Novel Phaeoacremonium species associated with necrotic wood of Prunus trees

Abstract

The genus Phaeoacremonium is associated with opportunistic human infections, as well as stunted growth and die-back of various woody hosts, especially grapevines. In this study, Phaeoacremonium species were isolated from necrotic woody tissue of Prunus spp. (plum, peach, nectarine and apricot) from different stone fruit growing areas in South Africa. Morphological and cultural characteristics as well as DNA sequence data (5.8S rDNA, ITS1, ITS2, β-tubulin, actin and 18S rDNA) were used to identify known, and describe novel species. From the total number of wood samples collected (257), 42 Phaeoacremonium isolates were obtained, from which 14 species were identified. Phaeoacremonium scolyti was most frequently isolated, and present on all Prunus species sampled, followed by Togninia minima (anamorph: Pm. aleophilum) and Pm. australiense. Almost all taxa isolated represent new records on Prunus. Furthermore, Pm. australiense,
Pm. iranianum, T. fraxinopennsylvanica and Pm. griseorubrum represent new records for South Africa, while Pm. griseorubrum, hitherto only known from humans, is newly reported from a plant host. Five species are newly described, two of which produce a Togninia sexual state. Togninia africana, T. griseo-olivacea and Pm. pallidum are newly described from Prunus armeniaca, while Pm. prunicolum and Pm. fuscum are described from Prunus salicina.

Full text

Persoonia 20, 2008: 87–102
www.persoonia.org doi:10.3767/003158508X324227
RESEARCH ARTICLE
INTRODUCTION
The genus Phaeoacremonium was established 12 years ago
(Crous et al. 1996) to accommodate cephalosporium-like fungi
known from grapevine (Petri 1912) and human infections (Ajello
et al. 1974). In spite of the exclusion of Pm. chlamydospora
(now Phaeomoniella chlamydospora, Crous & Gams 2000),
the number of known species increased quickly. The genus
Togninia was confirmed as teleomorph (Mostert et al. 2003), and
their phylogenetic position clarified within the Togniniaceae in
the Diaporthales (Réblová et al. 2004). Presently 23 Phaeoacre-
monium species have been described, of which 10 have been
linked to Togninia teleomorphs (Mostert et al. 2006a, Réblová
& Mostert 2007). Togninia species known to date have been
shown to have either a homo- or heterothallic mating strategy
(Mostert et al. 2003, 2006a, Rooney-Latham et al. 2005). In
species where the mating strategy has been studied in more
detail, like T. minima, both mating types have been found to
occur in the same field, and even the same grapevine (Mostert
et al. 2003, Pascoe et al. 2004, Rooney-Latham et al. 2005).
Recent research has focused on the development of genus- and
species-specific primers to facilitate early detection (Mostert
et al. 2006a, Aroca & Raposo 2007), and the development of
an online polyphasic identification system (www.cbs.knaw.nl/
phaeoacremonium/biolomics.aspx).
Phaeoacremonium species have been associated with human
infections, often skin- or nail-infections, so-called phaeohy-
phomycoses (Ajello et al. 1974, Guarro et al. 2003, Hemashet-
tar et al. 2006), as well as disease symptoms of a number of
woody hosts worldwide (Rumbos 1986, Di Marco et al. 2004,
Kubátová et al. 2004), especially with grapevine diseases such
as Petri disease and esca (Pascoe et al. 2004, Rooney-Latham
et al. 2005, Whiting et al. 2005, Mostert et al. 2006b, Aroca &
Raposo 2007). Only two Phaeoacremonium species are known
from Prunus. Phaeoacremonium aleophilum (T. minima) was
reported on Prunus pennsylvanica in Canada (Hausner et al.
1992) and P. armeniaca in South Africa (Mostert et al. 2006a).
Phaeoacremonium parasiticum (T. parasitica) was isolated from
wilting trees of P. armeniaca in Tunisia (Hawksworth et al. 1976)
and from P. avium in Greece (Rumbos 1986). Rumbos (1986)
described Pm. parasiticum as causal agent of a serious dieback
disease of cherry trees in different locations in Greece in the
1980s. Several cherry cultivars were found to be susceptible to
the disease, which caused leaf drop, wilting and wood discol-
ouration. In one orchard, were the fungus was closely associ-
ated with bark beetles (Scolytidae) and metallic wood-boring
beetles (Buprestidae), the majority of the trees were affected
and died. Phaeoacremonium parasiticum caused xylem lesions
in cherry, apricot, olive and peach trees (Rumbos 1986).
In South Africa, stone fruit orchards are often established in
close proximity of vineyards. Thirteen Phaeoacremonium spe-
cies have been reported from Vitis vinifera, eight of which are
known from South Africa, where T. minima was found on Vitis and
Prunus (Mostert et al. 2006a). It is possible that this pathogen
disseminates from one host to another. This phenomenon may
be more common among phytopathogenic ascomycetes than
previously accepted, as several species of Botryosphaeriaceae,
Novel Phaeoacremonium species associated with
necrotic wood of Prunus trees
U. Damm1,2, L. Mostert1, P.W. Crous1,2, P.H. Fourie1,3
1 Department of Plant Pathology, University of Stellenbosch, Private Bag
X1, Stellenbosch 7602, South Africa;
corresponding author e-mail: ulrikeda@yahoo.de.
2 CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Nether-
lands.
3 Citrus Research International, P.O. Box 2201, Stellenbosch 7602, South
Africa.
Key words
Diaporthales
molecular systematics
pathogenicity
Togninia
Togniniaceae
Abstract The genus Phaeoacremonium is associated with opportunistic human infections, as well as stunted
growth and die-back of various woody hosts, especially grapevines. In this study, Phaeoacremonium species were
isolated from necrotic woody tissue of Prunus spp. (plum, peach, nectarine and apricot) from different stone fruit
growing areas in South Africa. Morphological and cultural characteristics as well as DNA sequence data (5.8S
rDNA, ITS1, ITS2, β-tubulin, actin and 18S rDNA) were used to identify known, and describe novel species. From
the total number of wood samples collected (257), 42 Phaeoacremonium isolates were obtained, from which 14
species were identified. Phaeoacremonium scolyti was most frequently isolated, and present on all Prunus species
sampled, followed by Togninia minima (anamorph: Pm. aleophilum) and Pm. australiense. Almost all taxa isolated
represent new records on Prunus. Furthermore, Pm. australiense, Pm. iranianum, T. fraxinopennsylvanica and Pm.
griseorubrum represent new records for South Africa, while Pm. griseorubrum, hitherto only known from humans,
is newly reported from a plant host. Five species are newly described, two of which produce a Togninia sexual
state. Togninia africana, T. griseo-olivacea and Pm. pallidum are newly described from Prunus armeniaca, while
Pm. prunicolum and Pm. fuscum are described from Prunus salicina.
Article info Received: 9 May 2008; Accepted: 20 May 2008; Published: 24 May 2008.
© 2008 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures
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88 Persoonia – Volume 20, 2008
Table 1 Names, accession numbers and collection details of isolates studied.
Species Accession No.1 Host Location Pathotest2 GenBank accessions
ITS TUB ACT SSU
Phaeoacremonium STE-U 5960 Prunus salicina Paarl, Western Cape, South Africa EU128021 EU128069 EU128111
australiense STE-U 5961 P. salicina Paarl, Western Cape, South Africa x EU128022 EU128070 EU128112
STE-U 5839 P. salicina Paarl, Western Cape, South Africa x EU128023 EU128071 EU128113
STE-U 5838 P. salicina Paarl, Western Cape, South Africa EU128024 EU128072 EU128114 EU128055
STE-U 5959, CBS 120861 P. salicina Stellenbosch, Western Cape, South Africa EU128025 EU128073 EU128115 EU128054
Pm. fuscum STE-U 5969, CBS 120856* P. salicina Mookgopong, Limpopo, South Africa x EU128050 EU128098 EU128141 EU128059
STE-U 6366 P. salicina Mookgopong, Limpopo, South Africa EU128051 EU128199 EU128140
Pm. griseorubrum STE-U 5957, CBS 120860 P. salicina Paarl, Western Cape, South Africa x EU128026 EU128074 EU128116
STE-U 5958 P. salicina Paarl, Western Cape, South Africa x EU128027 EU128075 EU128117
Pm. iranianum STE-U 6092 P. armeniaca Robertson, Western Cape, South Africa x EU128028 EU128076 EU128118
STE-U 6179 P. armeniaca Montagu, Western Cape, South Africa x EU128029 EU128077 EU128119
STE-U 6091, CBS 120864 P. armeniaca Robertson, Western Cape, South Africa EU128030 EU128078 EU128120
Pm. pallidum STE-U 6104, CBS 120862* P. armeniaca Bonnievale, Western Cape, South Africa x EU128053 EU128103 EU128144 EU128061
Pm. prunicolum STE-U 5967, CBS 120858* P. salicina Mookgopong, Limpopo, South Africa x EU128047 EU128095 EU128137 EU128056
STE-U 5968 P. salicina Mookgopong, Limpopo, South Africa x EU128048 EU128096 EU128138 EU128057
Pm. scolyti STE-U 5955, CBS121755 P. persica var. Mookgopong, Limpopo, South Africa x EU128034 EU128082 EU128124
nucipersica
STE-U 6095, CBS 121438 P. armeniaca Robertson, Western Cape, South Africa EU128035 EU128083 EU128125
STE-U 6096 P. armeniaca Bonnievale, Western Cape, South Africa EU128036 EU128084 EU128126
STE-U 6097 P. persica Modimolle, Limpopo, South Africa EU128037 EU128085 EU128127
STE-U 6098, CBS121756 P. persica Modimolle, Limpopo, South Africa EU128038 EU128086 EU128128
STE-U 6099 P. persica Modimolle, Limpopo, South Africa EU128039 EU128087 EU128129
STE-U 6100 P. persica Modimolle, Limpopo, South Africa EU128040 EU128088 EU128130
STE-U 5834 P. salicina Stellenbosch, Western Cape, South Africa x EU128041 EU128089 EU128131
STE-U 5954, CBS 121439 P. salicina Paarl, Western Cape, South Africa EU128042 EU128090 EU128132
STE-U 5956 P. salicina Mookgopong, Limpopo, South Africa EU128043 EU128091 EU128133
Pm. subulatum STE-U 6094, CBS 120866 P. armeniaca Robertson, Western Cape, South Africa x EU128044 EU128092 EU128134
Togninia africana STE-U 6177, CBS 120863* P. armeniaca Montagu, Western Cape, South Africa x EU128052 EU128100 EU128142 EU128060
STE-U 6364 P. armeniaca Montagu, Western Cape, South Africa EU128101 EU128143
STE-U 6365 P. armeniaca Montagu, Western Cape, South Africa EU128102
T. fraxinopennsylvanica STE-U 6101, CBS 120865 P. salicina Franschhoek, Western Cape, South Africa x EU128031 EU128079 EU128121
(Pm. mortoniae) STE-U 6102 P. salicina Franschhoek, Western Cape, South Africa x EU128032 EU128080 EU128122
T. griseo-olivacea STE-U 5966, CBS 120857* P. armeniaca Mookgopong, Limpopo, South Africa x EU128049 EU128097 EU128139 EU128058
T. minima (Pm. aleophilum) STE-U 6088 P. armeniaca Robertson, Western Cape, South Africa EU128014 EU128062 EU128104
STE-U 6089, CBS 121434 P. armeniaca Bonnievale, Western Cape, South Africa EU128015 EU128063 EU128105
STE-U 6090 P. armeniaca Bonnievale, Western Cape, South Africa EU128016 EU128064 EU128106
STE-U 5836, CBS 121435 P. salicina Paarl, Western Cape, South Africa EU128017 EU128065 EU128107
STE-U 5962 P. salicina Paarl, Western Cape, South Africa EU128018 EU128066 EU128108
STE-U 5963 P. salicina Paarl, Western Cape, South Africa x EU128019 EU128067 EU128109
STE-U 5964, CBS 121436 P. persica Paarl, Western Cape, South Africa x EU128020 EU128068 EU128110
T. parasitica (Pm. parasiticum) STE-U 6093, CBS 121437 P. armeniaca Montagu, Western Cape, South Africa x EU128033 EU128081 EU128123
T. viticola (Pm. viticola) STE-U 5965, CBS 121440 P. salicina Paarl, Western Cape, South Africa x EU128045 EU128093 EU128135
STE-U 6180 P. salicina Franschoek, Western Cape, South Africa x EU128046 EU128094 EU128136
1 STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa; CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The
Netherlands.
2 Isolates studied in the pathogenicity test, * ex-type cultures.

