STUDIES IN MYCOLOGY 55: 163–173. 2006.
Re-evaluating the taxonomic status of Phaeoisariopsis griseola,
the causal agent of angular leaf spot of bean
Pedro W. Crous1*, Merion M. Liebenberg2, Uwe Braun3 and Johannes Z. Groenewald1
1Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands; 2ARC Grain Crops
Institute, P. Bag X1251, Potchefstroom 2520, South Africa; 3Martin-Luther-Universität, FB. Biologie, Institut für Geobotanik und Botanischer
Garten, Neuwerk 21, D-06099 Halle (Saale), Germany
*Correspondence: Pedro W. Crous, crous@cbs.knaw.nl
Abstract: Angular leaf spot of Phaseolus vulgaris is a serious disease caused by Phaeoisariopsis griseola, in which two major gene pools occur,
namely Andean and Middle-American. Sequence analysis of the SSU region of nrDNA revealed the genus Phaeoisariopsis to be indistinguishable
from other hyphomycete anamorph genera associated with Mycosphaerella, namely Pseudocercospora and Stigmina. A new combination is
therefore proposed in the genus Pseudocercospora, a name to be conserved over Phaeoisariopsis and Stigmina. Further comparisons by
means of morphology, cultural characteristics, and DNA sequence analysis of the ITS, calmodulin, and actin gene regions delineated two groups
within P. griseola, which are recognised as two formae, namely f. griseola and f. mesoamericana.
Taxonomic novelties: Pseudocercospora griseola (Sacc.) Crous & U. Braun comb. nov., P. griseola f. mesoamericana Crous & U. Braun f.
nov.
Key words: Ascomycetes, DNA sequence comparisons, Mycosphaerella, Phaeoisariopsis, Phaseolus vulgaris, Pseudocercospora,
systematics.
INTRODUCTION
Angular leaf spot (ALS) of beans (Phaseolus vulgaris)
is caused by Phaeoisariopsis griseola (Sacc.) Ferraris.
The disease is of major importance in tropical and
subtropical areas, causing yield losses of up to 80 %
(Schwartz et al. 1981, Saettler 1991, Liebenberg &
Pretorius 1997). The disease affects pods and foliage,
and is particularly destructive in warm, humid areas
(Saettler 1991). Pod symptoms consist of circular
to elliptical red-brown lesions, while leaf lesions
start as small, brown or grey spots that become
angular and necrotic, being confined by leaf veins.
Leaf spots eventually coalesce, causing premature
defoliation (Correa-Victoria et al. 1989, Saettler 1991).
Furthermore, the disease also affects the quality and
marketability of seed across bean-producing areas of
the world (Pastor-Corrales et al. 1998).
In the Great Lakes Region of Africa, losses
attributed to ALS have been estimated to be around
374 800 t (Wortmann et al. 1998). Disease control is
best achieved via the selection of resistant varieties.
Breeding for resistance against ALS is complicated,
as the pathogen is highly variable with regard to
pathogenicity, which means that durable resistance is
difficult to achieve (Pastor-Corrales et al. 1998). High
levels of pathogenic and genetic variation have been
reported in P. griseola by various authors (Guzmán et
al. 1995, Boshoff et al. 1996, Busogoro et al. 1999,
Mahuku et al. 2002, Wagara et al. 2004).
There are indications of at least two main,
morphologically distinguishable domestication events
for the common bean, which in turn gave rise to two
main gene pools, namely large-seeded beans of
Andean origin, and small to medium-sized beans of
Middle-American origin (Brown et al. 1982, Gepts &
Bliss 1985, 1986, Gepts et al. 1986, Koenig & Gepts
1989, Sprecher & Isleib 1989, Koenig et al. 1990, Singh
et al. 1991a, b, Miklas & Kelly 1992, Skroch et al. 1992,
Chacón et al. 2005).
Several fungal pathogens of P. vulgaris, in particular
Phaeoisariopsis griseola, causal organism of ALS,
Colletotrichum lindemuthianum (Sacc. & Magnus)
Briosi & Cavara, the causal organism of anthracnose,
and Uromyces appendiculatus (Pers. : Pers.) Unger
var. appendiculatus, the causal organism of bean
rust, have undergone parallel micro-evolution with the
host. Although there is considerable variation within
gene pools, differences are particularly evident when
the reactions of isolates to differential lines of known
Andean and Middle-American origin are compared.
Isolates originating from the Andes are virulent only
on large-seeded lines, whereas those originating from
countries such as Central America, Mexico, Bolivia and
Brazil are generally virulent on lines from both groups
(Steadman 1995, Liebenberg 1996, Pastor-Corrales
1996, Chacón et al. 1997, Araya & Steadman 1998,
Sandlin et al. 1999, Araya et al. 2004). Using isozyme
analysis, Correa-Victoria (1987) could distinguish two
groups in 55 P. griseola isolates from Africa, the U.S.A.
and Latin America. All 26 isolates from Africa clustered
in one group, whereas Latin American isolates clustered
in both groups. However, recently the presence of both
groups was reported from Africa (Liebenberg 1996,
Wagara et al. 2004), which was also supported by data
derived from isozyme analysis (Boshoff et al. 1996).
Guzmán et al. (1995) used RAPD analysis to divide 62
P. griseola isolates from Brazil, Wisconsin (U.S.A.) and
Malawi into two broad groups. Isolates in the Andean
group, collected predominantly from Andean bean
host genotypes, were more pathogenic on Andean
genotypes, whereas those from the second group,
163
CROUS ET AL.
originating predominantly from Middle-American bean
genotypes, were more pathogenic on Middle-American
bean genotypes. The 11 Brazilian isolates fell in the
second group, whereas 39 of the 42 Malawian isolates
belonged to the Andean group. This grouping reflects
the preference for small-seeded beans in Brazil, and
large-seeded beans in Malawi. A third, more virulent
group reported in Africa (CIAT 1996, Liebenberg 1996)
appears to be a variation of the Andean group (Mahuku
et al. 2002).
