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Circumscription of the anthracnose pathogens
Colletotrichum lindemuthianum
and
C. nigrum
Fang Liu
State Key Laboratory of Mycology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing,
100101, China; Microbiology, Department of Biology,
Utrecht University, Padualaan 8, 3584 CH Utrecht,
the Netherlands
Lei Cai
1
State Key Laboratory of Mycology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing,
100101, China
Pedro W. Crous
CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan
8, 3584 CT Utrecht, the Netherlands; Utrecht
University, Department of Biology, Microbiology,
Padualaan 8, 3584 CH Utrecht, the Netherlands;
Wageningen University and Research Centre (WUR),
Laboratory of Phytopathology, Droevendaalsesteeg 1,
6708 PB Wageningen, the Netherlands
Ulrike Damm
CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan
8, 3584 CT Utrecht, the Netherlands
Abstract
:The anthracnose pathogen of common
bean (
Phaseolus vulgaris
) is usually identified as
Colletotrichum lindemuthianum
, while anthracnose of
potato (
Solanum tuberosum
), peppers (
Capsicum
annuum
), tomato (
S. lycopersicum
) and several other
crop plants is often attributed to
C. coccodes
. In order
to study the phylogenetic relationships of these
important pathogens, we conducted a multigene
analysis (ITS, ACT, TUB2, CHS-1, GAPDH) of strains
previously identified as
C. lindemuthianum
,
C.
coccodes
and other related species, as well as repre-
sentative species of the major
Colletotrichum
species
complexes. Strains of
C. lindemuthianum
belonged to
a single clade; we selected an authentic specimen as
lectotype, and an appropriate specimen and culture
from the CBS collection to serve as epitype. Two
clades were resolved within
C. coccodes s. lat
.One
clade included the ex-neotype strain of
C. coccodes
on
Solanum
, while an epitype was selected for
C. nigrum
,
which represents the oldest name of the second clade,
which occurs on
Capsicum
,
Solanum
, as well as several
other host plants. Furthermore, we recognized
C.
lycopersici
as a synonym of
C. nigrum,
and
C.
biologicum
as a synonym of
C. coccodes
.
Key words: Ascomycota
,
Colletotrichum
, epitypifica-
tion, morphology, phylogeny, systematics
INTRODUCTION
Anthracnose is one of the most widespread and
economically important diseases of common bean
(
Phaseolus vulgaris
) (Del Rı´o et al. 2002, Gonc¸alves-
Vidigal et al. 2004, Kiryakov 2004, Mahuku and
Riascos 2004, Bardas et al. 2007, Bardas et al. 2009,
Munda et al. 2009), which can lead to significant
losses in bean production (Tu 1992), especially when
the climatic conditions favor disease development
(Gonza´lez et al. 1998). Lesions on stems and pods of
common bean are gray or brown, slightly sunken with
raised dark brown or reddish edges, frequently
bearing conspicuous salmon colored spore masses
(Briosi and Cavara 1889).
Bean anthracnose was first reported from Germany
and described as
Gloeosporium lindemuthianum
Sacc.
& Magnus (Saccardo 1878). Subsequently, Briosi and
Cavara (1889) transferred
Gm. lindemuthianum
to the
genus
Colletotrichum
. Von Arx (1957) regarded
C.
lindemuthianum
(Sacc. & Magnus) Briosi & Cavara as
a form of
C. gloeosporioides
, indistinguishable from it
but specialized to
Phaseolus vulgaris
.
Colletotrichum
lindemuthianum
has been considered closely related
to
C. orbiculare
,
C. trifolii
and
C. malvarum
(Pain et al.
1992, Sherriff et al. 1994, Bailey et al. 1996). Sherriff
et al. (1994) considered these species as conspecific,
while Liu et al. (2007) found the four species to be
distinct, representing a species complex. This com-
plex represents a basal position within the genus
Colletotrichum
(Farr et al. 2006, Cannon et al. 2012).
Farr and Rossman (2012) listed 214 records of
Colletotrichum
associated with
Phaseolus
spp., of which
142 refer to
C. lindemuthianum
. On the other hand,
C. lindemuthianum
was reported on a wide range of
other legumes, such as
Dolichos lablab
(Zhuang 2001),
Lotus corniculatus
(Mulenko et al. 2008),
Dolichos
sp.
(Lenne´ 1990),
Vicia faba
(Zhuang 2005) and
Vigna
sinensis
(Pande and Rao 1998).
While descriptions of
C. lindemuthianum
in litera-
tures agree with each other in colonial characteristics
in having slow growth rate and dark pigmentation,
but present considerable variation in conidial mor-
phology. According to von Arx (1957), conidia
measure 11–19 34–6 mm, form reddish droplets,
with abundant setae in culture that are often absent
Submitted 11 Aug 2012; accepted for publication 14 Nov 2012.
1
Corresponding author, E-mail: mrcailei@gmail.com
Mycologia,
105(4), 2013, pp. 844–860. DOI: 10.3852/12-315
#2013 by The Mycological Society of America, Lawrence, KS 66044-8897
844
on the host plant. While Sutton (1980) described
C.
lindemuthianum
as having conidia that measure 9.5–
11.5 33.5–4.5 mm, form in honey- or pale-colored
masses, with few appressoria and setae.
Very few studies of
C. lindemuthianum
have
included DNA sequence analysis. Prior to our study,
there were only 14 ITS sequences and a few sequences
of other gene regions, e.g. Mat1-2 and glutamine
synthetase, lodged in GenBank. A comparison of
these ITS sequences with other
Colletotrichum
species
revealed that they belong to different species com-
plexes (Cannon et al. 2012). Some of these ITS
sequences, e.g. EF608059 (Huang et al. 2010) and
JN198431 (Wu L and Qin L, unpubl. data), belong to
the
C. boninense
species complex (Damm et al.
2012b).
A recent study by Liu et al. (2011) characterized
and neotypified
C. coccodes
(Wallr.) S. Hughes., which
is known to represent an important pathogen of
potato. Farr and Rossman (2012) listed over 30 plant
hosts for
C. coccodes
, including many reports on
peppers (
Capsicum annuum
) and tomato (
Solanum
lycopersicum
syn.
Lycopersicon esculentum
). How many
of these records are really attributable to
C. coccodes
remains unknown. For example, Thaung (2008)
reported
C. coccodes
as pathogen of
Camellia thea
.
However, none of our isolates, nor sequences from
this host on GenBank relate to
C. coccodes
(unpubl.
data); most of them belong to the
C. gloeosporioides
species complex.