89
U. Damm et al.: Phaeoacremonium on Prunus wood
Cryphonectriaceae and Valsaceae have been dispersed from
branches and stems of fruit trees to other woody hosts in the
vicinity (Adams et al. 2005, Crous et al. 2006b, Gryzenhout et
al. 2006, Damm et al. 2007).
The comparatively slow-growing and, until recently, relatively
unknown species of Phaeoacremonium were probably often
excluded from surveys of fungi on woody plants in South Af-
rica, and subsequently only Phaeomoniella chlamydospora is
listed in the most recent compilation of phytopathogenic fungi
from South Africa (Crous et al. 2000). Because of the highly
diverse, endemic vegetation and different climatic regions, more
than 200 000 species of fungi have been estimated to occur
in South Africa (Crous et al. 2006a), which was acknowledged
by the authors as rather conservative. As a recent study on
Botryosphaeriaceae has shown (Damm et al. 2007), Prunus
represents a rich catch-crop for many of these fungi, and thus
would also be a good host to sample for novel species of
Phaeoacremonium. Therefore, the aim of the present study
is to determine the diversity of Phaeoacremonium species on
Prunus wood in South Africa and to describe five new species
isolated from P. armeniaca and P. salicina. A further aim of this
study was to determine which species of Phaeoacremonium,
formerly known from grapevines, would have Prunus spp. as
alternate hosts.
MATERIAL AND METHODS
Isolates
Branches with wood symptoms (e.g. die-back, canker, necrosis)
were sampled from plum (Prunus salicina), peach (P. persica),
nectarine (P. persica var. nucipersica) and apricot (P. armeni-
aca) orchards in the Western Cape and the Limpopo province
of South Africa. Wood pieces with necrosis symptoms were
prepared according to Damm et al. (2007) and incubated on
potato-dextrose agar (2 % PDA; Biolab, Midrand, South Africa,
supplemented with 100 mg/L streptomycin sulphate and 100
mg ampicillin) and synthetic nutrient-poor agar medium (SNA;
Nirenberg 1976) supplemented with 100 mg penicillin G, 50 mg
streptomycin sulphate, 10 mg chlortetracycline hydrochloride
(pH 6), under cool fluorescent white light at 25 °C. Single-
conidial isolates were obtained from all strains for further study.
Reference strains are maintained in the culture collection of
the Department of Plant Pathology, University of Stellenbosch
(STE-U), Stellenbosch, South Africa, and the Centraalbureau
voor Schimmelcultures (CBS) Utrecht, The Netherlands. Iso-
lates used for morphological and sequence analysis are pre-
sented in Table 1.
Morphology
The Phaeoacremonium anamorphs were morphologically char-
acterised on malt extract plates (MEA; 2 % malt extract, Oxoid
Ltd., England; 1.5 % agar, Difco, USA) incubated at 25 °C in the
dark for 2 – 3 wks as described in Mostert et al. (2006a). Teleo-
morph structures were described from PDA and SNA plates
incubated for 3 or 2 mo at 25 °C in the laboratory under diffuse
daylight. Vertical sections through perithecia and photographs
of characteristic structures were made as described in Damm
et al. (2007). Colony characters and pigment production on
MEA, PDA and oatmeal agar (OA; Gams et al. 2007) incubated
at 25 ºC were noted after 8 and 16 d. Colony colours were
determined using the colour charts of Rayner (1970). Cardinal
temperatures for growth were determined by incubating MEA
plates in the dark at temperatures ranging from 5 to 40 °C in
5 °C intervals, also including 37 °C, emulating human body
temperature. Radial growth was measured after 8 d at 25 °C.
DNA isolation, amplification and analysis
Genomic DNA of all isolates was isolated from fungal mycelium
grown on PDA plates, placed in a 1.5 mL tube with glass beads
and 600 µL hexadecyltrimethyl ammonium bromide (CTAB)
extraction buffer (0.2 M Tris, 1.4 M NaCl, 20 mM EDTA, 0.2
g/ L CTAB) and crushed 3 min at 30 vibrations per second in
a Retsch Mixer Mill MM301 (Retsch, Haan, Germany). Before
adding 400 µL chloroform : isoamylalcohol (24 : 1), the tube
was placed in a 65 °C water bath for 15 min. The fungal matrix
was spun down for 5 min at 15 800 x g. The watery superna-
tant was transferred into a new centrifuge tube and cold am-
monium acetate solution (final concentration 2.5 M) and 600
µL cold isopropanol were added. After 15 min incubation at
room temperature, the precipitate was spun down for 5 min at
15 800 x g and the supernatant discarded. One millilitre cold
70 % ethanol was added to the pellet, spun down for 5 min at
15 800 x g and the supernatant discarded. The DNA pellet was
dried and resuspended in 100 µL ddH2O.
The 5.8S ribosomal gene with the two flanking internal tran-
scribed spacers (ITS1 and ITS2), the β-tubulin gene (TUB),
the actin gene (ACT) and a partial sequence of the 18S rDNA
gene (SSU) were amplified and sequenced using the primer
pairs ITS-1F (Gardes & Bruns 1993) + ITS-4 (White et al.
1990), primers T1 (O’Donnell & Cigelnik 1997) + Bt2b (Glass
& Donaldson 1995), ACT-512F + ACT-783R (Carbone & Kohn
1999) and NS1 + NS8 (White et al. 1990), according to the
conditions and protocols explained in Mostert et al. (2006a). Ad-
ditional primers used for sequencing the SSU were: NS2, NS3,
NS4, NS5 (White et al. 1990). The ITS region was sequenced
for preliminary identification of the fungi isolated from Prunus
wood. Even though the ITS region has shown not to be robust
for all species determination in the genus Phaeoacremonium
(Groenewald et al. 2001, Mostert et al. 2005), we did found it
valuable information for future ITS comparisons and lodged it
in GenBank (Table 1).
The sequences generated in this study and additional
sequences obtained from GenBank (www.ncbi.nlm.gov)
were manually aligned using Sequence Alignment Editor
v. 2.0a11 (Rambaut 2002). Pleurostomophora richardsiae
(CBS 270.33) and Wuestneia molokaiensis (CBS 114877)
were used as outgroup in the TUB and ACT phylogenies, while
Cochliobolus sativus (U42479) and Pleospora betae (U3466)
were used as outgroup in the SSU phylogeny. Two introns,
only present in the outgroups (sequence positions 205 – 267,
388–421) were excluded from the SSU analysis. Phylogenetic
analyses were performed using PAUP (Phylogenetic Analysis
Using Parsimony) v. 4.0b10 (Swofford 2003). The TUB and
ACT data were analysed for each region separately, as well as
with a combined data set. Alignment gaps in all analyses were
treated as missing data and all characters were unordered and

