is additional evidence
for a ‘killer’ mutant. In our experience, in older specimens
of Hypocrea it is not unusual to find some asci with aborted
ascospores. Based on the study of available herbarium spe-
cimens of f. octospora and literature, we conclude that this
forma falls within the normal circumscription of H. lixii.
The holotype of H. lixii is a collection made in Papua New
Guinea, and no anamorph is associated with this specimen.
Because stroma morphology in Hypocrea can be shared by
more than one species, anamorph morphology can be critical
to species recognition. Therefore, in the interest of nomen-
clatural stability we designate a specimen from Thailand (BPI
745654, culture G.J.S. 97-96, CBS 110080, ATCC MYA-
2478), which we have cultured, and deposited in culture
collections, to be the epitype of H. lixii. The designated epi-
type is on the same substratum (Ganoderma) as the holotype.
Taxonomy
Hypocrea lixii Pat., Rev. Mycol. Toulouse 13: 138. 1891
Figs 1–9
=Hypocrea lentiformis Rehm, Hedwigia 37: 193. 1898.
= Chromocrea nigricans Imai, Trans. Sapporo Nat. Hist. Soc. 14:
102. 1935.
≡Hypocrea nigricans (Imai) Doi, Bull. Natl. Sci. Mus. Tokyo 15:
732. 1972.
= Hypocrea nigricans f. octospora Doi, Bull. Natl. Sci. Mus. 15:
734. 1972.
Anamorph: Trichoderma harzianum Rifai, Mycol. Pap. 116:
38. 1969 (for illustrations see BISSETT 1991, Figs 56–63; SA-
MUELS et al. 2002, Figs 6–16).
=Trichoderma inhamatum Veerkamp & W. Gams, Caldasia 13:
710. 1983.
Stromata solitary or aggregated, pulvinate, nearly circular in
outline, (0.3)1.0–1.1(3.0) mm diam (n = 154), (440)730–785
(1400) µm high (n = 139), broadly attached, surface smooth,
sometimes with slight perithecial protuberations, dark brown
or green, almost black, changing color from dark green to
brown in KOH, if stromata dark brown then KOH–, ostiolar
openings not obvious due to the dark color of the stromata.
Stroma surface (8)17–20(45) µm thick (n = 176), formed of
angular cells, compacted, pigmented brown (KOH-) or dark
green (KOH+), (2.7)7.8–8.5(17) µm diam (n = 398), walls
(0.5)1.0 (2.0) µm thick (n = 294). Tissue immediately below
the stromatal surface of compact to loose textura angularis to
t. epidermoidea, colorless or slightly pigmented, (2.0)7.0–7.5
(22.0) µm diam (n = 366), walls (0.2)0.5–0.6(1.0) µm thick
(n = 321). Internal tissue below the perithecia formed of an-
gular cells, colorless, KOH–, (5.0)13–14(31) µm diam, walls
(0.4)0.7–0.8(1.5) µm thick (n = 365). Perithecia completely
immersed in stroma, generally closely aggregated, sometimes
widely spaced, subglobose, (153)231–244(336) µm high, (75)
142–152(259) µm wide (n = 142), wall composed of com-
pacted cells, KOH+ or –, (6.0)12.5–13.2(25.0) µm thick (n =
154), ostiolar canal (44)63–68(101) µm long (n = 125). Asci
cylindrical, (44)73–78(138) x (3.0)4.5–4.7(6.5) µm (n = 372),
ascospores uniseriate. Part-ascospores green, warted, dimor-
phic, distal part globose to subglobose, (3.0)4.3–4.4(5.6) x
(2.8)3.9–4.0(5.2) µm, proximal part wedge-shaped to cylin-
drical, (3.4)4.5–4.6(6.5) µm (n = 472).
Habitat: Fungicolous, corticolous, lignicolous; infrequently
on rotting leaves of palms.
Known distribution: Cosmopolitan.
Holotype: PAPUA NEW GUINEA,on hymenium of Ganoderma
pourii, Jul. 1891, Lix (FH!).
284 Hypcrea lixii – Trichoderma harzianum
©DGfM 2002

Mycological Progress 1(3) / 2002 285
©DGfM 2002
Additional specimens examined
AUSTRIA, Niederösterreich, 23rd district of Vienna, Mauer Wald,
on decorticated wood and black pyrenomycete, 3 Oct. 1998, W. Jak-
litsch (G.J.S. 98-183, BPI 841387). – BRAZIL, Sta. Catharina State,
on leaves of Euterpe sp., Aug. 1888, Ule (HBG #812, ISOTYPE of
H. lentiformis). – FRANCE, Pyrénées Atlantiques, 64 Oloron, Forêt
de Josbaig, on decorticated wood of Fagus sp., 13 Sep. 1992, F. Can-
doussau (G.J.S. 92-110, BPI 802854). – GERMANY, Thuringia,
Weimar, Belvedere, on decaying wood of Pinus sylvestris, 8 Oct.