Capsicum annuum
is often severely affected by
anthracnose, which causes serious yield losses in many
countries (Oh and Kim 2007). Many
Colletotrichum
species reported from
Capsicum annuum
belong to
the
C. acutatum
species complex (
C. acutatum
,
C.
brisbanense
,
C. nymphaeae
,
C. scovillei
) (Than et al.
2008a, b; Damm et al. 2012a),
C. boninense
species
complex (
C. novae-zelandiae
) (Damm et al. 2012b)
and
C. gloeosporioides
species complexes (
C. siamense
)
(Weir et al. 2012) as well as
C. truncatum
(synonym:
C. capsici
) (Pakdeevaraporn et al. 2005, Sharma et al.
2005, Damm et al. 2009). There are also many reports
of other
Colletotrichum
species from
Capsicum
annuum
(Farr and Rossman 2012), especially of
C.
coccodes
and
C. nigrum
. The latter is of uncertain
status and listed as doubtful species by Hyde et al.
(2009).
Colletotrichum nigrum
was placed in synonymy
with
C. gloeosporioides
by von Arx (1957), who later
resurrected it as a species restricted to
Capsicum
(von
Arx 1981).
Colletotrichum coccodes
is also reported as a devas-
tating pathogen of tomato (
Solanum lycopersicum
)
causing anthracnose on fruits (Hughes 1958; Chesters
and Hornby 1965a, b; Chapin et al. 2006; Alkan et al.
2008; Ben-Daniel and Bar-Zvi 2009). There are four
other
Colletotrichum
species on tomato listed in the
USDA database,
C. dematium
,
C. gloeosporioides
,
C.
lycopersici
and
C. nigrum
(Farr and Rossman 2012).
No sequence data of
C. lycopersici
and
C. nigrum
are
available in GenBank. In a preliminary study, the ITS
sequences of most of the strains in the CBS collection
originally identified as
C. lycopersici
and
C. nigrum
cluster with
C. coccodes
(unpubl. data).
The names
C. lindemuthianum
,
C. lycopersici
and
C.
nigrum
are of uncertain application because no ex-
type or authentic cultures were retained. The aims of
this study are therefore, to investigate the phyloge-
netic relationships of
C. lindemuthianum
and
C.
coccodes
and related strains to other species in
Colletotrichum
based on a multilocus phylogeny, to
locate type or authentic material of
C. lindemuthia-
num
,
C. lycopersici
,
C. nigrum
, and to select epitype
specimens with living cultures derived from them to
fix the application of these names.
MATERIALS AND METHODS
Isolates.—
Isolates previously identified as
C. lindemuthia-
num
and
C. coccodes
as well as other related species were
obtained from the culture collection of the CBS-KNAW
Fungal Biodiversity Centre, Utrecht, the Netherlands
(CBS). Type specimens of the species studied are located
in the fungaria of the CBS, the Herbarium Hamburgense
(HBG), Germany and the US National Fungus Collections
(BPI), Beltsville, Maryland, USA. Cultures derived from the
epitypes and neotypes, as well as all other isolates used for
morphological and phylogenetic analyses are maintained in
the CBS culture collection and presented in TABLE I.
Morphological analysis.—
To enhance sporulation, 5 mm
diam plugs from the margin of actively growing cultures
were transferred to the center of 9- cm diam Petri dishes
containing synthetic nutrient-poor agar medium (SNA;
Nirenberg 1976) amended with autoclaved filter paper
and double-autoclaved stems of
Anthriscus sylvestris
placed
onto the agar surface. The strains were also studied after
growth on oatmeal agar (OA) (Crous et al. 2009) and on
autoclaved pods of common bean (
Phaseolus vulgaris
).
Cultures were incubated at 20 C under near UV light with a
12 h photoperiod for 10 d. Measurements and photographs
of characteristic structures were made according to methods
described by Damm et al. (2007). Appressoria on hyphae
were observed on the reverse side of colonies grown on SNA
plates. Appressoria were also induced using a slide culture
technique (Cai et al. 2009). Microscopic preparations were
made in clear lactic acid, with 30 measurements per
structure, and observed with a Nikon SMZ1000 dissecting
microscope (DM) or with a Nikon Eclipse 80i microscope
using differential interference contrast (DIC) illumination.
Colony characters and pigment production on SNA and OA
incubated at 20 C were noted after 10 d. Colony colors were
rated according to Rayner (1970). Growth rates were
measured after 7 and 10 d.
LIU ET AL.: EPITYPIFICATION OF TWO
C
OLLETOTRICHUM
SPECIES 845
TABLE I. Strains of
Colletotrichum
studied in this paper with details about host/substrate and location, and accession numbers of reference sequences from GenBank
Species Accession no.
a
Host/substrate Locality
GenBank accession nos.
ITS ACT TUB2 CHS-1 GAPDH HIS3
b
C. acutatum
CBS 112996, ATCC 56816*
Carica
sp. Australia JQ005776 JQ005839 JQ005860 JQ005797 JQ948677 JQ005818
C. anthrisci
CBS 125334*
Anthriscus sylvestris
The Netherlands GU227845 GU227943 GU228139 GU228335 GU228237 GU228041
C. anthrisci
CBS 125335
Anthriscus sylvestris
The Netherlands GU227846 GU227944 GU228140 GU228336 GU228238 GU228042
C. boninense
CBS 123755, MAFF
305972*
Crinum asiaticum
var. sinicum
Japan JQ005153 JQ005501 JQ005588 JQ005327 JQ005240 JQ005414
C. boninense
CBS 128526
Dacrycarpus
dacrydioides
, leaf
endophyte
New Zealand JQ005162 JQ005510 JQ005596 JQ005336 JQ005249 JQ005423
C. circinans
CBS 111.21
Allium cepa
USA GU227854 GU227952 GU228148 GU228344 GU228246 GU228050
C. circinans
CBS 221.81*
Allium cepa
Serbia GU227855 GU227953 GU228149 GU228345 GU228247 GU228051
C. cliviae
CBS 125375, CSSK4*
Clivia miniata
China JX519223 JX519240 JX519249 JX519232 JX546611 JX560963
C. coccodes
CBS 103.16
Solanum tuberosum
The Netherlands JX546820 JX546628 JX546866 JX546674 JX546724 JX546772
C. coccodes
CBS 121.24 unknown USA JX546821 JX546629 JX546867 JX546675 JX546725 JX546773
C. coccodes
CBS 122.25
Solanum tuberosum
India? JX546822 JX546630 JX546868 JX546676 JX546726 JX546774
C. coccodes
CBS 125.57
Solanum tuberosum
unknown JX546823 JX546631 JX546869 JX546677 JX546727 JX546775
C. coccodes
CBS 126.57
Beta vulgaris
unknown JX546824 JX546632 JX546870 JX546678 JX546728 JX546776
C. coccodes
CBS 134.30 unknown unknown JX546825 JX546633 JX546871 JX546679 JX546729 JX546777
C. coccodes
CBS 150.33
Anthurium
sp. Germany JX546826 JX546634 JX546872 JX546680 JX546730 JX546778
C. coccodes
CBS 164.49
Solanum tuberosum
The Netherlands JQ005775 JQ005838 JQ005859 JQ005796 HM171672 JQ005817
C. coccodes
CBS 369.75*
Solanum tuberosum
The Netherlands HM171679 HM171667 JX546873 JX546681 HM171673 JX546779
C. coccodes
CBS 527.77
Solanum lycopersicum
Bulgaria JX546827 JX546635 JX546874 JX546682 JX546731 JX546780
C. coccodes
CBS 528.77 soil The Netherlands JX546828 JX546636 JX546875 JX546683 JX546732 JX546781
C. coccodes
CBS 641.97
Globodera rostochiensis
Switzerland JX546829 JX546637 JX546876 JX546684 JX546733 JX546782
C. coccodes
CBS 109213, BBA 62126
Solanum tuberosum
Germany JX546830 JX546638 JX546877 JX546685 JX546734 JX546783
C. coccodes
CBS 112987, IMI 61249
Solanum lycopersicum
Zimbabwe JX546831 JX546639 JX546878 JX546686 JX546735 JX546784
C. coccodes
CBS 125342, MUCL 8255
Capsicum
sp. Yugoslavia JX546832 JX546640 JX546879 JX546687 JX546736 JX546785
C. coccodes
CBS 125352, CCF 3825
Cucurbita pepo
,
rotten (fruit?)