90 Persoonia – Volume 20, 2008
of equal weight. Maximum parsimony analysis was performed
using the heuristic search option with 100 random sequence
additions and tree bisection and reconstruction (TBR) as the
branch-swapping algorithm. The robustness of the trees ob-
tained was evaluated by 1 000 bootstrap replications with 100
random sequence additions (Hillis & Bull 1993). Tree length,
consistency index (CI), retention index (RI), rescaled consist-
ency index (RC) and homoplasy index (HI) were calculated for
the resulting tree. A partition homogeneity test with the same
search criteria was conducted in PAUP to examine the possibil-
ity of a joint analysis of the TUB and ACT data sets. Sequences
derived in this study were lodged at GenBank (Table 1) and the
alignments in TreeBASE.
Cochliobolus sativus U42479
Pleospora betae U43466
Togninia minima AY761068
Togninia viticola DQ173147
Togninia griseo-olivacea EU128058
Phaeoacremonium fuscum EU128059
Phaeoacremonium pallidum EU128061
Togninia novae-zealandiae AY761069
Phaeoacremonium prunicolum EU128056
Togninia africana EU128060
Gnomoniella fraxini AF277106
Gnomonia setacea AF277121
Cryphonectria parasitica L42441
Endothia gyrosa L42443
Leucostoma persoonii M83259
Diaporthe phaseolorum L36985
Pleurostomophora richardsiae AY761066
Pleurostomophora repens AY761067
Pleurostoma ootheca AY761074
Calosphaeria pulchella AY761071
Togniniella acerosa AY761072
Togniniella acerosa AY761073
Madurella mycetomatis AF527811
Chaetomium elatum M83257
Lasiosphaeria ovina AY083799
Sordaria fimicola X69851
Kionochaeta ramifera AB003788
Chaetosphaeria curvispora AY502933
Camarops microspora Z49783
Cryptendoxyla hypophloia AF096175
Albertiniella polyporicola AF096170
Magnaporthe grisea AB026819
Gaeumannomyces graminis AF277125
Ceratosphaeria lampadophora AY761088
Sporothrix schenckii M85053
Sporothrix sp. AF267227
Ophiostoma stenoceras M85054
5 changes
Togniniaceae
DIAPORTHALES
CALOSPHAERIALES
SORDARIALES
CHAETOSPHAERIALES
BOLINIALES
Cephalothecaceae
Magnaporthaceae
Ophiostomatales
100
65
87
57
63
100
100
69
67
74
100
62
100
78
90
100
78
98
99
100
51
92
100
99
74
67
Fig. 1 One of 12 most parsimonious trees obtained from heuristic searches of the SSU gene sequences (Length = 591 steps, CI = 0.633, RI = 0.772,
RC = 0.489, HI = 0.367). Bootstrap support values (1 000 replicates) above 50 % are shown at the nodes. Cochliobolus sativus U42479 and Pleospora betae
U3466 were used as outgroups. Isolates analysed in this study are emphasised in bold.

91
U. Damm et al.: Phaeoacremonium on Prunus wood
CBS 114877 Wuestneia molokaiensis
CBS 270.33 Pleurostomophora richardsiae
CBS 109479*
CBS 113597*
STE-U 6096
STE-U 6095
STE-U 5956
STE-U 6100
STE-U 5834
STE-U 5954
STE-U 5955
STE-U 6097
STE-U 6098
STE-U 6099
CBS 111657 *
STE-U 5957
STE-U 5958
CBS 110627 *
STE-U 6094
CBS 113584 *
CBS 113589*
STE-U 5961
STE-U 5839
STE-U 5960
STE-U 5959
STE-U 5838
CBS 110573*
CBS 498.94 *
CBS 110034 *
CBS 860.73 *
STE-U 6093
CBS 110156*
STE-U 6101
CBS 101585 *
STE-U 6177*
STE-U 6364
STE-U 5967*
STE-U 5968
CBS 777.83*
STE-U 5966*
CBS 114992*
CBS 101738*
STE-U 5965
STE-U 6180
CBS 112949*
CBS 111586*
STE-U 6104*
CBS 246.91*
STE-U 5963
STE-U 6088
STE-U 5964
STE-U 5836
STE-U 5962
STE-U 6089
STE-U 6090
STE-U 6091
STE-U 6092
STE-U 6179
CBS 101357*
STE-U 5969*
STE-U 6366
CBS 651.85*
CBS 117115 *
CBS 391.71*
CBS 337.90*
10 changes
Pm. scolyti
T. minima
Pm. iranianum
Pm. theobromatis
T. austroafricana
Pm. angustius
T. viticola
T. argentinensis
T. fraxinopennsylvanica
T. novae-zealandiae
Pm. venezuelense
T. vibratilis
Pm. inflatipes
Pm. tardigrescens
Pm. amstelodamense
Pm. griseorubrum
Pm. australiense
Pm. subulatum
Pm. alvesii
T. rubrigena
T. parasitica
T. krajdenii
Pm. sphinctrophorum
T. africana
Pm. prunicolum
T. griseo-olivacea
Pm. pallidum
Pm. fuscum
93
97
94
100
100
84
97
99
100
100
100
82
65
98
84
99
97
75
100
100
94 100
89
98
100
83
82
95
99
100
95
60
100
94 90
67
100
100
100
100
97
100
93
99
97
Fig. 2 One of 312 most parsimonious trees obtained from heuristic searches of a combined alignment of the TUB and ACT gene sequences (Length = 1807
steps, CI = 0.522, RI = 0.833, RC = 0.435, HI = 0.478). Bootstrap support values (1 000 replicates) above 60 % are shown at the nodes. Pleurostomophora
richardsiae and Wuestneia molokaiensis were used as outgroups. Isolates analysed in this study are emphasised in bold. Ex-type strains are indicated with
asterisks.