Buruchara (1983) observed differences in conidial
size and amount of septation between isolates. However,
he concluded that, due to the extent of variation within
groups, these characteristics could not be used for
grouping isolates. Several authors have attempted to
associate lesion size with pathogenicity differences.
Verma & Sharma (1984) observed two types of lesions
in the field that differed in size, but found no significant
differences in the number and size of lesions caused
by the two groups of isolates, or in their radial growth in
culture. Lesion size can vary considerably, but CorreaVictoria (1987) found no significant correlation between
disease severity and lesion size, and no correlation
between spore production and lesion size, but reported
it to be highly dependent on the host cultivar (CorreaVictoria 1987). Lesion size may be affected by the
interaction between host gene pool and pathogen origin
(Liebenberg et al. 1996). These phenomena gave rise
to questions as to the extent of differences between the
Andean and Middle-American groups.
Ferraris (1909) erected the genus Phaeoisariopsis
Ferraris for four Isariopsis-like species, including
Isariopsis griseola Sacc. (Saccardo 1878), the type
species, characterised by having synnematous
conidiophore fascicles and pigmented conidiophores
and conidia. In subsequent years several diverse
elements were included in the genus (Ellis 1971, 1976,
von Arx 1983). Chupp (1954) described a bean pathogen
in his monograph under Cercospora columnaris Ellis
& Everh., but cited the older name Phaeoisariopsis
griseola as synonym. In his notes he stressed to favour
the retention of Phaeoisariopsis. Deighton (1990)
reassessed the genus, and considered the synnematous
arrangement of conidiophores to be unsuitable as sole
character for generic differentiation. Subsequently he
confined Phaeoisariopsis to a few species similar to P.
griseola, having non-geniculate conidiogenous cells
with flattened, but conspicuous scars. Deighton placed
species with conspicuously geniculate conidiogenous
cells and thickened, darkened scars in Passalora Fr.,
whereas taxa with quite inconspicuous conidiogenous
loci were reallocated to Pseudocercospora Speg. Von
Arx (1983) and Braun (1992, 1995a, b) preferred to
maintain Phaeoisariopsis, based on synnematous
conidiomata, but confined it to species with conspicuous
(slightly thickened, not darkened) conidiogenous loci.
The primary aim of the present study was to
resolve the generic status of Phaeoisariopsis within
Mycosphaerella Johanson, for which a subset of
isolates were subjected to DNA sequence analysis of
the SSU region. A further aim was to compare isolates
of the Andean and Middle-American groups to address
164
the question if they represent two groups or species.
For this purpose isolates were compared by means
of morphology, cultural characteristics, and DNA
sequence analysis of their internal transcribed spacer
region (ITS-1, ITS-2 and 5.8S), calmodulin, and actin
regions.
MATERIALS AND METHODS
Isolates
Phaseolus leaves exhibiting ALS symptoms, collected
in Africa and South America, were studied (Table 1).
Single-conidial cultures were established on 2 % malt
extract agar (MEA) (Biolab, Midrand, South Africa) as
outlined by Crous (1998). Colonies were subcultured
onto 2 % potato-dextrose agar (PDA; Gams et al.
1998) and incubated at 25 °C under continuous nearultraviolet light to promote sporulation.
DNA phylogeny
Genomic DNA was isolated from fungal mycelium
grown on MEA in Petri dishes and the ITS, actin
(ACT) and calmodulin (CAL) regions were amplified
and sequenced using the protocols and primers as
described by Crous et al. (2004). The 5’ end of the 18S
rRNA gene (SSU) was amplified and sequenced as
described by Braun et al. (2003).
The nucleotide sequences generated in this study
were added to other sequences obtained from GenBank
(http://www.ncbi.nlm.nih.gov) and the alignment
was assembled using Sequence Alignment Editor v.
2.0a11 (Rambaut 2002) with manual improvement of
the alignment where necessary. Sequence data were
analysed as explained in Braun et al. (2003) using
PAUP (Phylogenetic Analysis Using Parsimony) v.
4.0b10 (Swofford 2002) with both neighbour-joining
and parsimony algorithms. Neighbour-joining analyses
were conducted with the uncorrected (“p”), the Kimura
2-parameter and the HKY85 substitution models in
PAUP. When they were encountered, ties were broken
randomly. For parsimony analysis, alignment gaps were
treated as new character states and all characters were
unordered and of equal weight. Heuristic searches were
performed with 10 random taxon additions. A partition
homogeneity test (Farris et al. 1994) was conducted in
PAUP to consider the feasibility of combining the ITS,
actin and calmodulin data sets Sequence data were
deposited in GenBank (Table 1) and the alignments in
TreeBASE (S1507, M2709-10).
Determination of virulence phenotypes
The monoconidial isolates studied (Table 1) have
previously been subjected to virulence phenotype
characterisation on ALS differential lines from both
the large- and small-seeded gene pools, as published
previously (Liebenberg 1996, Mahuku et al. 2002).
Morphology and cultural characteristics
Wherever possible, thirty measurements (× 1000
magnification) were made of structures mounted in
ANGULAR LEAF SPOT OF BEAN
lactic acid, and the extremes of spore measurements
given in parentheses. Colony colours (surface and
reverse) were assessed after 14 d on PDA at 25 °C
in the dark, using the colour charts of Rayner (1970).