Czech Republic JX546833 JX546641 JX546880 JX546688 JX546737 JX546786
C. coccodes
CBS 125963, NB 596
Solanum lycopersicum
The Netherlands JX546834 JX546642 JX546881 JX546689 JX546738 JX546787
C. coccodes
CBS 126378, C 101
Solanum tuberosum
South Africa JX546835 JX546643 JX546882 JX546690 JX546739 JX546788
C. coccodes
IMI 345429, CPC 20243
Heterodera pallida
Irland JX546836 JX546644 JX546883 JX546691 JX546740 JX546789
C. coccodes
IMI 78352, CPC 16810
Solanum tuberosum
UK JX546837 JX546645 JX546884 JX546692 JX546741 JX546790
C. dracaenophilum
CBS 118199*
Dracaena sanderana
,
living leaves
China JX519222 JX519238 JX519247 JX519230 JX546707 JX546756
C. fructi
CBS 346.37*
Malus sylvestris
USA GU227844 GU227942 GU228138 GU228334 GU228236 GU228040
C. gloeosporioides
CBS 953.97, CBS 112999,
IMI 356878*
Citrus sinensis
Italy GQ485605 GQ856782 GQ849434 GQ856733 GQ856762 JQ005413
C. gloeosporioides
CORCG5
Vanda
sp. China HM034809 HM034801 HM034811 HM034805 HM034807 —
C. hippeastri
CBS 125377, CSSG2
Hippeastrum vittatum
China GQ485598 GQ856789 GQ849445 GQ856726 GQ856765 JQ005491
846 MYCOLOGIA
TABLE I. Continued
Species Accession no.
a
Host/substrate Locality
GenBank accession nos.
ITS ACT TUB2 CHS-1 GAPDH HIS3
b
C. hippeastri
CBS 125376, CSSG1*
Hippeastrum vittatum
China GQ485599 GQ856788 GQ849446 GQ856725 GQ856764 JQ005492
C. karstii
CBS 132134,
CGMCC3.14194*
Vanda
sp. China HM585409 HM581995 HM585428 HM582023 HM585391 —
C. lindemuthianum
CBS 131.57
Phaseolus vulgaris
USA JX546805 JX546613 JX546851 JX546659 JX546708 JX546757
C. lindemuthianum
CBS 132.57
Phaseolus vulgaris
USA JX546806 JX546614 JX546852 JX546660 JX546709 JX546758
C. lindemuthianum
CBS 133.57
Phaseolus vulgaris
USA JX546807 JX546615 JX546853 JX546661 JX546710 JX546759
C. lindemuthianum
CBS 143.31
Phaseolus vulgaris
Germany JX546808 JX546616 JX546854 JX546662 JX546711 JX546760
C. lindemuthianum
CBS 144.31*
Phaseolus vulgaris
Germany JQ005779 JQ005842 JQ005863 JQ005800 JX546712 JQ005821
C. lindemuthianum
CBS 146.31
Phaseolus vulgaris
Germany JX546809 JX546617 JX546855 JX546663 JX546713 JX546761
C. lindemuthianum
CBS 147.31
Phaseolus vulgaris
Germany JX546810 JX546618 JX546856 JX546664 JX546714 JX546762
C. lindemuthianum
CBS 150.28
Phaseolus vulgaris
Germany JX546811 JX546619 JX546857 JX546665 JX546715 JX546763
C. lindemuthianum
CBS 151.28
Phaseolus vulgaris
Germany GU227800 GU227898 GU228094 GU228290 GU228192 GU227996
C. lindemuthianum
CBS 151.56, IMI
063364, ATCC
12611, UCLAF 230
Phaseolus vulgaris
,
pod
France JX546812 JX546620 JX546858 JX546666 JX546716 JX546764
C. lindemuthianum
CBS 152.28
Phaseolus vulgaris
The Netherlands JX546813 JX546621 JX546859 JX546667 JX546717 JX546765
C. lindemuthianum
CBS 153.28
Phaseolus vulgaris
The Netherlands JX546814 JX546622 JX546860 JX546668 JX546718 JX546766
C. lindemuthianum
CBS 523.97, LARS 798
Phaseolus coccineus
Costa Rica JX546815 JX546623 JX546861 JX546669 JX546719 JX546767
C. lindemuthianum
CBS 524.97, LARS 800
Phaseolus coccineus
Costa Rica JX546816 JX546624 JX546862 JX546670 JX546720 JX546768
C. lindemuthianum
CBS 569.97,
ATCC56897, LARS 9
Phaseolus vulgaris
Europe JX546817 JX546625 JX546863 JX546671 JX546721 JX546769
C. lindemuthianum
CBS 571.97, LARS 83
Phaseolus vulgaris
Brazil JX546818 JX546626 JX546864 JX546672 JX546722 JX546770
C. lindemuthianum
CBS 130841, ClKY1
Phaseolus vulgaris
USA JX546819 JX546627 JX546865 JX546673 JX546723 JX546771
C. lineola
CBS 125339
Apiaceae
sp. Czech Republic GU227830 GU227928 GU228124 GU228320 GU228222 GU228026
C. lineola
CBS 125337*
Apiaceae
sp. Czech Republic GU227829 GU227927 GU228123 GU228319 GU228221 GU228025
C. liriopes
CBS 122747
Liriope muscari
Mexico GU227805 GU227903 GU228099 GU228295 GU228197 GU228001
C. liriopes
CBS 119444*
Liriope muscari
Mexico GU227804 GU227902 GU228098 GU228294 GU228196 GU228000
C. nigrum
CBS 169.49*
Capsicum
sp. Argentina JX546838 JX546646 JX546885 JX546693 JX546742 JX546791
C. nigrum
CBS 174.59, ATCC
12521, SC 2145
Solanum
lycopersicum
unknown JX546839 JX546647 JX546886 JX546694 JX546743 JX546792
C. nigrum
CBS 175.59, ATCC 12624
Solanum lycopersicum
? unknown JX546840 JX546648 JX546887 JX546695 JX546744 JX546793
C. nigrum
CBS 288.81
Helianthus tuberosus
Serbia and
Montenegro
JX546841 JX546649 JX546888 JX546696 JX546745 JX546794
C. nigrum
CBS 127562, CPC 16433
Cichorium intybus
Chile JX546842 JX546650 JX546889 JX546697 JX546746 JX546795
C. nigrum
CBS 128507, ICMP
12927, C1147-2
Capsicum annuum
New Zealand JX546843 JX546651 JX546890 JX546698 JX546747 JX546796
C. nigrum
CBS 128553, ICMP
12929, C1167-1
Solanum
lycopersicum
New Zealand JX546844 JX546652 JX546891 JX546699 JX546748 JX546797
LIU ET AL.: EPITYPIFICATION OF TWO
C
OLLETOTRICHUM
SPECIES 847
TABLE I. Continued
Species Accession no.