92 Persoonia – Volume 20, 2008
Pathogenicity tests
Preliminary pathogenicity tests were conducted with 14 taxa on
detached apricot (cv. ‘Belida’, 4-year-old trees) and plum (cv.
‘Southern Bell’, 5-year-old trees) shoots. Depending on strain
availability, one or two isolates per taxon were used and treated
as sub-samples in the statistical analysis. Fresh vegetative
shoots were collected from the trees shortly after harvest, cut
into 12 cm pieces (5–8 mm diam), treated and inoculated with
colonised agar plugs from 2-wk-old PDA cultures according to
Damm et al. (2007), except for the surface sterilisation (40 s
in 0.1 % solution of a patented didecyldimethylammonium
chloride formulation, Sporekill, ICA International Chemicals Pty.
Ltd., Stellenbosch, South Africa). Acremonium strictum (STE-
U 6296) and uncolonised PDA plugs were used as negative
controls. Shoots were incubated at 25 °C in moist chambers
(> 93 % RH) for 2 wks, after which the bark was peeled off
and lesions visible on the xylem tissue were measured. Each
treatment combination consisted of one shoot, which was rep-
licated four times in each of three blocks (= moist chambers).
Re-isolations were made from the leading edges of lesions and
the resulting cultures identified. The layout of the trial was a
randomised block design. Lesion length data were subjected
to analyses of variance using SAS v. 8.1 (SAS Institute, Cary,
North Carolina USA) and Student’s t-test for Least Significant
Difference was calculated at the 5 % significance level to com-
pare the treatment means for the different taxa.
RESULTS
Phylogenetic analysis
Five SSU sequences produced in this study were added to the
32 sequences obtained from GenBank comprising an alignment
of 2266 characters including the gaps, of which 244 characters
were parsimony-informative, 80 variable (parsimony-uninforma-
tive) and 1942 constant. The heuristic search of the SSU data
resulted in 12 most parsimonious trees (Length = 591 steps,
CI = 0.633, RI = 0.772, RC = 0.489, HI = 0.367), of which one is
shown in Fig. 1. The clades represent eight classes or families,
respectively, within the Sordariomycetes. The unknown Phaeoa-
cremonium species found on Prunus trees grouped with species
of Togniniaceae, Togninia minima, T. novae-zealandiae and
T. viticola, forming the Togniniaceae-subclade (87 % bootstrap
support). This subclade grouped with other Diaporthales (65 %)
forming a sister clade to the Calosphaeriales.
The partition homogeneity test (p-value = 0.192) led us to
combine the TUB and ACT data sets (633 characters in data
Fig. 3 Phaeoacremonium fuscum. a−f. Aerial structures on MEA; a. conidiophores; b. type III phialides; c. conidiophore and type II phialide (indicated by
arrow head) ; d. type I phialides; e. type II phialides; f. conidia. — g, h. Structures on the surface of and in MEA; g. adelophialides; h. conidia; all from CBS
H-19944 (holotype); a−h: DIC. — Scale bar: a = 10 µm, applies to a−h.
c
b
da
gh
ef

93
U. Damm et al.: Phaeoacremonium on Prunus wood
set 1, 298 in data set 2). A selection of 40 isolates was used
for the phylogenetic analysis, with further 25 sequences being
added from GenBank. The data set contained 931 characters
including the gaps, of which 431 were parsimony-informative,
107 were variable and parsimony-uninformative, and 393 were
constant. After a heuristic search, 312 most parsimonious trees
with the same overall topology (differences only within species)
were retained (Length = 1807 steps, CI = 0.522, RI = 0.833,
RC = 0.435, HI = 0.478), of which one is shown in Fig. 2. The
majority of the isolates grouped with known species of Tognin-
iaceae: 10 of the isolates with Pm. scolyti (97 % bootstrap
support), two with Pm. griseorubrum (99 %), one with Pm.
subulatum (100 %), five with Pm. australiense (99 %), one with
T. parasitica (100 %), one with T. fraxinopennsylvanica (100 %),
two with T. viticola (97 %), seven with T. minima (100 %) and
three with Pm. iranianum (100 %). A further eight isolates
did not group with any known species. Isolates STE-U 6177
and 6364, as well as 5967 and 5968, formed two clades with
100 % bootstrap support that formed a sister group (90 %) to
T. novae-zealandiae, T. fraxinopennsylvanica and T. argentin-
ensis (94 %). Isolate STE-U 5966 grouped with these species
(98 %) but formed a separate lineage. Isolate STE-U 6104 also
formed a separate lineage in a clade (97 %) with Pm. angus-
tius, T. viticola, T. austroafricana and Pm. theobromatis. Two
isolates, STE-U 5969 and 6366, formed a sister group next to
Pm. venezuelense (100 %).
Taxonomy
The 42 strains of Phaeoacremonium isolated from stone fruit
wood (Table 1) could be assigned to 14 species based on the
DNA sequence data generated and their morphology. Five
species proved distinct from known species and are newly de-
scribed below. Mostert et al. (2006a) developed a polyphasic,
online identification system for species recognition (www.cbs.
knaw.nl/phaeoacremonium/biolomics.aspx). The latter key has
been updated to include all taxonomic novelties described in
this study.
Phaeoacremonium fuscum L. Mostert, Damm & Crous, sp. nov.
— MycoBank 505140; Fig. 3
Phaeoacremonio venezuelensi simile, sed coloniis in cultura (OA) fuscis-
nigris vel isabellinis, conidiophoris brevioribus et phialibus typorum I et II.
Etymology. Named after its dark brown colonies (fuscus Lat. = dark
brown).
Aerial structures — Mycelium consisting of branched, sep-
tate hyphae that occur singly or in bundles of up to 10; hyphae
tuberculate with warts up to 2 µm diam, verruculose, orange-
brown and 1– 2 µm wide. Conidiophores short and usually
unbranched, up to 3-septate, bearing 1–2 terminal phialides,
sometimes showing percurrent rejuvenation, (14–)17–28(–40)
(av. 23) µm long and (1.5 –)2 (– 2.5) (av. 2) µm wide. Phialides
terminal or lateral, mostly monophialidic, sometimes poly-
phialidic, sparsely tuberculate to verruculose, orange-brown,
sometimes hyaline; collarettes, slightly flaring, 1– 2 µm long
and 2 – 2.5 µm wide; type I phialides cylindrical, occasionally
widened at the base, tapering towards the apex, (2 –)4–7(– 8)
× 1–1.5(– 2) (av. 5 × 1.5) µm; type II phialides subcylindrical or
navicular, some elongate-ampulliform and attenuated at the
base, tapering towards the apex, (7–)9–11(–12) × 1.5– 2(–2.5)
(av. 10 × 2) µm; type III phialides mostly subcylindrical, some
navicular, 13 –17(– 20) × (1.5–) 2(–2.5) (av. 15 × 2) µm, gradu-
ally tapering towards the apex. Type I and II phialides most
common. Conidia hyaline, oblong-ellipsoidal some reniform,
(3.5–)4 – 5 × (1–)1.5(–2) (av. 4 × 1.5) µm, L /W = 3.4.
On surface or submerged in the agar — Phialides pale or-
ange-brown or hyaline, cylindrical, 1–4(–7) × 1–1.5(–2) (av. 3
× 1) µm. Conidia hyaline, cylindrical or allantoid, (4 –)5–7(–8)
× (1–)1.5–2 (av. 6 × 2) µm, L/W = 3.3.
Cultural characteristics — Colonies reaching a radius of
13.5–14 mm after 8 d at 25 °C. Minimum temperature for
growth 10 °C, optimum 30 °C, maximum 37 °C. Colonies on
MEA flat, mostly felty with a few woolly tufts, with entire margin;
after 8 d and 16 d colonies dark mouse-grey (13'''''k) to greyish
sepia (15''''I) becoming buff (19''d) towards the margin above,
reverse same. Colonies on PDA flat, felty to powdery, with entire
margin; after 8 d and 16 d fawn (13'''i) to vinaceous-buff (17''i),
similar in reverse, becoming sepia (15''m) to fawn (13'''i) after
16 d. Colonies on OA flat, felty to woolly, with entire margin;
after 8 d brown-vinaceous (5'''m) to isabelline (17''i), after 16 d
fuscous-black (7''''k) to isabelline (17''i) above. A pale brown
pigment produced after 16 d on PDA.
Specimen examined. South AfricA, Limpopo province, Mookgopong, from
small dark brown central V-shaped necrosis close to canker developing from
old pruning wound in wood of Prunus salicina, 31 Aug. 2004, U. Damm, CBS
H-19944 holotype, culture ex-type CBS 120856 = STE-U 5969.
Notes — The various species that have grey-brown colonies
and a growth rate that falls in the range of Pm. fuscum include
Pm. inflatipes, Pm. iranianum, Pm. krajdenii, Pm. sphinctropho-
rum and Pm. venezuelense (Mostert et al. 2006a). Of these,
Pm. venezuelense also has orange-brown mycelium, but can
be distinguished by the predominance of type III phialides and
hyaline phialides on and in the agar, in comparison with the
predominance of phialide type I and II and often pale orange-
brown phialides of Pm. fuscum. Furthermore, the maximum
growth temperature of Pm. fuscum was at 37 °C, in comparison
with 40 °C in the case of Pm. venezuelense.
Phaeoacremonium pallidum Damm, L. Mostert & Crous, sp. nov.
— MycoBank 505141; Fig. 4
Phaeoacremonio angustio simile, sed conidiis latioribus, in cultura (OA) coloniis
albis, ad 20 °C optime crescentibus.
Etymology. Named after its pale colonies (pallidus Lat. = pale).
Aerial structures — Aerial mycelium sparse, consisting of
branched, septate hyphae that occur singly or in bundles of up to
21; hyphae tuberculate with warts up to 2 µm diam, verruculose
to smooth, hyaline and 1–2.5 µm wide. Conidiophores short
and usually unbranched, up to 3-septate, bearing one terminal
phialide, (14–)17–36 (– 40) (av. 27) µm long and 1.5–2 (av. 2)
µm wide. Phialides terminal or lateral, mostly monophialidic,
sometimes polyphialidic, sparsely tuberculate to verruculose,
hyaline; collarettes slightly flaring, 1–1.5 µm long and 1 µm wide;
type I phialides most predominant, cylindrical, occasionally wid-
ened at the base, tapering towards the apex, (1.5 –)3 – 6 (–7) ×