Cardinal temperatures for growth (from 9–33 °C,
in 3° intervals) were determined on PDA plates as
explained in Crous (1998). All cultures obtained in
this study are maintained in the culture collection of
the Centraalbureau voor Schimmelcultures (CBS) in
Utrecht, the Netherlands (Table 1).
these characters 38 are parsimony-informative, 57 are
variable and parsimony-uninformative, and 934 are
constant. Neighbour-joining analysis using the three
substitution models on the sequence data yielded trees
with identical topologies (data not shown). The same
overall topology was also obtained with the parsimony
analysis, which yielded 13 most parsimonious trees
(TL = 135 steps; CI = 0.807; RI = 0.809; RC = 0.653),
one of which is shown in Fig. 1. In this tree, species
of Pseudocercospora and Stigmina form a welldefined clade (bootstrap support value of 83 %) within
Mycosphaerella.
The ITS region was sequenced to provide better
resolution of the order of the species within the
Pseudocercospora clade. The manually adjusted ITS
sequence alignment contains 45 isolates (including the
two outgroups) and 499 characters including alignment
gaps; of these characters 168 are parsimony-informative,
25 are variable and parsimony-uninformative, and 306
RESULTS
DNA phylogeny
The manually adjusted SSU sequence alignment
contains 29 isolates (including the two outgroups)
and 1029 characters including alignment gaps; of
Cladosporium cladosporioides AY251091
Cladosporium herbarum AY251096
Dissoconium dekkeri AY251101
Pseudocladosporium hachijoense AY251100
Mycosphaerella populorum AF271130
Mycosphaerella sp. AY251116
56
Mycosphaerella punctiformis AY490775
Septoria tritici AY251117
Passalora fulva AY251109
100
Pseudocercospora cruenta AY251105
83
Pseudocercospora vitis CPC 11595
85
CBS 194.47
CBS 880.72
Pseudocercospora griseola
CPC 10779
Pseudocercospora protearum var. leucadendri AY251107
“Stigmina” platani CPC 4299
61
Cercospora zebrina AY251104
Septoria epambrosiae AF279583
Mycosphaerella latebrosa AY251114
Mycosphaerella sp. AY251115
83 Ramulispora sorghi AY251110
Ramulispora sorghi AY251111
Passalora janseana AY251103
53
1 change
Ramularia sp. AY251112
56
62
Septoria rosae AY251113
Batcheloromyces proteae AY251102
Mycosphaerella ohnowa AY251119
Trimmatostroma macowanii AY251118
Mycosphaerella nubilosa AY251120
Fig. 1. One of 15 most parsimonious trees obtained from a heuristic search with 10 random taxon additions of the 18S rRNA gene sequence
alignment. The scale bar shows a single change and bootstrap support values from 1000 replicates are shown at the nodes. Thickened lines
indicate the strict consensus branches and the tree was rooted to two Cladosporium species.
165
Species
Accession number1
Host
Virulence type
Origin
Collector
GenBank numbers2
Cladosporium herbarum
CBS 572.78
Polyporus radiatus
—
Russia
—
DQ289799, DQ289831, —, DQ289866
Davidiella tassiana
CPC 11600
Delphinium barbeyi
—
U.S.A.
A. Ramalay
DQ289800, DQ289832, —, DQ289867
Pseudocercospora griseola f. griseola
CBS 194.47; ATCC 22393
Phaseolus vulgaris
—
Portugal
—
DQ289801, DQ289833, DQ289861, DQ289868
CBS 880.72
Phaseolus vulgaris
—
Netherlands
H. A. v. Kesteren
DQ289802, DQ289834, DQ289862, DQ289869
CPC 5592; Pg97MZ41
Phaseolus vulgaris
Andes
Zambia
R. Buruchara
DQ289803, DQ289835, —, DQ289870
CPC 5594; Pg97LB48
Phaseolus vulgaris
Andes
South Africa
M.M. Liebenberg
DQ289804, DQ289836, —, DQ289871
CPC 10457; Pg97MZ64
Phaseolus vulgaris
Andes
Zambia
R. Buruchara
DQ289805, DQ289837, —, DQ289872
CPC 10458; Pg96CE7
Phaseolus vulgaris
Andes
South Africa
M.M. Liebenberg
DQ289806, DQ289838, —, DQ289873
CPC 10459; Pg97CE78
Phaseolus vulgaris
Andes
South Africa
M.M. Liebenberg
DQ289807, DQ289839, —, DQ289874
CPC 10460; Pg97AT101
Phaseolus vulgaris
Andes
Tanzania
F.S. Ngulu; C. Mushi
DQ289808, DQ289840, —, DQ289875
CPC 10464; Pg97CE105
Phaseolus vulgaris
Andes
—
—
DQ289809, DQ289841, —, DQ289876
CPC 10465; Pg97CE106
Phaseolus vulgaris
Andes
—
—
DQ289810, DQ289842, —, DQ289877
CPC 10467; Pg97MZ42
Phaseolus vulgaris
Andes
Zambia
R. Buruchara
DQ289811, DQ289843, —, DQ289878
CPC 10468; Pg97AT95
Phaseolus vulgaris
Andes
Tanzania
F.S. Ngulu; C. Mushi
DQ289812, DQ289844, —, DQ289879
CPC 10469; Pg97KZ44
Phaseolus vulgaris
Andes
Zambia
R. Buruchara
DQ289813, DQ289845, —, DQ289880
CPC 10477; Pg97CE23
Phaseolus vulgaris
Andes
South Africa
M.M. Liebenberg
DQ289814, DQ289846, —, DQ289881
CPC 10480; Pg96VI90
Phaseolus vulgaris
Andes