a
Host/substrate Locality
GenBank accession nos.
ITS ACT TUB2 CHS-1 GAPDH HIS3
b
C. nigrum
CBS 132450, Hu 20
Solanum lycopersicum
USA JX546845 JX546653 JX546892 JX546700 JX546749 JX546798
C. nigrum
CBS 132451, Sa 3
Solanum lycopersicum
USA JX546846 JX546654 JX546893 JX546701 JX546750 JX546799
C. nigrum
IMI 17310, CPC 20244,
NCTC 1130
Solanum lycopersicum
USA JX546847 JX546655 JX546894 JX546702 JX546751 JX546800
C. nigrum
IMI 352646, CPC 19381
Lens culinaris
Canada JX546848 JX546656 JX546895 JX546703 JX546752 JX546801
C. nigrum
IMI 363581, CPC 18133
Fragaria
sp. UK? JX546849 JX546657 JX546896 JX546704 JX546753 JX546802
C. nigrum
IMI 363582, CPC 18083
Fragaria
sp. UK? JX546850 JX546658 JX546897 JX546705 JX546754 JX546803
C. oncidii
CBS 129828*
Oncidium
sp., leaf Germany JQ005169 JQ005517 JQ005603 JQ005343 JQ005256 JQ005430
C. oncidii
CBS 130242
Oncidium
sp., leaf Germany JQ005170 JQ005518 JQ005604 JQ005344 JQ005257 JQ005431
C. rusci
CBS 119206* Ruscus sp. Italy GU227818 GU227916 GU228112 GU228308 GU228210 GU228014
C. simmondsii
CBS 122122, BRIP 28519*
Carica papaya
Australia GU183331 GQ849454 GU183289 GQ856735 GQ856763 JQ949267
C. tofieldiae
CBS 495.85
Tofieldia calyculata
Switzerland GU227801 GU227899 GU228095 GU228291 GU228193 GU227997
C. tofieldiae
CBS 168.49
Lupinus polyphyllus
Germany GU227802 GU227900 GU228096 GU228292 GU228194 GU227998
C. torulosum
CBS 128544*
Solanum melongena
New Zealand JQ005164 JQ005512 JQ005598 JQ005338 JQ005251 JQ005425
C. torulosum
CBS 102667
Passiflora edulis
, leaf blotch New Zealand JQ005165 JQ005513 JQ005599 JQ005339 JQ005252 JQ005426
C. trichellum
CBS 118198
Hedera
sp. UK GU227813 GU227911 GU228107 GU228303 GU228205 GU228009
C. trichellum
CBS 217.64
Hedera helix
Germany GU227812 GU227910 GU228106 GU228302 GU228204 GU228008
C. truncatum
CBS 120709
Capsicum frutescens
India GU227877 GU227975 GU228171 GU228367 GU228269 GU228073
C. truncatum
CBS 151.35*
Phaseolus lunatus
USA GU227862 GU227960 GU228156 GU228352 GU228254 GU228058
C. verruculosum
IMI 45525
Crotalaria juncea
Zimbabwe GU227806 GU227904 GU228100 GU228296 GU228198 GU228002
C. yunnanense
CBS 132135,
AS 3.9617*
Buxus
sp. China JX546804 JX519239 JX519248 JX519231 JX546706 JX546755
Monilochaetes
infuscans
CBS 869.96
Ipomoea batatas
South Africa JQ005780 JQ005843 JQ005864 JQ005801 JX546612 JQ005822
a
CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, the Netherlands; IMI: Culture collection of CABI Europe
UK Centre, Egham, UK; BRIP: Plant Pathology Herbarium, Department of Employment, Economic, Development and Innovation, Queensland, Australia; ATCC:
American Type Culture Collection; ICMP: International Collection of Microorganisms from Plants, Auckland, New Zealand; MAFF: MAFF Genebank Project, Ministry of
Agriculture, Forestry and Fisheries, Tsukuba, Japan; CGMCC, AS: China General Microbiological Culture Collection. Strains studied in this paper are in boldface.
* ex-holotype or ex-epitype cultures.
b
HIS3 gene was not used in multilocus phylogenetic analysis.
848 MYCOLOGIA
Sexual compatibility.—
Seventeen isolates of
C. lindemuthia-
num
(TABLE I) were crossed with each other in all possible
combinations on SNA medium amended with sterile stems of
Anthriscus sylvestris
, pine needles and autoclaved bean pods.
In all tests, two isolates were placed on opposite sides of a
piece of
A. sylvestris
, pine needles or bean pods. Control
tests, where isolates were crossed with themselves, were
undertaken to determine whether strains had a heterothallic
or homothallic mating system. The plates were incubated at
20 C under near UV light with a 12 h photoperiod for 6 wk.
Matings were regarded as successful when isolate combina-
tions produced perithecia and ascospores.