1990, G. Arnold (G.J.S. 90-254, BPI 1109306). – INDONESIA,
North Sulawesi, Dumoga-Bone National Park, between Madison’s
Camp and ‘1440’ Camp, on decaying wood, 5 Oct. 1985, G.J. Sa-
muels 2161 (G.J.S. 85-119, NY). – PAPUA NEW GUINEA, New
Britain Island, Rabaul, on decaying palm leaves, 2 Jan. 1970, Y. Doi
(NY, PARATYPE of H. nigricans f. octospora NS-1323, TNS.D-
723, TNS-F-191628). – SWITZERLAND, on artificially inoculated
Pinus sp. blocks, O. Petrini (G.J.S. 92-135, BPI 802883). – THAI-
LAND, Saraburi Province, Khao Yai National Park, Wang Jumpee
Trail, on hymenium of Ganoderma sp., 31 Jul. 1997, K. Põldmaa,
P. Chaverri, G.J. Samuels 8233 (BPI, EPITYPE of H. lixii, BPI
745654, G.J.S. 97-96, ATCC MYA-2478, CBS 110080); vicinity of
park headquarters, on bark and corticioid basidiomycete, 30 Jul. 1997,
K. Põldmaa, P. Chaverri, S. Sivichai, G.J. Samuels 8201 (G.J.S. 97-
106, BPI 745629). – UNITED STATES OF AMERICA, Alabama:
Winston County, W.B. Bankhead National Forest, Sipsey Wilderness
Area, Sipsey Recreation River along trail, on decorticated wood, 25
Sep. 1992, C.T. Rogerson, S.M. Huhndorf, G.J. Samuels (G.J.S. 92-
Figs 1-9: Hypocrea lixii (Epitype BPI 745654). Fig 1. Stroma. Fig 2. Section of stroma. Fig 3. Perithecium. Fig 4. Outer tissue
of stroma. Fig 5. Top view of stroma surface cells. Fig 6. Tissue immediately below the stromatal surface. Fig 7. Inner tissue
below perithecia. Fig 8. Ascus and ascospores. Fig 9. Ascospore (notice the warted ornamentation). Bars: Fig 1 = 1 mm, Fig 2
= 250 µm, Fig 3 = 25 µm, Figs 4–7 = 15 µm, Figs 8, 9 = 5 µm.

286 Hypcrea lixii – Trichoderma harzianum
100, BPI 802845). Illinois: Shawnee National Forest, vicinity of Po-
mona, Pomona Natural Bridge, on decorticated wood and basi-
diomycete, 30 Sep. 1994, W. Sundberg, G.J. Samuels (G.J.S. 94-53,
BPI 749348). Maryland: Prince George County, E of Largo, in old
growth forest at Church Rd., on bark of decaying wood, 11 Oct. 1991,
A.Y. Rossman, S.E. Rehner, F.A. Uecker, G.J. Samuels (G.J.S. 91-
138, BPI 1112907). North Carolina: Macon County, Blue Valley,
off Clear Creek Rd., along overflow Creek, on bark of decaying
wood (probably on dematiaceous mycelia and lichen), 16 Oct. 1990,
Y. Doi, A.Y. Rossman, G.J. Samuels (G.J.S. 90-127, BPI 1109390).
Wisconsin: Sand County, Aldo Leopold Reserve, on decorticated
wood (probably growing on dematiaceous mycelia), 23 Jun. 1990,
G. J. Samuels (G. J. S. 90-22, BPI 802600).
Acknowledgments
This research was supported by NSF-PEET grant 9712308 to
the Pennsylvania State University, Dept. of Plant Pathology,
“Monographic Studies of Hypocrealean Fungi: Hypocrea and
Hypomyces”. Also a grant from the National Geographic So-
ciety to G.J.S. provided support for fieldwork in Indonesia, in
collaboration with Royal Entomological Society ‘Project Wal-
lace.’ We appreciate the assistance given by Dr. Nigel Hywel-
Jones (Bangkok) in support of our fieldwork in Thailand. We
thank our colleagues Günter Arnold, Françoise Candoussau
and Walter Jaklitsch for providing us with European collec-
tions of Hypocrea lixii. Revisions and comments given by Drs.
Amy Y. Rossman and Elwin L. Stewart were very helpful.
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Accepted: 17.5.2002