South Africa
M.M. Liebenberg
DQ289815, DQ289847, —, DQ289882
CPC 10481; Pg95GT5
Phaseolus vulgaris
Andes
South Africa
A.J. Liebenberg
DQ289816, DQ289848, —, DQ289883
CPC 10484; Pg95CE7
Phaseolus vulgaris
Andes
South Africa
M.M. Liebenberg
DQ289817, DQ289849, —, DQ289884
CPC 10779
Phaseolus vulgaris
—
Korea
H.D. Shin
DQ289818, DQ289850, DQ289863, DQ289885
CPC 12238; Pg350
Phaseolus vulgaris
Andes
Colombia
G. Mahuku
DQ289819, DQ289851, —, DQ289886
CPC 12239; Pg3
Phaseolus vulgaris
Andes
Colombia
G. Mahuku
DQ289820, DQ289852, —, DQ289887
CPC 12240; Pg266
Phaseolus vulgaris
Andes
Colombia
G. Mahuku
DQ289821, DQ289853, —, DQ289888
CPC 5596; Pg99GT4
Phaseolus vulgaris
Middel-Amerikaans
South Africa
A.J. Liebenberg
DQ289822, DQ289854, —, DQ289889
CPC 5597; Pg97TM109
Phaseolus vulgaris
Middel-Amerikaans
Malawi
A.J. Liebenberg
DQ289823, DQ289855, —, DQ289890
CPC 10463; Pg96GT35
Phaseolus vulgaris
Middel-Amerikaans
South Africa
M.M. Liebenberg
DQ289824, DQ289856, —, DQ289891
CPC 10474; Pg96GT32
Phaseolus vulgaris
Middel-Amerikaans
South Africa
M.M. Liebenberg
DQ289825, DQ289857, —, DQ289892
CPC 10479; Pg99CE5
Phaseolus vulgaris
Middel-Amerikaans
South Africa
M.M. Liebenberg
DQ289826, DQ289858, —, DQ289893
CPC 12241; Pg8
Phaseolus vulgaris
Middel-Amerikaans
Honduras
G. Mahuku
DQ289827, DQ289859, —, DQ289894
CPC 12242; Pg32
Phaseolus vulgaris
Middel-Amerikaans
Colombia
G. Mahuku
DQ289828, DQ289860, —, DQ289895
Pseudocercospora vitis
CPC 11595
Vitis vinifera
—
Korea
H.D. Shin
DQ289829, —, DQ289864, —
CPC 11660
Vitis flexuosa
—
Korea
H.D. Shin
DQ289830, —, —, —
“Stigmina” platani
CBS 110755; CPC 4299; IMI 136770
Platanus orientalis
—
India
—
AY260090, —, DQ289865, —
(ITS, CAL, SSU, ACT)
Ps. griseola f. mesoamericana
1ATCC: American Type Culture Collection, Virginia, U.S.A.; CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; IMI: International Mycological Institute, CABI-
Bioscience, Egham, U.K.
2ITS: internal transcribed spacer region, CAL: partial calmodulin gene, SSU: partial 18S rRNA gene, ACT: partial actin gene. All DQ numbers refer to newly generated sequences.
CROUS ET AL.
166
Table 1. Isolates used for sequence analysis.
ANGULAR LEAF SPOT OF BEAN
are constant. Neighbour-joining analysis using the
three substitution models on the sequence data yielded
trees with identical topologies (data not shown). Only
the order and grouping of the deeper nodes differed
between the neighbour-joining and parsimony analyses
(data not shown). Parsimony analysis yielded 13 most
parsimonious trees (TL = 293 steps; CI = 0.816; RI =
0.918; RC = 0.749), one of which is shown in Fig. 2. In
this tree, isolates of Ps. griseola are grouped together
with a bootstrap support value of 100 %, with the MiddleAmerican isolates (Ps. griseola f. mesoamericana)
grouping together with a bootstrap support value of 84
%. Also in the tree are other Pseudocercospora species
10 changes
(89 % bootstrap support), two strains of Ps. vitis (type
species of Pseudocercospora, 95 % bootstrap support)
and a basal well-defined clade (bootstrap support value
of 100 %) of two GenBank sequences of Stigmina
platani.
To determine whether Ps. griseola isolates from
Middle-American and Andean origin can be distinguished
phylogenetically, the ACT (235 characters) and CAL
(316 characters) sequences were combined with the ITS
sequences. The partition homogeneity test showed that
the three loci were combinable into a single analysis (P
= 0.6550). The manually adjusted combined alignment
consists of 1050 bases (including alignment gaps) and
Cladosporium herbarum CBS 572.78
Davidiella tassiana CPC 11600
CPC 12239
CPC 5592
CPC 5594
CPC 10457
CPC 10458
CPC 10459
100 CPC 10460
CPC 10464
CPC 10465
CPC 10467
CPC 10468
Ps. griseola f. griseola
CPC 10469
CPC 10477
66 CPC 10480
CPC 10481
CBS 880.72
CBS 194.47
CPC 10484
CPC 10779
CPC 12238
CPC 12240
89
AF222834
CPC 10474
CPC 5596
CPC 5597
Ps. griseola f. mesoamericana
84 CPC 12242
CPC 10463
CPC 10479
CPC 12241
Ps. protearum var. leucadendri AY260089
97 Ps. basiramifera AF309595
100
Ps. paraguayensis AF309596
89 53
100 AY266148
Ps. musae
AY266149
Ps. eucalyptorum AF309598
Ps. eucalyptorum AF309599
68
56
Ps. natalensis AF309594
Ps. robusta AF309597
Ps. syzygiicola AF309600
CPC 11595
Ps. vitis
95 CPC 11660
100
AF222849
“Stigmina” platani
AY260090
Fig. 2. One of 13 most parsimonious trees obtained from a heuristic search with 10 random taxon additions of the ITS sequence alignment.