Phylogenetic analyses.—
Genomic DNA of the isolates was
extracted using the method of Damm et al. (2008). Six loci
including the 5.8S nuclear ribosomal gene with the two
flanking internal transcribed spacers (ITS), a 200-bp intron of
the glyceraldehyde-3-phosphate dehydrogenase (
GAPDH
), a
partial sequence of the actin (
ACT
), chitin synthase 1 (
CHS-
1
), beta-tubulin (
TUB2
) and histon3 (
HIS3
)genewere
amplified and sequenced using the primer pairs ITS1F
(Gardes and Bruns 1993) +ITS4 (White et al. 1990), GDF1
+GDR1 (Guerber et al. 2003), ACT-512F +ACT-783R
(Carbone and Kohn 1999), CHS-354R +CHS-79F (Carbone
and Kohn 1999), T1 (O’Donnell and Cigelnik 1997) +Bt-2b
(Glass and Donaldson 1995) and CYLH3F +CYLH3R (Crous
et al. 2004), respectively. The PCR protocols were performed
as described by Damm et al. (2009). The DNA sequences
obtained from forward and reverse primers were used to
obtain consensus sequences using MEGA5, and subsequent
alignments were generated using MAFFT v.6 (Katoh and Toh
2010), and manually edited using BioEdit (Hall 1999).
A maximum parsimony analysis was performed on the
multilocus alignment (ITS,
ACT, TUB2, CHS-1, GAPDH
)
using PAUP v.4.0b10 (Swofford 2002). Ambiguously aligned
regions were excluded from all analyses. Unweighted parsi-
mony (UP) analysis was performed. Trees were inferred using
the heuristic search option with TBR branch swapping and
1000 random sequence additions. Maxtrees were unlimited,
branches of zero length were collapsed and all multiple
parsimonious trees were saved. Clade stability was assessed in a
bootstrap analysis with 1000 replicates, each with 10 replicates
of random stepwise addition of taxa. A second phylogenetic
analysis using a Markov Chain Monte Carlo (MCMC)
algorithm was done to generate trees with Bayesian posterior
probabilities in MrBayes v.3.2.1 (Ronquist and Huelsenbeck
2003). Nucleotide substitution models were determined using
MrModeltest v.2.3 (Nylander 2004) for each gene region and
included in the analyses. Two analyses of four MCMC chains
were run from random trees for ten millions generations and
sampled every 1000 generations. The first 25%of trees were
discarded as the burn-in phase of each analysis and posterior
probabilities determined from the remaining trees. Sequences
derived in this study were deposited in GenBank, and the
alignment in TreeBASE (S13363).
RESULTS
The phylogenetic analysis included 83 ingroup
strains, with
Monilochaetes infuscans
(CBS 869.96) as
outgroup. The dataset of five genes (
ACT, CHS-1,
GAPDH
,ITS,
TUB2
) comprised 1471 characters
including the alignment gaps, of which 683 characters
were parsimony-informative, 103 parsimony-uninfor-
mative and 685 constant. Parsimony analysis resulted
in nine most parsimonious trees, one of them
(Length 52978, CI 50.547, RI 50.911, RC 5
0.498) is shown in FIG. 1. The Bayesian tree agreed
with both the tree topology and bootstrap supports of
the trees obtained with maximum parsimony, Bayes-
ian posterior probability values $0.95 are shown as
thickened branches on the phylogenetic tree.
The strains studied in this paper formed two main
clades in the multigene phylogeny (FIG. 1). One of
these clades constitutes the upper part of the
phylogenetic tree with isolates previously identified
as
C. coccodes
and related strains comprising two
subclades. The first subclade (
C. coccodes
), with a
bootstrap support/Bayesian posterior probability val-
ue of 94/0.99 respectively, contains 20 strains
including the ex-neotype strain of
C. coccodes
(CBS
369.75), while the other 13 strains form a sister clade
(98/1.00) to
C. coccodes
. Both clades have close
relationship with a species complex formed by species
such as
C. tofieldiae
,
C. liriopes
and
C. verruculosum
.
Seventeen isolates from
Phaseolus
spp. identified as
C.
lindemuthianum
form a single clade (100/1.00),
which is basal to all other clades in the genus.
TAXONOMY
Based on DNA sequence data and morphology, the 17
strains of
C. lindemuthianum
studied belong to a
single species, while a second species
C. nigrum
was
revealed closely related to
C. coccodes
.Both
C.
lindemuthianum
and
C. nigrum
are characterized
and epitypified below.
Colletotrichum lindemuthianum (Sacc. & Magnus)
Briosi & Cavara,
Funghi Parass. Piante Colt. od Utili
,
Fasc. 2:no. 50. 1889. FIG.2
Basionym:
Gloeosporium lindemuthianum
Sacc. &
Magnus,
Michelia
1:129. 1878.
On SNA: Vegetative hyphae1–10 mm diam, hyaline to
pale brown, smooth-walled, septate, branched. Chla-
mydospores not observed. Conidiomata appearing as
accumulations of conidia on the surface of the
medium, conidiophores and setae formed directly on
hyphae. Setae not formed in ex-epitype strain, setae of
strain CBS 146.31, CBS 523.97 and CBS 130841 straight
or flexuous, pale brown, 1–4-septate, 53–87.5 mm long,
basal cell cylindrical, 3.5–5 mm diam, tip rounded.
Conidiophores hyaline, 23–51 mm long, branched, 0–3-
septate. Conidiogenous cells hyaline, smooth-walled,
LIU ET AL.: EPITYPIFICATION OF TWO
C
OLLETOTRICHUM
SPECIES 849
FIG. 1. One of nine most parsimonious trees obtained from a heuristic search of combined
ACT, CHS-1, GAPDH
, ITS and
TUB2
gene sequences of
Colletotrichum
species. Bootstrap support values (1000 replicates) above 50%are shown at the nodes.
Bayesian posterior probability values $0.95 are emphasized by thickened branches. The tree is rooted with
Monilochaetes
infuscans
. Numbers of ex-holotype, ex-epitype and ex-neotype strains are emphasized in bold. Strain numbers of strains
studied are followed by host species and country of origin.
850 MYCOLOGIA
cylindrical to ampulliform, 8–14 34–5 mm, opening 1–
1.5 mm diam, collarette 0.5 mm, periclinal thickening
not observed. Conidia hyaline, smooth-walled, asep-
tate, guttulate, cylindrical, both ends obtuse or with an
acute base, (10.5–)11.5–13.5(–14.5) 3(3–)3.5–4(–4.5)
mm, mean 6SD 512.6 61.1 33.8 60.4 mm, L/W
ratio 53.3. Appressoria not formed in ex-epitype
strain, appressoria of strain CBS 146.31 single or in
loose groups, (pale to) medium brown, smooth-walled,
outline subglobose, ovoid to ellipsoidal, with entire or
undulate edge, (5–)5.5–8.5(–12) 3(4.5–)5–6.5(–7)
mm, mean 6SD 5761.7 35.6 60.7 mm, L/W ratio 5
1.3.