94 Persoonia – Volume 20, 2008
1–1.5 (av. 4 × 1) µm; type II phialides, subcylindrical or navicular,
tapering towards the apex, (7–)9–11(–12) × 1.5– 2 (av. 10 × 1.5)
µm; type III phialides cylindrical or subcylindrical, 16–19 × 1 (av.
18 × 1) µm, gradually tapering towards the apex. Conidia hya-
line, oblong-ellipsoidal or allantoid to reniform, (3.5–)4 – 6(–7)
× 1.5–2 (av. 5 × 2) µm, L/W = 2.6.
On surface or submerged in the agar — Phialides hyaline,
cylindrical to subcylindrical, (1–)2 – 6 (–10) × 1–1.5 (av. 4 × 1)
µm. Conidia hyaline, allantoid or cylindrical, (4 –)6–8(–11) ×
(1.5–)2(–2.5) (av. 7 × 2) µm, L/W = 3.6.
Cultural characteristics — Colonies reaching a radius of
7.5–8.5 mm after 8 d at 25 °C. Minimum temperature for
growth 10 °C, optimum 20 °C, maximum 30 °C. Colonies on
MEA flat, mostly felty with very little aerial mycelium, appearing
yeast-like, with entire margin; after 8 d and 16 d colonies buff
(19''d), similar in reverse. Colonies on PDA flat, felty, with entire
to lobate margin; after 8 d buff (19''d), reverse same. Colonies
on OA flat, felty with entire margin; after 8 d and 16 d white
above.
Specimen examined. South AfricA, Western Cape province, Bonnievale,
from irregular necrosis with dark brown annual rings close to pruning wound in
wood of Prunus armeniaca, 23 Aug. 2005, U. Damm, CBS H-19945 holotype,
culture ex-type CBS 120862 = STE-U 6104.
Notes — Colonies do not have a distinct colour ranging
from buff (on MEA and PDA) to white (on OA). Of the various
pale-coloured species, Pm. pallidum resembles Pm. angustius,
especially in the predominance of the type I phialide and the
shape of the type II phialides that are subcylindrical or navicular
(Mostert et al. 2006a). Phaeoacremonium pallidum and T. vibra-
tilis can be distinguished by their optimum growth temperature
at 20 °C (Réblová & Mostert 2007). Phaeoacremonium pallidum
can be distinguished by the presence of all three phialide types
in comparison with Phaeoacremonium vibratilis having type I
and type II phialides.
Phaeoacremonium prunicolum L. Mostert, Damm & Crous,
sp. nov. — MycoBank 505139; Fig. 5
Phaeoacremonio novae-zealandiae simile, sed conidiis longioribus, in cultura
(OA) pigmento flavido nullo.
Etymology. Named after its host, Prunus.
Aerial structures — Mycelium consisting of branched, sep-
tate hyphae that occur singly or in bundles of up to 11; hyphae
tuberculate with warts up to 2 µm diam, verruculose, medium
brown and 1– 2 µm wide. Conidiophores short and usually
Fig. 4 Phaeoacremonium pallidum. a−e. Aerial structures on MEA; a. conidiophores; b. type III phialides; c. type II phialides (arrow head indicates polyphialide);
d. type I phialides; e. conidia. — f, g. Structures on the surface of and in MEA; f. conidia; g. adelophialides with conidia; all from CBS H-19945 (holotype). a−g:
DIC. — Scale bar: a = 10 µm, applies to a−g.
c
b
d
a
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U. Damm et al.: Phaeoacremonium on Prunus wood
unbranched, up to 2-septate, mostly bearing one terminal phi-
alide, 14– 37(–70) (av. 26) µm long and 1.5– 3 (av. 2) µm wide.
Phialides terminal or lateral, sometimes polyphialidic, sparsely
tuberculate to verruculose or smooth, pale brown to subhyaline;
collarettes slightly flaring, 1–1.5 µm long and 1–1.5 µm wide;
type I phialides cylindrical, occasionally widened at the base,
tapering towards the apex, (2 –)3 – 6(–9) × 1–1.5 (av. 5 × 1) µm;
type II phialides elongate-ampulliform and attenuated at the
base, or navicular, tapering towards the apex, (7–)9 –12 (–13)
× 1.5–2 (– 2.5) (av. 11 × 2) µm; type III phialides subcylindrical
to navicular, (13–)14 –18 (–21) × (1.5 –)2(– 3) (av. 16 × 2) µm,
gradually tapering towards the apex. Type I and III phialides
most common. Conidia hyaline, oblong-ellipsoidal, cylindrical or
allantoid, 5–7(–8) × 1–1.5(– 2) (av. 6 × 1.5) µm, L/W = 3.9.
On surface or submerged in the agar — Phialides hyaline,
cylindrical, (1–)2–6(–10) × 1(–1.5) (av. 4 × 1) µm. Conidia
hyaline, cylindrical or allantoid, 6 – 8(–10) × 1.5(– 2) (av. 7 ×
1.5) µm, L/ W = 4.6.
Cultural characteristics — Colonies reaching a radius of
8.5–10 mm in 8 d at 25 °C. Minimum temperature for growth
10 °C, optimum 25 °C, maximum 30 °C. Colonies on MEA flat,
mostly felty to short woolly, with entire margin; after 8 d and
16 d colonies olivaceous grey (21'''''i) to buff (19''d), similar in
reverse. Colony characters on PDA similar to those on MEA.
Colonies on OA flat, felty with woolly tufts, with entire margin;
after 8 d pale buff (19''f), after 16 d olivaceous green (23''') to
pale buff (19''f).
Specimens examined. South AfricA, Limpopo province, Mookgopong,
from irregularly roundish, reddish to greenish brown necrosis in wood of
Prunus salicina close to pruning wound, 31 Aug. 2004, U. Damm, CBS H-
19943 holotype, culture ex-type CBS 120858 = STE-U 5967; Mookgopong,
from reddish brown V-shaped necrosis in wood of Prunus salicina close to
pruning wound, 31 Aug. 2004, U. Damm, STE-U 5968.
Notes — Phaeoacremonium prunicolum can be distinguished
by having olive-grey colonies on MEA, PDA as well as OA.
Similar species such as Pm. novae-zealandiae also have olive-
grey colonies on MEA, but can be differentiated by its ability to
produce a yellow pigment on OA (Mostert et al. 2006a), whereas
this is absent in Pm. prunicolum.
Togninia africana Damm, L. Mostert & Crous, sp. nov. — Myco-
Bank 505138; Fig. 6
Anamorph. Phaeoacremonium sp.
Togniniae viticolae similis, sed peritheciis majoribus et ascosporis gut-
tulatis.
Etymology. Named after the continent of origin, Africa.
Fig. 5 Phaeoacremonium prunicolum. a –e. Aerial structures on MEA; a. conidiophores (arrow head indicates polyphialide); b. type III phialides; c. type II
phialides; d. type I phialides; e. conidia. — f, g. Structures on the surface of and in MEA; f. adelophialides with conidia; g. conidia; all from CBS H-19943
(holotype); a−g: DIC. — Scale bar: a = 10 µm, applies to a −g.
c
b
d
a
g
e f