The scale bar shows 10 changes and bootstrap support values from 1000 replicates are shown at the nodes. Thickened lines indicate the strict
consensus branches and the tree was rooted to Cladosporium herbarum and Davidiella tassiana.
167
CROUS ET AL.
52 / 71 % bootstrap support, respectively), which is
the result of three characters that changed in the CAL
sequence of isolates CPC 12238 and CPC 12239
(99.04 % sequence similarity to the other Ps. griseola
f. griseola isolates).
30 isolates (including the two outgroups). Of the 1050
characters, 288 are parsimony-informative, 42 were
variable and parsimony-uninformative, and 720 were
constant. The topologies of the trees obtained from
the neighbour-joining analyses were identical to each
other and also to that obtained from the parsimony
analysis (data not shown). Parsimony analysis of the
combined data yielded three most parsimonious trees
(TL = 353 steps; CI = 0.994; RI = 0.994; RC = 0.988),
one of which is shown in Fig. 3. The tree shows two
distinct clades, namely Ps. griseola f. griseola and what
we call here the Ps. griseola f. mesoamericana clade.
Bootstrapping using parsimony results in support
values of 53 % and 71 % for each clade, respectively.
These values increase to 62 % and 98 %, respectively,
if neighbour-joining with the HKY85 substitution model
is used for bootstrapping. The Ps. griseola f. griseola
clade is further split into two groups (62 / 95 % and
Taxonomy
Pseudocercospora griseola (Sacc.) Crous & U.
Braun, comb. nov. MycoBank MB500855. Fig. 4.
Basionum: Isariopsis griseola Sacc., Michelia 1: 273. 1878.
≡ Phaeoisariopsis griseola (Sacc.) Ferraris, Ann. Mycol. 7: 273.
1909.
≡ Lindaumyces griseolus (Sacc.) Gonz. Frag. (as “g riseola”),
Mem. R. Acad. Ci. Exact. Madrid, Ser. 2, 6: 339. 1927.
≡ Cercospora griseola (Sacc.) Ragunath. & K. Ramakr., J.
Madras Univ. 35–36: 11. (1965–1966) 1968.
= Cylindrosporium phaseoli (Cylindrospora) Rabenh., Klotzschii
Herbarium vivum mycologicum, Editio nova, Series Prima,
Centuria 4, No. 327, Dresden 1856, nom. nud., also Bot. Zeitung
15(6): 94. 1857, nom. nud. and Flora 15(9): 134. 1857, nom.
nud.
Cladosporium herbarum CBS 572.78
Davidiella tassiana CPC 11600
CPC 5592
CPC 5594
CPC 10457
CPC 10458
CPC 10459
CPC 10460
CPC 10464
CPC 10465
CPC 10467
62 / 95
CPC 10468
CPC 10469
Ps. griseola f. griseola
CPC 10477
CPC 10480
CPC 10481
53 / 62 CBS 880.72
CBS 194.47
CPC 10484
CPC 10779
CPC 12240
100 / 100
CPC 12238
CPC 12239
10 changes
CPC 10474
52 / 71
CPC 5596
CPC 5597
71 / 98
CPC 12242
Ps. griseola f. mesoamericana
CPC 10463
CPC 10479
CPC 12241
Fig. 3. One of three most parsimonious trees obtained from a heuristic search with 10 random taxon additions of a combined ITS, actin and
calmodulin sequence alignment. The scale bar shows 10 changes and bootstrap support values from 1000 replicates are shown at the nodes
(values from parsimony before the slash and neighbour-joining with the HKY85 substitution model after the slash). Thickened lines indicate
branches found in the strict consensus parsimony tree and the tree was rooted to Cladosporium herbarum and Davidiella tassiana.
168
ANGULAR LEAF SPOT OF BEAN
= Graphium laxum Ellis, Bull. Torrey Bot. Club 8: 64. 1881.
≡ Isariopsis laxa (Ellis) Sacc., Syll. Fung. 4: 631. 1886.
≡ Phaeoisariopsis laxa (Ellis) S.C. Jong & E.F. Morris,
Mycopathol. Mycol. Appl. 34: 269. 1968.
= Cercospora solimanii Speg. (solimani), Anales Soc. Ci. Argent.
16: 167. 1883.
= Cercospora columnaris Ellis & Everh. (as “columnare”), Proc.
Acad. Nat. Sci. Philadelphia 46: 380. 1894.
≡ Pseudocercospora columnaris (Ellis & Everh.) J.M. Yen, in
Yen & Lim, Gard. Bull., Singapore 33: 172. 1980.
= Arthrobotryum puttemansii Henn., Hedwigia 41: 309. 1902.
= Cercospora stuhlmannii Henn., Bot. Jahrb. Syst. 33: 40.
1904.
Syntypes: on Phaseolus vulgaris, Italy, Selva, Aug.
1877, Saccardo, Mycotheca Veneta 1247 (e.g., B,
HAL, PAD).
Formae novae:
Pseudocercospora griseola (Sacc.) Crous & U.
Braun, f. griseola
Specimen examined: Tanzania, on Phaseolus vulgaris, F.S. Ngulu &
C. Mushi, CBS H-19683, epitype designated here, CBS 119906 =
CPC 10468. culture ex-epitype. The epithet “griseola” was based on
European material, and from our analysis, it appears that European
material is representative of P. griseola f. griseola.
Pseudocercospora griseola (Sacc.) Crous & U.
Braun, f. mesoamericana Crous & U. Braun f. nov.
MycoBank MB500856.
Differt a f. griseola variatione virulentiae majore, culturis crescentibus
ad ≥ 30 °C.
Morphologically similar to P. griseola f. griseola, but
distinct by having a broader range of virulence on
different bean types, and being able to grow at or above
30 °C, which is not the case for f. griseola.