On
Anthriscus
stem: Conidiomata acervular, conid-
iophores and setae formed from a cushion of pale
brown, angular cells, 2.5–6 mm diam. Setae pale to
medium brown, 1–4-septate, 30–93 mm long, basal cell
cylindrical, inflated or constricted, 3.5–5 mm diam, tip
round. Conidiophores hyaline to pale brown, smooth-
walled, branched, 1-septate, up to 20 mmlong.
FIG.2.
Colletotrichum lindemuthianum
(a–c, f–h, n–o from ex-epitype strain CBS 144.31, d–e from CBS 130841, i–m from
strain CBS 146.31). a–b. Acervuli; c. Setae; d. Tip of seta; e. Basal part of seta; f–h. Conidiophores; i–m. Appressoria; n–o.
conidia; a, c, g, n: from
Anthriscus
stem; b, d–e, f, h, i–m, o: from SNA. a–b: DM; c–o: DIC. — Scale bars: a 5100 mm; c 510 mm;
a applies to a–b; c applies to c–o.
LIU ET AL.: EPITYPIFICATION OF TWO
C
OLLETOTRICHUM
SPECIES 851
Conidiogenous cells hyaline to pale brown, smooth,
8–13.5 33–5 mm, opening 1–1.5 mm diam, collarette
0.5 mm, periclinal thickening sometimes visible.
Conidia hyaline, smooth-walled, aseptate, guttulate,
cylindrical, both ends obtuse or with base acute,
(10.5–)11.5–14(–15.5) 3(3–)3.5–4(–4.5) mm, mean
6SD 512.9 61.2 33.8 60.4 mm, L/W ratio 53.4.
Culture characteristics: Colonies on SNA flat with
entire margin, medium hyaline, filter paper gray
olivaceous, acervuli aggregated close to the
Anthriscus
stem, aerial mycelium lacking, 21–24 mm in 7 d (29–
31 mm in 10 d). Colonies on OA flat with entire
margin, surface gray olivaceous to olivaceous black,
covered by long hyaline and sparse aerial mycelium,
conidial masses whitish, abundant, reverse olivaceous
gray to iron gray, 26–27 mm in 7 d (41–43 mm after
10 d). Conidia in masses whitish to pale salmon.
Materials examined
. BRAZIL. On
Phaseolus vulgaris
, Mar
1997,
J.A. Bailey
, culture
CBS 571.97
5
Lars 83
. COSTA
RICA. On
P. coccineus
, Feb 1997,
J.A. Bailey
, culture
CBS
523.97
5
LARS 798
;On
P. coccineus
, Feb 1997,
J.A. Bailey
,
culture
CBS 524.97
5
LARS 800
. EUROPE. Unknown
country. On
P. vulgaris
, Mar 1997,
J.A. Bailey
, culture
CBS
569.97
5
LARS 9
5
ATCC 56897
. FRANCE. Paris, Roussel
Lab. Ltd. On pod of
P. vulgaris
, Jul 1956,
Uclaf
, culture
CBS
151.56
5
IMI 063364
5
ATCC 12611
5
UCLAF 230
.
GERMANY. Bonn, Poppelsdorf, fruit and vegetable garden
of the Agricultural Institute Poppelsdorf. On pods of
Phaseolus vulgaris
, 23 Aug 1875,
H. Lindemuth
(
HBG26/
2270
, ex herb. P. Magnus–syntype of
Gloeosporium linde-
muthianum
, lectotype here designated); Bonn. On
P.
vulgaris
, Aug 1931,
E. Schaffnit
(
CBS H-20954
, epitype
here designated, culture ex-epitype
CBS 144.31
); Scho¨nin-
gen. On
P. vulgaris
, Feb 1928,
E. Schaffnit
, culture
CBS
150.28
; Scho¨ningen. On
P. vulgaris
, Feb 1928,
E. Schaffnit
,
culture
CBS 151.28
; Scho¨ningen. On
P. vulgaris
, Feb 1928,
E. Schaffnit,
culture
CBS 153.28
; Berlin, Malchow. On
P.
vulgaris
, Aug 1931,
E. Schaffnit
, culture
CBS 143.31
;
Dresden. On
P. vulgaris
, Aug 1931,
E. Schaffnit
, culture
CBS 146.31
; Kirchwa¨rder near Hamburg. On
P. vulgaris
,
Aug 1931,
E. Schaffnit
, culture
CBS 147.31
. THE NETHER-
LANDS. Amsterdam. On
P. vulgaris
, Feb 1928,
E. Schaffnit
,
culture
CBS 152.28
. USA. MARYLAND: Takoma park. On
P.
vulgaris
, Jul 1906,
C.L. Shear
(
BPI 597216
); WASHINGTON:
District of Columbia. On
P. vulgaris
, Dec 1908,
C.L. Shear
(
BPI 597217
); VIRGINIA: Arlington. On
Phaseolus
sp., Jul
1942,
C.L. Shear
(
BPI 597215
); KENTUCKY. On
P. vulgaris
,
2007, unknown collector, culture
CBS 130841
5
C1KY1
;
NEW YORK: Ithaca, Cornell University. On
P. vulgaris
,Jan
1957, unknown collector (isolated by W.H. Burkholder),
culture
CBS 131.57
; NEW YORK: Ithaca, Cornell University.
On
P. vulgaris
, Jan 1957, unknown collector (isolated by
W.H. Burkholder), culture
CBS 132.57
; NEW YORK: Ithaca,
Cornell University. On
P. vulgaris
, Jan 1957, unknown
collector (isolated by W.H. Burkholder), culture
CBS 133.57
.
Notes:
Gloeosporium lindemuthianum
was described
from common beans collected in 1875 from Poppels-
dorf, near Bonn, Germany and in the same year in
Padova, Italy (Saccardo 1878). We could not locate the
specimen from Italy, but we located the authentic
herbarium specimens of
Gm. lindemuthianum
in the
Herbarium Hamburgense (HBG). Two of these spec-
imens were collected by H. Lindemuth prior to the
description of
Gm. lindemuthianum.
The label of one of
them contains the same collection data as the original
description, and was therefore selected as lectotype.