96 Persoonia – Volume 20, 2008
Fig. 6 Togninia africana teleomorph and anamorph states. a. Perithecia on SNA; b. peridium; c. ascospores; d. longitudinal section through perithecium;
e. asci attached to ascogenous hyphae and paraphyses; f. asci; g. asci attached to ascogenous hyphae and paraphyses (remnant bases indicated by arrow
head). — h–m. Aerial structures on MEA; h, i. conidiophores; j. type III phialides; k. type II phialides; l. type I phialides; m. conidia. — n, o. Structures on the
surface of and in MEA; n. adelophialides; o. conidia; all from CBS H-19942 (holotype); a: DM, b –o: DIC. — Scale bars: a = 500 µm; b = 20 µm; c = 2.5 µm;
d = 200 µm; f = 5 µm; e, g, h = 10 µm; h applies to h– o.
c
b
d
a
g
h i
j
k
m
n
l
e f
o

97
U. Damm et al.: Phaeoacremonium on Prunus wood
Ascomata — Perithecia formed on SNA containing the pieces
of necrotic wood after 2 mo of incubation; non-stromatic, solitary,
superficial to semi-immersed, subglobose to obpyriform, (215–)
270–395(– 440) µm diam, basal part (270 –)315– 440 (– 460)
µm tall. Wall consisting of two regions of textura angularis: outer
region dark brown, individual cells hardly visible, 15 – 25 µm
thick; inner region pale brown becoming hyaline towards the
centre, 5 –7 cell layers and 10 –15 µm thick. Surface covered
with brown, septate hyphal appendages that become hyaline
towards the tip. Perithecial neck curved, 1 per perithecium,
550–1000 (av. 720) µm long, 70–130 µm wide at the base, 35–
65 µm wide at the tip. Paraphyses hyaline, septate, unbranched,
cylindrical with round tips, slightly constricted at septa, 30–130
(av. 80) µm long, narrowing from 3– 6.5 µm at the base to 1.5– 4
µm at the apex, persistent. Asci arising in acropetal succession
from sympodially proliferating ascogenous hyphae that appear
spicate when mature, hyaline, clavate, with bluntly rounded
apex and base, (16 –) 20 – 25.5(–26) × (3.5 –) 4 –5(– 5.5) (av.
22.5 × 4.5) µm. Ascogenous hyphae hyaline, branched, smooth-
walled, 15–28 × 1.5 –2.5 µm, remnant bases 4– 6.5 × 2–3.5 µm.
Ascospores aseptate, hyaline, smooth-walled, ellipsoidal to
subcylindrical with rounded ends, sometimes slightly bent,
containing small guttules at the ends, biseriate, (2.5–)3.5 –
4.5(– 5.5) × 1.5 – 2(–2.5) (av. 4 × 1.8) µm.
Aerial structures — Mycelium consisting of branched, sep-
tate hyphae that occur singly or in bundles of up to 22; hyphae
tuberculate, with warts up to 2 µm diam, verruculose to smooth,
mostly hyaline, some pale brown, 1–3 µm wide. Conidiophores
short and usually unbranched, up to 2-septate, sometimes
constricted at septa, bearing one terminal phialide, sometimes
showing percurrent rejuvenation, (13–)15 – 23(– 24) (av. 19)
µm long and 2 µm wide. Phialides terminal or lateral, mostly
monophialidic, sometimes polyphialidic, sparsely tuberculate to
verruculose, hyaline, sometimes subhyaline; collarettes slightly
flaring, 1–2 µm long and 1 µm wide; type I phialides most pre-
dominant, cylindrical, occasionally widened at the base, tapering
towards the apex, (2–)3 – 5(–7) × 1–1.5(–2) (av. 4 × 1) µm; type
II phialides subcylindrical, some elongate-ampulliform and at-
tenuated at the base, (4–)7–11(–12) × (1–)1.5– 2(–3) (av. 9 × 2)
µm; type III phialides subcylindrical or navicular, 13–20(–28)
× (1.5–) 2(– 2.5) (av. 16 × 2) µm. Type II and III phialides often
strongly tapered towards the apex. Conidia hyaline, cylindrical or
allantoid, (4.5–)5–8(–12) × 1.5– 2 (av. 7 × 1.5) µm, L/ W = 4.
On surface or submerged in the agar — Phialides hyaline,
cylindrical, 1–7(–18) × 1–1.5(– 2) (av. 4 × 1) µm. Conidia hyaline,
cylindrical or allantoid, (5–)6 – 9(–12) × 1.5 – 2 (av. 8 × 2) µm,
L/ W = 4.3.
Cultural characteristics — Colonies reaching a radius of
9–9.5 mm after 8 d at 25 °C. Minimum temperature for growth
10 °C, optimum 25 °C, maximum 30 °C. Colonies on MEA flat,
mostly felty with a few woolly tufts, with entire margin; after 8 d
and 16 d colonies buff (19''d), similar in reverse. Colonies on
PDA flat, felty, with entire margin; after 8 d buff (19''d), similar
in reverse; after 16 d vinaceous-buff (15'''d) becoming buff
(19''d) with straw undertone, reverse honey (19''b) to buff (19''d).
Colonies on OA flat, felty to woolly, with entire margin; after 8 d
and 16 d olivaceous-buff (21'''d) to buff (19''d) with straw (21'd)
undertone above. Prominent yellow pigment produced on MEA,
OA and after 16 d also on PDA.
Specimen examined. South AfricA, Western Cape province, Montagu,
from greenish brown V-shaped necrosis in wood under canker developing
from a broken-off twig of Prunus armeniaca, 23 Aug. 2005, U. Damm, CBS
H-19942 holotype, culture ex-type CBS 120863 = STE-U 6177.
Notes — Togninia africana can be distinguished by its buff
coloured colonies together with the production of a yellow pig-
ment on MEA. Phaeoacremonium angustius is similar to the
Phaeoacremonium anamorph of T. africana in the pale coloured
colonies, predominance of the type I phialide and production of
yellow pigment on OA. However, Phaeoacremonium angustius
forms yellow-white to grey-red colonies on OA (Mostert et al.
2006a) in comparison with olivaceous-buff colonies of T. afri-
cana. The type II and III phialides taper sharply towards the
apex, similar to the subulate phialides found in Pm. subulatum.
Only T. viticola has both perithecia that often exceed 300 µm
diam and mainly ellipsoidal ascospores as found in T. africana.
However, T. viticola has one to three necks per perithecium and
ascospores that can also be curved, whereas T. africana has
one neck per perithecium and ascospores that are predomi-
nantly straight.
Togninia griseo-olivacea Damm, L. Mostert & Crous, sp. nov.
— MycoBank 505137; Fig. 7
Anamorph. Phaeoacremonium sp.
Togniniae fraxinopennsylvanicae similis, sed peritheciis minoribus cum collo
breviore.
Etymology. Named after its greyish olivaceous colonies (griseolus-oliva-
ceus Lat. = greyish olivaceous).
Ascomata — Single-conidial isolates gave rise to perithecia
on PDA after 3 mo; non-stromatic, solitary, superficial, globose
to subglobose, dark brown, (150 –) 225 µm diam, (150–)200
µm tall (dimensions of only one mature perithecium available;
measurements of immature perithecia in brackets). Wall consist-
ing of two regions of textura angularis: outer region dark brown,
4–7 cells and 5–15 µm thick; inner region pale brown becoming
hyaline towards the centrum, 5 – 6 cell layers and 10 – 20 µm
thick. Surface covered with brown, septate hyphal appendages.
Perithecial neck black, straight, 1 per perithecium, (200 –)460
µm long, (40–)50 µm wide at the base, (25 –)37 µm wide at the
tip, dividing into two near the tip. Paraphyses hyaline, septate,
unbranched, cylindrical with rounded tips, slightly constricted at
septa, 30–70 (av. 45) µm long, 2–4.5 µm wide at the base. Asci
arising in acropetal succession from sympodially proliferating
ascogenous hyphae that appear spicate when mature, hyaline,
clavate, rounded apex and base, 15 –16 × 3– 4 (av. 15.5 × 3.5)
µm. Ascogenous hyphae hyaline, branched, smooth-walled,
13–23 × 2– 3 µm, remnant bases only occasionally observed,
6–9(–12) × 3 –4 (– 5) µm. Ascospores aseptate, hyaline,
smooth-walled, ellipsoidal to reniform with rounded ends, some-
times containing small guttules at the ends, biseriate, 3– 5(–6)
× 1.5–2 (av. 3.5 × 1.5) µm.
Aerial structures — Mycelium consisting of branched, septate
hyphae that occur singly or in bundles of up to 17; hyphae tuber-
culate with warts up to 2 µm diam, verruculose to verrucose,
medium brown, 1–2 µm wide. Conidiophores short and usually
unbranched, up to 2-septate, bearing one terminal phialide,
(20–)22 – 35(–42) (av. 29) µm long and 1.5–2 (av. 2) µm wide.