Specimen examined: South Africa, on Phaseolus vulgaris, M.M.
Liebenberg, CBS H-19684, holotype, culture ex-type CBS 119113
= CPC 10463.
Descriptions (selection): Gonzáles Fragoso (1927:
339), Chupp (1954: 295, as Cercospora columnaris),
Ellis (1971: 269), Shin & Kim (2001: 151–153).
Illustrations (selection): Saccardo, Fungi italici, Pl. 838,
Padova 1881; Briosi & Cavara, Funghi parassiti delle
piante coltivate od utili, Fasc. I, No. 17, figs 1–2, Pavia
1888; Gonzáles Fragoso (1927: 340, fig. 79); Ellis
(1971: 269, fig. 183); Deighton (1990: 1098, figs 2–3);
Shin & Kim (2001: 153, fig. 65).
Description in vivo: On leaves, petioles, stems and
pods; leaf spots amphigenous, angular–irregular, rarely
subcircular–elliptical, mostly vein-limited, 1–8 mm wide,
finally sometimes confluent, forming larger patches,
brown, ranging from pale olivaceous, olivaceousbrown, yellowish brown, greyish brown to dark brown,
on pods often reddish brown and more regular,
subcircular–elliptical, margin indefinite, only delimited
by veins, or surrounded by a narrow, dark brown border
or marginal line. Caespituli on petioles, pods, stems and
leaves, amphigenous, mostly hypophyllous, usually
scattered, occasionally aggregated, conspicuous,
punctiform, dark brown to blackish grey. Mycelium
internal. Stromata almost lacking to well-developed,
subglobose, depressed to lacrimoid, up to 70 µm
diam, brown. Conidiophores numerous, up to approx.
40, in dense fascicles, often forming synnematous
conidiomata, erumpent, 100–500 × 20–70 µm, rarely
longer, olivaceous-brown, composed of a more or
less firm stipe of closely appressed conidiophores
and a terminal, loose capitulum, i.e. conidiophores
splaying out at the end of the conidiomata, free ends
usually up to 100 µm long, individual conidiophores
filiform, appressed threads 2–5 µm wide, up to 7 µm
wide towards the apex, pluriseptate, subhyaline to
olivaceous-brown, thin-walled, occasionally becoming
rough-walled with age. Conidiogenous cells integrated,
terminal, 20–100 µm long, subcylindrical to subclavate,
usually not or only barely geniculate, but moderately
geniculate in some collections; conidiogenous
loci terminal and lateral, quite inconspicuous to
subconspicuous, i.e. unthickened or almost so, but
slightly darkened-refractive, in surface view visible
as minute circles, 1.5–2.5 µm diam, usually flat, nonprotruding. Conidia solitary, obclavate-cylindrical,
broadly subfusiform, short conidia sometimes ellipsoidovoid to short cylindrical, straight to curved, 20–75(–85)
× 4–9 µm, (0–)1–5(–6)-septate, usually not constricted
at the septa, rarely with slight constrictions, subhyaline
to pale olivaceous or olivaceous-brown, thin-walled,
smooth, sometimes rough-walled, with obtuse apex,
and obconically truncate to rounded base, 1.5–2.5(–
3) µm wide, hila unthickened or almost so, at most
somewhat refractive.
Cultural characteristics: Forma griseola; on OA colonies
flat to slightly erumpent, spreading with moderate
aerial mycelium; margins smooth, regular, surface with
patches of olivaceous-grey and smoke-grey to dirtywhite; on PDA erumpent with moderate aerial mycelium,
surface pale olivaceous-grey to olivaceous-grey in the
central part; margin iron-grey, and also iron-grey in
reverse. Cardinal temperature requirements for growth:
minimum 6 > °C, optimum = 24 °C, maximum < 30 °C.
Forma mesoamericana; on OA flat to slightly erumpent,
spreading, with moderate aerial mycelium; margins
irregular, feathery to smooth, even; surface with the
central part dirty-white to pale or darker olivaceous-grey,
outer region iron-grey; on PDA spreading, erumpent,
with moderate aerial mycelium; surface olivaceousgrey in the central part; outer region and reverse irongrey, margins feathery, irregular. Cardinal temperature
requirements for growth: minimum 6 > °C, optimum 24
°C, maximum > 30 °C.
Herbarium specimens examined: On Lablab niger, Japan, Tokyo,
Toyoda, Itino-machi, Minamitama-gun, 9 Aug. 1962, S. Takamoto
(IMI 96372). On Phaseolus vulgaris, Italy, Selva, Aug. 1877, Sacc.,
Mycoth. Ven. 1247 (HAL), type of Isariopsis griseola; Italy, Pavia,
Casatisma e Albaredo Arnaboldi, 1888, Briosi & Cavara, Funghi
parass. 17 (HAL); Russia, Czernigov, Borzova, Aug. 1914, G.
Nevodovsky, Petr. Mycoth. gen. 249 (B); South Korea, Chunchon,
7 Oct. 2003, H.D. Shin (HAL). On Phaseolus sp., Brazil, São Paulo,
Botanical Garden, 26 Dec. 1901, Puttemans, No. 413 (B), type of
Arthrobotryum puttemansii; Italy, Bugellae et Vercellis, Cesati,
Rabenh., Herb. mycol., Ed. 2, No. 327 (HAL), type of Cylindrosporium
phaseoli; USA, N.J., Newfield, 27 Sep. 1894, J.B. Ellis (NY), type
of Cercospora columnaris. Unidentified host (Phaseolus sp.?),
Paraguay, Caá-guazú, Jan. 1882, B. Balansa, No. 3492 (LSP 918),
type of Cercospora solimanii.
169
CROUS ET AL.