Conidia on the lectotype specimen are hyaline and
cylindrical, 11.5–21 33.5–5 mm, mean 6SD 515.5 6
1.8 34.5 60.4 mm, and formed light salmon to pink
masses. The conidial size is concordance with the
original description (16–19 34.5–5.5 mm) (Saccardo
1878) and von Arx (1957) (11–19 34–6 mm), but
larger than that from Sutton (1980, 1992) (9.5–11.5 3
3.5–4.5 mm). Conidial dimensions of the ex-epitype
strain CBS 144.31 formed on SNA,
Anthriscus
stems
and autoclaved bean pods (9.5–22 33.5–5.5 mm,
mean 6SD 513 62.1 34.6 60.4 mm) are also
concordance with the conidial dimensions of the
lectotype specimen and original description.
The two
C. lindemuthianum
strains from
P.
coccineus
(CBS 523.97 and CBS 524.97) differed from
the strains from
P. vulgaris
in the morphology of
their conidiophores. In addition to the conidiophores
typical for
C. lindemuthianum
described above,
conidia were also formed from up to 89 mm long
hyphae that resemble a transitional stage between
setae and conidiophores, with a conidiogenous cell
that is usually cylindrical and 20–28.5 mm long. Both
types of conidiophores are branched and septate.
Sometimes setae and conidiophores extend from the
same node. The phylogeny of the multigene dataset
generated in this study only supports one species
(FIG. 1), suggesting that these morphological differ-
ences fall within the variability of the species.
Shear and Wood (1913) first induced the sexual
stage of
C. lindemuthianum
,
Glomerella lindemuthiana
,
by incubating a strain from Takoma Park, Maryland,
USA on living bean pod tissue with acervuli on corn
meal agar. However, Shear and Wood (1913) failed to
designate a type specimen for
Glomerella lindemuthi-
ana
and only provided a morphological description.
Three
Glomerella lindemuthiana
specimens (plant
materials) collected by Shear in the United States in
the years 1906 (BPI 597216), 1908 (BPI 597217) and
1942 (BPI 597215) are available in the BPI herbari-
um, one of them (BPI 597217) was collected in
Washington. This specimen contained cylindrical
conidia, measuring 10–14.5 34–5.5 mm, mean 6SD
512.2 61.2 34.7 60.3 mm, L/W ratio 52.6 and
septate setae measuring 40–120 mmlong,which
agrees with those of the ex-epitype culture (CBS
144.31). However, ascomata were not observed from
BPI597217.
852 MYCOLOGIA
In this study, several methods were unsuccessfully
tested to induce the teleomorph stage of
C.
lindemuthianum
. Previous morphological descrip-
tions for teleomorph are doubtful and conflict with
each other. Kimati and Galli (1970) induced an
ascogenous stage of
C. lindemuthianum
by pairing
isolates from
Phaseolus
sp. from Brazil in semi-sythetic
medium, and was referred as
Glomerella cingulata
f.
sp.
phaseoli
. They observed two types of asci and
ascospores: (1–)4(–8)-spored asci, with mean asco-
spores dimension 20 36.5 mm, and 8-spored asci, with
ellipsoidal ascospores, measuring on average 10 3
4mm, which indicates that another fungus may be
involved. In addition, fig.1 of Rodrı´guez-Guerra et al.
(2005) shows a sexual stage with 8-spored asci and
ascospores that are longer and wider (15.5–29 34.5–
7mm) than those formed in 8-spored asci described by
Kimati and Galli (1970) (av. 10 34mm). In spite of
this, the occurrence of sexual reproduction in
Ga.
lindemuthiana
under field conditions has not yet
been observed. Furthermore, most of the previous
studies reporting sexual stages lack molecular data,
and the few studies that include them show diver-
gence from
C. lindemuthianum.
For example, one of
the six RAPD groups found by Talamini et al. (2006)
formed a sexual stage (referred to as
Glomerella
cingulata
f. sp.
phaseoli
) and was divergent from the
other groups. Phylogenies based on ITS and HMG
sequences by Barcelos et al. (2011) show that strains
from common beans with and without sexual stages
belong to different clades within
Colletotrichum
.We
are not aware of any data that shows
C. lindemuthia-
num
s. str. forms a sexual stage. We assume that sexual
stages linked to
C. lindemuthianum
in the past belong
to other species. This would explain why attempts to
repeat inducing sexual stage of
C. lindemuthianum
failed (Edgerton 1915, Muller 1927, Batista and
Chaves 1982, Bryson 1990).
Another species described as causing anthracnose
of
Phaseolus vulgaris
is
C. caulicolum
. However the
conidia of
C. caulicolum
are falcate and measure 18–
30 33.5–4 mm (Heald and Wolf 1911). This species
may be a synonym of
C. truncatum
, which has also
been reported from beans (Damm et al. 2009).
Colletotrichum coccodes (Wallr.) S. Hughes,
Can. J.
Bot.
36:754. 1958.
Basionym:
Chaetomium coccodes
Wallr., Fl. Crypt.
Germ. (Nu¨ rnberg) 2:265. 1833.
5
Colletotrichum biologicum
Chaudhuri,
Ann. Bot.
38:735.
1924.
For neotype and description of
C. coccodes
refer to
Liu et al. (2011).
Materials examined
. BULGARIA. Shumen. On
Solanum
tuberosum
, 1976,
E. llieva
,
CBS H-10565
, culture
CBS
527.77
. CZECH REPUBLIC. Prague. On
Cucurbita pepo
,
unknown collector and collection date (isolated by A.
Kuba´tova´), culture
CBS 125352
5
CCF 3825
. INDIA? On
dying stem of
Solanum tuberosum
, unknown collection date
and collector (deposited in CBS collection Feb. 1925 by
H.
Chaudhuri
), culture
CBS 122.25
;On
Solanum tuberosum
,
unknown collector and collection date (isolated by H.
Nirenberg), culture
CBS 109213
5
BBA 62126
;Mu¨nster.
On leaf of
Anthurium
sp., unknown collection date,
P.
Kotthoff
,
CBS H-10654
, culture
CBS 150.33
. IRELAND. On
Heterodera pallida
, unknown collector and collection date,
culture
IMI 345429
5
CPC 20243
. THE NETHERLANDS.
On
Solanum tuberosum
, unknown collector and collection
date (isolated by H.M. Quanjer), culture
CBS 103.16
;On
stem of
Solanum tuberosum
, unknown collector and
collection date (isolated by Naktuinbouw), culture
CBS
125963
5
NB 596
; Groningen. On tuber of
Solanum
tuberosum
, 1975,
G. Jager
,
CBS H-10573
, culture
CBS
369.75
; Wageningen. On
Solanum tuberosum
, unknown
collector and collection date, culture
CBS 164.49
; From
soil, Oct 1977,
Q. Jager
,
CBS H-10575
,
CBS H-10571
,
culture
CBS 528.77
. SOUTH AFRICA. University of
Stellenbosch. On
Solanum tuberosum
, unknown collector
and collection date, culture
CBS 126378
5
C101
.