98 Persoonia – Volume 20, 2008
Fig. 7 Togninia griseo-olivacea teleomorph and anamorph states. a. Perithecium on PDA; b. longitudinal section through perithecium; c. peridium; d, e. asci
attached to ascogenous hyphae; f. ascospores; g. asci; h. paraphyses; i. asci attached to ascogenous hyphae. — j– p. Aerial structures on MEA; j. ring-like
growth of mycelium with conidiophore (indicated by arrow head); k. conidiophores; l. type III phialides; m. type II phialides; n, o. type I phialides; p. conidia.
— q, r. Structures on the surface of and in MEA; q. adelophialides with conidia; r. conidia; all from CBS H-19941 (holotype). a: DM, b −r: DIC. — Scale bars:
a = 100 µm; b = 50 µm; c– i = 5 µm; j = 10 µm; j applies to j– r.
c
b da
g
h
i
j
k
mn
l
e
f
q
r
p
o

99
U. Damm et al.: Phaeoacremonium on Prunus wood
Phialides terminal or lateral, mostly monophialidic, sometimes
polyphialidic, sparsely tuberculate to verruculose, medium to
pale brown, sometimes hyaline; collarettes slightly flaring, 1 µm
long and 1– 2 µm wide; type I phialides predominant, cylin-
drical, occasionally widened at the base, tapering towards
the apex, (2 –) 3 – 6(– 9) × 1–1.5 (– 2) (av. 4 × 1) µm; type II
phialides, elongate-ampulliform and attenuated at the base,
or subcylindrical, tapering towards the apex, (7–)9–12(–13) ×
1.5–2(– 2.5) (av. 11 × 2) µm; type III phialides mostly subcylin-
drical, some navicular, (13–)14–18(– 20) × (1.5 –)2 (av. 16 × 2)
µm, gradually tapering towards the apex. Conidia subhyaline,
oblong-ellipsoidal or reniform, (3.5–)4 – 5 (– 6) × 1.5– 2 (av. 5 ×
1.5) µm, L/ W = 2.8.
On surface or submerged in the agar — Phialides hyaline,
cylindrical, (1.5 –)2–6(–10) × 1(–1.5) (av. 4 × 1) µm. Conidia
hyaline, cylindrical, reniform or allantoid, (5–)6 –7(– 8) × 1.5– 2
(av. 6 × 2) µm, L/W = 3.6.
Cultural characteristics — Colonies reaching a radius of
9–9.5 mm in 8 d at 25 °C. Minimum temperature for growth
10 °C, optimum 25 °C, maximum 30 °C. Colonies on MEA flat,
mostly felty, becoming woolly with age, with entire margin; after
8 d colonies buff (19''d) above, in reverse olivaceous (21''k)
to buff (19''d); after 16 d mouse-grey (13'''''i) to buff (19''d)
above, similar in reverse. Colonies on PDA flat, felty with a few
woolly tufts, with entire margin; after 8 d buff (19''d) with a few
olivaceous (21''k) spots above, similar in reverse; after 16 d
olivaceous (21''k) ring in centre becoming buff (19''d) towards
margin, similar in reverse. Colonies on OA flat, felty with woolly
tufts and entire margin; after 8 d and 16 d isabelline (17''i) to
buff (19''d) above.
Specimen examined. South AfricA, Limpopo province, Mookgopong,
from reddish brown to greenish irregular necrosis with darker discoloured
annual rings in wood of Prunus armeniaca close to pruning wound with resin
exudation, 31 Aug. 2004, U. Damm, CBS H-19941 holotype, culture ex-type
CBS 120857 = STE-U 5966.
Notes — The various Togninia and Phaeoacremonium spe-
cies that have brownish coloured colony centres with a broad
buff ring towards the margin on MEA include Pm. austra-
liense, T. novae-zealandiae and T. parasitica. Togninia griseo-
olivacea can be distinguished from Pm. australiense and
T. novae-zealandiae by not producing a yellow pigment in
OA and, additionally, from T. novae-zealandiae by forming
shorter asci. Togninia griseo-olivacea differs from T. parasitica
in the shape of the ascospores that are usually allantoid in
T. parasitica and ellipsoidal to reniform in T. griseo-olivacea.
The Phaeoacremonium anamorph of T. griseo-olivacea does
not have such long conidiophores as T. parasitica (av. length
47 µm), prominent warts (up to 3 µm diam) or the predominant
type III phialides found in T. parasitica (Mostert et al. 2006a).
The Togninia species that have relatively short asci and short
oblong-ellipsoidal ascospores with ranges that overlap with
that of T. griseo-olivacea include T. argentinensis, T. austro-
africana, T. fraxinopennsylvanica and T. novae-zealandiae.
The size ranges of the asci and ascospores mostly resemble
that of T. fraxinopennsylvanica. Togninia griseo-olivacea can
be distinguished by having on average smaller perithecia and
a shorter neck length.
Pathogenicity
All the isolates had been obtained from discoloured wood inside
living branches of trees of different Prunus species. In cross-
section, the symptomatic wood had either irregularly shaped
or V-shaped necrotic lesions and were situated close to old
pruning wounds and /or cankers, sometimes also associated
with gummosis. In plum wood, such lesions were often reddish
brown in the centre and greenish towards the margin. Phaeoa-
cremonium species were mostly isolated from these lesions in
combination with other fungi, for example Botryosphaeriaceae
and Schizophyllum commune.
Fungal species Mean of lesion length (mm)1 Mean of re-isolation frequency (%)
Plum Apricot Plum Apricot
Togninia parasitica 55.0 a 63.2 a 75 8
Phaeoacremonium iranianum 36.1 bc 56.5 ab 80 38
Pm. subulatum 33.5 bc 57.7 ab 83 8
Pm. griseorubrum 32.8 bc 57.2 ab 70 55
T. africana 33.1 bc 53.9 ab 75 50
T. griseo-olivacea 39.4 b 38.8 bcd 92 50
T. minima 37.8 bc 45.7 abc 75 29
T. viticola 35.8 bc 47.6 abc 84 8
Pm. prunicolum 35.2 bc 33.4 cd 84 25
T. fraxinopennsylvanica 33.9 bc 45.7 abc 80 13
Pm. scolyti 33.2 bc 50.6 abc 80 17
Pm. australiense 33.0 bc 44.2 abcd 54 38
Pm. pallidum 31.5 bcd 38.7 bcd 25 0
Pm. fuscum 25.1 cd 39.3 bcd 100 8
Acremonium strictum 19.8 de 32.4 cd 25 0
Agar plug 12.6 e 24.8 d – –
LSD (P < 0.05) 12.0 19.9
1 Means followed by the same letter are not significantly different (P < 0.05).
Table 2 Means of lesion lengths caused by different Phaeoacremonium species on detached green plum and apricot
shoots, and mean re-isolation frequencies of these species from observed lesions.