Fig. 4. Pseudocercospora griseola. A–C. Leaf disease symptoms. D. Lesions on bean pod. E–G. Fasciculate conidiophores. H–J. Conidiogenous
cells giving rise to conidia. K–O. Conidia. Scale bars: F = 8 µm, G = 200 µm, H = 10 µm.
Hosts and distribution: Lablab niger, ?L. purpureus,
?Lathyrus odoratus, ?Macroptilium atropurpureum,
Phaseolus acutifolius, P. aureus, P. coccineus, P.
lunatus, P. pubescens, P. vulgaris, Vigna angularis,
V. mungo, V. radiata, V. sinensis, V. unguiculata
(Leguminosae), worldwide, including Angola, Argentina,
Armenia, Australia, Austria, Bhutan, Brazil, Bulgaria,
Burundi, Cameroon, Canada, China, Colombia,
Congo, Costa Rica, Croatia, Cuba, Dominican Republ.,
Ecuador, El Salvador, Ethiopia, Fiji, France, Georgia,
Germany, Ghana, Great Britain, Greece, Guatemala,
Haiti, Hungary, Jamaica, Japan, India, Indonesia, Iran,
Ireland, Israel, Italy, Ivory Coast, Jamaica, Japan, Kenya,
Korea, Laos, Latvia, Malawi, Madagascar, Malaysia,
Mauritius, Mexico, Mozambique, Nepal, Netherlands,
Netherlands Antilles, New Caledonia, New Zealand,
170
Nicaragua, Nigeria, Norfolk Island, Panama, Papua New
Guinea, Paraguay, Peru, Philippines, Poland, Portugal,
Puerto Rico, Réunion, Romania, Russia, Rwanda, Saint
Helena, Senegal, Sierra Leone, Singapore, Slovenia,
Solomon Islands, Somalia, South Africa, Spain, Sudan,
Suriname, Swaziland, Switzerland, Taiwan, Tanzania,
Thailand, Trinidad and Tobago, Turkey, Uganda,
Ukraine, U.S.A. (CT, DE, Eastern states, FL, HI, IN, MA,
MD, ME, MI, MS, NC, NH, NJ, NY, OK, PA, SC, TX, VA,
WI), Vanuatu, Venezuela, Virgin Islands, Yugoslavia,
Zambia, Zimbabwe (Crous & Braun 2003).
Notes: As a consequence of molecular sequence
analyses (Figs 1–3), and re-examination and
reassessments of the synnematous conidiomata and
scar and hilum structures (Fig. 4, see Discussion),
ANGULAR LEAF SPOT OF BEAN
Phaeoisariopsis griseola proved to be congeneric
with Pseudocercospora. The proposed assignment
of this species to Pseudocercospora presupposes
acceptance of a formal proposal to conserve the latter
genus against the older names Phaeoisariopsis and
Stigmina (Braun & Crous 2006). All other taxa formerly
placed in Phaeoisariopsis have already been treated
and reallocated elsewhere (Crous & Braun 2003).
Cylindrosporium phaseoli Rabenh. is the oldest
name coined for this species, which appeared first on the
printed label of ‘Rabenh., Herb. mycol. 327, 1856’. This
name was repeated in Fürnrohr (1857), Schlechtendal
(1857) and Saccardo (1884), but in all cases without
any description (nom. nud.). Gonzáles Fragoso (1927:
339) was the first author who correctly cited this name
as synonym of Phaeoisariopsis griseola, which we
confirm after having re-examined Rabenhorst’s original
material.
Deighton (1990) reduced Cercospora solimanii
Speg. to synonymy with Ph. griseola, but without any
comments and references. Braun (2000) examined
type material of this species and confirmed Deighton’s
(1990) synonymy.
Although there are two clear entities associated with
the angular leaf spot disease of bean on pathological
or molecular grounds, we were unable to find enough
morphological, cultural or phylogenetic support to
separate these as two species. Because isolates
can readily be classed as either one or the other type
based on their host reaction on differential cultivars, we
have chosen to designate them as formae of the same
species.
DISCUSSION
A primary aim of the present study was to determine
the species status of the Andean and Middle-American
groups of the angular leaf spot pathogen of beans.
Because we have been unable to obtain good
morphological differences between the two groups
(other than cardinal temperatures for growth), nor
clear phylogenetic support for the separation based on
various gene loci, we have chosen to recognise these
two operational units as formae of the same species,
namely f. griseola and f. mesoamericana.
Two basic characters have in the past been
used for the discrimination of Phaeoisariopsis and
Pseudocercospora, namely the structure of the
conidiomata and the type of conidiogenous loci and
conidial hila. In molecular studies, the conidiomatal
structures were shown to be unreliable at the genus
level for anamorphs of Mycosphaerella. This is aptly
illustrated by the examples of Septoria Sacc. (pycnidia)
and Phloeospora Wallr. (acervuli) (Verkley et al. 2004),
Colletogloeopsis Crous & M.J. Wingf. (acervuli) and
Phaeophleospora-like species with aseptate conidia
and pycnidia (Cortinas et al. 2005), Ramularia Unger
(normal fascicles) and Phacellium Bonord. (synnemata)
(Crous et al., unpubl. data), which are all irregularly
scattered among the cladogrames. The coelomycete
genus Septoria (pycnidia) always clusters basal to
Cercospora Fresen. (fasciculate hyphomycete) (Crous
et al. 2000, 2001). The presence of synnemata is
thus insufficient to separate Phaeoisariopsis from
Pseudocercospora (Crous et al. 2001, Crous & Braun
2003). Furthermore, Pseudocercospora already includes
some synnematous species [e.g. the type species, P.
vitis (Lév.) Speg.]. Several species originally placed in
Phaeoisariopsis, but with inconspicuous conidial scars,
have already been reallocated in Pseudocercospora
(Deighton 1990). There are also some other genera
of hyphomycetes with synnematous as well as nonsynnematous species, e.g., Spiropes Cif. (Ellis 1971).