100 Persoonia – Volume 20, 2008
Analyses of variance of the lesion length data on apricot and
plum cane sections indicated a significant treatment effect
(P < 0.0001; Anova tables not shown). All species, except Pm.
fuscum and Pm. pallidum, caused lesions in the xylem of plum
shoots that were significantly longer than the controls (Table 2).
The re-isolation frequencies from plum were between 70 % and
100 % for all fungi, except Pm. australiense and Pm. palli-
dum. Five species caused lesions on apricot shoots that were
significantly longer than the controls: T. parasitica, Pm. ira-
nianum, Pm. subulatum, Pm. griseorubrum and T. africana. Addi-
tionally, T. parasitica, Pm. iranianum and Pm. griseorubrum also
caused lesions that were visible on the bark surface of more
than half of the apricot canes; mostly dark brown rings around
the inoculation site. Other species formed surface lesions on
apricot canes less frequently. On plum, surface lesions on the
bark were only occasionally observed. Most of the species were
re-isolated from apricot wood in frequencies below 50 %. No
Phaeoacremonium species were isolated from the negative
controls. Togninia parasitica was the species that induced the
longest lesions on plum and apricot wood.
DISCUSSION
Although Phaeoacremonium species have previously been
relatively unknown from stone fruit trees, this study reveals
these hosts to harbour a broad diversity (14 species) and abun-
dance (present in 33 of the 257 specimens, with 3 specimens
occupied by more than one Phaeoacremonium species) of this
genus from Prunus trees in South Africa. Most species were
found in plum (8 species) and apricot (8 species) wood, while
peach and nectarine were rarely colonised by Phaeoacremo-
nium (2 species). However, there were no Phaeoacremonium
species known on Prunus salicina, P. persica and P. persica
var. nucipersica before the onset of this study. Most species
found on P. armeniaca also represent new reports on Prunus,
except for T. parasitica and T. minima (Hawksworth et al. 1976,
Mostert et al. 2006a). We also observed regional differences.
While the species found in the Cape Winelands (Paarl, Stellen-
bosch, Franschhoek) comprised only known species, three of
the four species found in the Limpopo province were new to
science. Reasons for this could be the relative remoteness of
the area compared to the Western Cape province (hosts not
previously sampled for microfungi), and the different climate
(summer-rainfall area vs winter-rainfall area).
The dominant species on stone fruit trees were Pm. scolyti and
T. minima. Togninia minima is known as one of the causal organ-
isms of Petri disease and esca on grapevines, and has previ-
ously been found on V. vinifera in South Africa (Groenewald et
al. 2001, Mostert et al. 2003). In this study, T. minima was found
on three Prunus species in the Western Cape province of South
Africa. Phaeoacremonium scolyti is also known on V. vinifera
in South Africa (Mostert et al. 2003). In our study, the fungus
had the broadest host range and was found on all Prunus spe-
cies sampled. According to Mostert et al. (2006b), Pm. scolyti
could be dispersed between woody hosts by bark beetles, as
it has previously been isolated from beetles (Kubátová et al.
2004). Rumbos (1986) assumed Pm. parasiticum to be spread
throughout a cherry orchard by bark and wood-boring beetles.
Phaeoacremonium scolyti was also the only species that oc-
curred in different orchards in the Western Cape and Limpopo
provinces of South Africa.
Notwithstanding the new taxa, several known species were
also found on Prunus, four of which comprise new reports for
South Africa. Phaeoacremonium australiense, Pm. iranianum
and T. fraxinopennsylvanica had been previously reported from
grapevines in other countries (Groenewald et al. 2001, Mostert
et al. 2006a, Gramaje et al. 2007). This study shows, however,
that these fungi also occur on Prunus species in South Africa.
Phaeoacremonium australiense was even quite common in one
orchard in the Western Cape. Phaeoacremonium griseorubrum,
which was previously known only from human infections in
Japan and the United States (Mostert et al. 2005), was found
here to also occur in wood of Prunus salicina in South Africa.
All Phaeoacremonium spp. were associated with wood decay
symptoms on Prunus trees. According to the pathogenicity test,
most species were shown to be potentially pathogenic to plum,
while only a few species were shown to be potentially pathogenic
to apricot. The species most commonly isolated from Prunus
wood, Pm. scolyti, was not the most virulent species. Three of
the species, Pm. subulatum, T. parasitica and T. viticola, had
been tested on grapevines in greenhouse and field experiments
(Halleen et al. 2007). While in our study, T. parasitica was the
most virulent Phaeoacremonium species on apricot and plum
wood, it was less virulent on grapevines trunks than T. viticola
and Pm. subulatum (Halleen et al. 2007). The relevance of
T. parasitica in die-back disease on Prunus species is uncertain,
since only one isolate was obtained. Rumbos (1986) showed
Pm. parasiticum to be pathogenic on cherry, apricot, olive and
peach. Togninia minima and T. parasitica also caused discol-
orations in wood of potted kiwifruit vines (Di Marco et al. 2004).
Associated field symptoms and the pathogenicity test indicate
a possible pathogenic relationship of these Phaeoacremonium
species and Prunus trees.
Only one of the five new Phaeoacremonium species, namely
Pm. fuscum, had a maximum growth temperature of 37 °C in
comparison with 30 °C for the other species. The ability to grow
at 37 °C suggests that it has the potential to survive at human
body temperature, while the other species appear to be strictly
plant-associated taxa.
Phaeoacremonium species are commonly isolated from healthy
(Halleen et al. 2003) and symptomatic grapevines (Mostert et al.
2006a). In grapevines they occur in association with other fungi,
namely Phaeomoniella chlamydospora, Fomitiporia species
and to a lesser extent, Stereum hirsutum (Larignon & Dubos
1997, Mugnai et al. 1999, Fischer 2002). In this study, Phaeoa-
cremonium species have mostly been found in combination
with other fungi. Because Phaeoacremonium species that had
frequently been isolated from diseased vines only gave a weak
host response in pathogenicity trials on grapevines, Halleen et
al. (2007) assumed that they might not be able to cause disease
on their own, but required synergism with other fungi of this
disease complex. Some of these fungi have been shown to be
associated with stress-related diseases (Ferreira et al. 1999),
and Halleen et al. (2007) only observed a clear disease expres-
sion in a field-trial monitored over a longer period.

101
U. Damm et al.: Phaeoacremonium on Prunus wood
Except for Pm. griseorubrum, all known Phaeoacremonium
species found on Prunus, had previously been isolated from
Vitis vinifera (Crous et al. 1996, Groenewald et al. 2001, Mostert
et al. 2005, 2006a). Phaeoacremonium species are known
as causal organisms of Petri disease, destructive grapevine
trunk disease (decline, die-back), and young grapevine decline
(Scheck et al. 1998). Petri disease is considered as a major
reason for the death of vines in nurseries and young vineyards
in the Western Cape province of South Africa (Halleen et al.
2003). Togninia minima is well-known on Vitis from South Africa
(Mostert et al. 2003), and T. minima, T. parasitica, T. viticola
and Pm. subulatum have been shown to be true wood colonis-
ers and vascular pathogens of grapevines (Sparapano et al.
2001, Halleen et al. 2007). Based on the results obtained in
the present study on different Prunus species, Phaeoacremo-
nium species seem to lack host-specificity. Since a number of
fungi, which have previously been reported to be pathogenic
to grapevines were isolated from the wood of Prunus spp.,
stone fruit orchards should be considered as potential inoculum
sources of grapevine trunk disease pathogens. Pathogenic or
saprobic survival of these grapevine trunk disease pathogens in
stone fruit orchards could have serious implications for disease
management practices employed on farms where vineyards
are planted adjacent to fruit tree orchards.
Acknowledgements The authors acknowledge the University of Stellen-
bosch, National Research Foundation, THRIP, Winetech and the Deciduous
Fruit Producer’s Trust for financial support. Prof. Uwe Braun, Martin-Luther-
Universität Halle-Wittenberg, Institut für Geobotanik und Botanischer Garten,
is kindly thanked for providing the Latin diagnoses.
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