The structure of the conidiogenous loci and conidial
hila represent another important character used for the
distinction of Phaeoisariopsis and Pseudocercospora.
Prior to the introduction of the scar structure as basic
feature in the taxonomy of cercosporoid genera (Deighton
1967, 1973, 1974, 1976), Phaeoisariopsis was mainly
or even solely based on the synnematous arrangement
of the conidiophores, combined with pigmented conidia
formed singly. Therefore, it was hardly surprising that
Sawada (1922) transferred Septonema vitis Lév., the
type species of Pseudocercospora, to Phaeoisariopsis,
and thus reduced Pseudocercospora to synonymy with
Phaeoisariopsis. The heterogeneity of Phaeoisariopsis
is also reflected by the exclusion of all species, except
for the type species, I. griseola, originally placed in this
genus by Ferraris (1909): Isariopsis grayiana Ellis (≡
Fusicladium grayianum (Ellis) Deighton & M.B. Ellis),
I. mexicana Ellis & Everh. (≡ Exosporium mexicanum
(Ellis & Everh.) M.B. Ellis) and I. pilosa Earle (=
Morrisographium persicae (Schwein.) Deighton)
(see Deighton 1990). Von Arx (1983), Deighton
(1990) and Braun (1992, 1995, 1998) considered the
conidiogenous loci and conidial hila in Phaeoisariopsis
to be conspicuous or at least subconspicuous, i.e.,
barely to slightly thickened and darkened. However, Yen
(Yen & Lim 1980) already placed the ALS pathogen in
Pseudocercospora (conidiogenous loci inconspicuous),
although the wrong combination [Pseudocercospora
columnaris (Ellis & Everh.) J.M. Yen] was introduced,
and the correct basionym, Isariopsis griseola, cited as
synonym. The inclusion of Phaeoisariopsis griseola
in Pseudocercospora (Sawada 1922) thus reduces
Pseudocercospora to synonymy with Phaeoisariopsis.
We have re-examined the scars and hila in Ph. griseola
in detail, based on a wide range of samples in vivo and
in vitro, including type material of Isariopsis griseola,
Cylindrosporium phaseoli, Cercospora columnaris
and C. solimanii. The conidiogenous cells are usually
not or barely geniculate, the conidiogenous cells are
terminal to lateral, non-protruding, quite inconspicuous
to subconspicuous, i.e. unthickened or almost so,
but slightly darkened-refractive. There are collections
with completely inconspicuous conidiogenous loci,
e.g. the types of Isariopsis griseola and Cercospora
solimanii. In other samples, the loci range from
being quite inconspicuous to subconspicuous. The
African collection illustrated by Deighton (1990) is
an example of subconspicuous loci. However, as
demonstrated earlier by molecular examinations, taxa
171
CROUS ET AL.
with subconspicuous loci and hila (unthicked or almost
so, but slightly darkened-refractive or only the ultimate
rim slightly thickened and darkened) clustered together
with Pseudocercospora species, so that further
segregate-genera like Paracercospora Deighton and
Pseudophaeoramularia U. Braun had to be reduced to
synonymy with Pseudocercospora (Crous et al. 2000,
2001; Crous & Braun 2003).
Based on the molecular data presented here, the
type species of Pseudocercospora (P. vitis) clusters
with the type of Phaeoisariopsis (P. griseola), and the
type of Stigmina Sacc. [S. platani (Fuckel) Sacc.]. The
close affinity of these three genera underlines earlier
suspicions of mycologists that criteria such as 1) slightly
thickened conidial hila and scars, 2) synnematous to
fasciculate to sporodochial conidiomata, 3) transverse
to muriformly septate conidia, 4) euseptate to
distoseptate conidia, 5) smooth percurrent proliferations
and sympodial proliferation, versus irregular, rough
percurrent proliferations on conidiogenous cells, are
an insufficient basis to separate anamorph genera in
Mycosphaerella.
Given the fact that these three genera represent
anamorph forms of Mycosphaerella, and that they
phylogenetically reside in the same clade, the next
predicament arises as to what name should be
applied: Pseudocercospora (1910; 1171 names),
Phaeoisariopsis (1909, 65 names), or Stigmina
(1880, 161 names). Although Stigmina is the oldest
name, Pseudocercospora is the most commonly
used, and many species of Stigmina in fact represent
other fungi. Phaeoisariopsis, which also is older than
Pseudocercospora, has been reduced to its type
species, with most other species being placed in either
Passalora or Pseudocercospora. Stigmina predates
Phaeoisariopsis. If the Code of Botanical Nomenclature
were to be strictly applied, all species in this complex
should be transferred to Stigmina. As the latter is a
poorly resolved, still heterogeneous genus, we choose
to avoid this upheaval, and support conservation of
the commonly used and accepted generic name,
Pseudocercospora (Braun & Crous 2006). The latter
genus should be used for the whole complex of
hyphomycetes formerly placed in Phaeoisariopsis
and some of Stigmina. A formal conservation proposal
to this extent has been prepared for Taxon (Braun &
Crous 2006).
ACKNOWLEDGEMENTS
Mrs Marizeth Groenewald (CBS) is thanked for conducting the
growth study on isolates of P. griseola, Miss Malou van der Horst
(CBS) for sequencing some isolates, Dr Walter Gams (CBS) for
his comments on nomenclatural issues, and Miss Marjan Vermaas
(CBS) for preparing the photo plate. Dr George Mahuku of the
Centro Internacional de Agricultura Tropical (CIAT), Colombia, is
thanked for providing isolates from south and central America.
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