SWITZERLAND. On
Globodera rostochiensis
,unknown
collector and collection date (isolated by A. Papert), culture
CBS 641.97
. UK. On
Solanum tuberosum
, unknown collec-
tor and collection date, culture
IMI 78352
5
CPC 16810
.
UNKNOWN LOCATION. On
Solanum tuberosum
, unknown
collector and collection date (isolated by G. Van den Ende),
culture
CBS 125.57
;On
Beta vulgaris
, unknown collector
and collection date (isolated by G. Van den Ende), culture
CBS 126.57
; Unknown substrate, unknown collector and
collection date (isolated by F. Labrousse), culture
CBS
134.30
. USA. Unknown substrate, unknown collector and
collection date (isolated by C.L. Shear), culture
CBS
121.24
. YUGOSLAVIA. Novisad. On
Capsicum
sp., 1965,
M. Acimovic
,culture
CBS 125342
5
MUCL 8255
.
ZIMBABWE. On
Solanum tuberosum
, unknown collection
date,
P. Cannon
, culture
CBS 112897
5
IMI 61249
.
Notes:
Colletotrichum biologicum
was first reported
from the dying stalks of potatoes (
Solanum tuberosum
)
in the garden of the ‘‘Biologische Reichsanstalt fu¨r
Forst- und Landwirtschaft’’ at Berlin-Dahlem, Ger-
many (Chaudhuri 1924). In February 1925, Chaud-
huri deposited one culture in the CBS collection
(CBS 122.25). According to the CBS database this
strain is from India, which is doubtful. It is more likely
that this is a strain from the original collection from
Germany that H. Chaudhuri sent to CBS after
returning to India. The strain is included in the
current study and clusters with the ex-neotype strain
of
C. coccodes
(CBS 369.75) in FIG. 1, which indicates
that
C. biologicum
is the synonym of
C. coccodes
.
Colletotrichum coccodes
, originally described from
potato black dot as
Chaetomium coccodes
Wallr. (Wall-
roth 1833), is not host-specific. In our study, several
strains from other hosts cluster in the
C. coccodes
LIU ET AL.: EPITYPIFICATION OF TWO
C
OLLETOTRICHUM
SPECIES 853
FIG.3.
Colletotrichum nigrum
(a–f, h–q from strain CBS 128507, g from strain CBS 132451). a–c. Acervuli; d. Tip of seta; e.
Basal part of seta; f–h. Conidiophores; i–o. Appressoria; p–q. Conidia; a, d–f, i–k, q: from
Anthriscus
stem; b–c, g–h, l–p: from
SNA. a–c: DM; d–p: DIC. — Scale bars: a 5100 mm; h 510 mm; a applies to a–c; c applies to d–q.
854 MYCOLOGIA
clade, e.g. strains from
Beta vulgaris
,
Curcubita pepo
and
Lycopersicon esculentum
. These strains form larger
conidia (average 17.5–19.5 33.5 mm on SNA, average
19–20 33.5 mmon
Anthriscus
stem) than those from
potato (average 12 33.5 mm on SNA, average 14–16
34mmon
Anthriscus
stem).
Colletotrichum nigrum Ellis & Halst., Bull. Torrey
Bot. Club 18:15. 1891. FIG.3
5
Colletotrichum lycopersici
Chester, Bull. Torrey Bot.
Club 18:372. 1891.
On SNA: Vegetative hyphae hyaline to medium
brown, smooth-walled, septate, branched, 1–7 mm
diam. Chlamydospores not observed. Conidiomata
acervular, or appearing as accumulation of conidia on
surface of medium, conidiophores formed from a
cushion of pale brown, angular cells or directly on
hyphae. Setae not observed. Conidiophores hyaline,
branched, septate. Conidiogenous cells hyaline,
smooth-walled, cylindrical, 15.5–31.5 31.5–3.5 mm,
opening 1–1.5 mm diam. Conidia hyaline, smooth-
walled, aseptate, guttulate, cylindrical, apex subacute
or obtuse, (9–)15–20(–24) 3(3.0–)3.5–4(–4.5) mm,
mean 6SD 517.5 62.5 3460.4 mm, L/W ratio 5
4.4. Appressoria single, brown, smooth-walled, glo-
bose, obvoid, clavate, or irregular outline, with an
entire or undulate edge, (4.5–)7–13(–21) 3(4.5–)
5.5–7.5(–9) mm, mean 6SD 510 62.9 36.5 6
1.0 mm, L/W ratio 51.5.
On
Anthriscus
stem: Conidiomata acervular, com-
pact fruiting structures composed of cushions of
brown to black angular cells, or appearing as
accumulation of conidia on stem. Setae abundant,
straight or flexuous, gradually tapering to the tip, 1–5-
septate, medium brown, 38–116 mm long, basal cell
cylindrical, inflated or constricted, 3–7 mm diam, tip
usually acute. Conidiophores hyaline to pale brown,
branched, septate. Conidiogenous cells hyaline to
pale brown, cylindrical to ampulliform, 7–12.5 32.5–
4.5 mm, opening 1–2 mm diam. Conidia hyaline,
smooth-walled, aseptate, guttulate, cylindrical, apex
subacute or obtuse, (13.5–)17.5–21.5 (–32) 3(3–)
3.5–4(–4.5) mm, mean 6SD 519.5 61.9 33.5 6
0.3 mm, L/W ratio 55.6.
Culture characteristics: Colonies on SNA flat with
entire margin, hyaline, aerial mycelium lacking,
abundant salmon or black acervuli aggregated on
Anthriscus
stem and filter paper, few acervuli on
medium, 40–48 mm in 7 d (58–66 mm in 10 d).
Colonies on OA flat with entire margin, entirely
covert with dense, smoke gray aerial mycelium,
reverse mouse gray to dark mouse gray, 42–43 mm
in 7 d (59–61 mm in 10 d). Conidia in masses salmon.
Materials examined
. ARGENTINA. On
Capsicum
sp.,
unknown collection date and collector (
CBS H-21032
,
epitype here designated, ex-epitype culture
CBS 169.49
).
Quarantine interception. On
Fragaria
sp., petiole, unknown
collection date and collector (deposited in IMI collection
1994 with no. B11/12), culture
IMI 363582
5
CPC 18083
;
On
Fragaria
sp., petiole, unknown collection date and
collector (deposited in IMI collection 1994 with no. A10/
11), culture
IMI 363581
5
CPC 18133
. CANADA. On
Lens
culinare
, 1992, unknown collector, culture
IMI 352646
5
CPC 19381
. CHILE. San Ignacio. On
Cichorium intybus
,
unknown collection date,
A. Schilder
, culture
CBS 127562
5
CPC 16433
. INDONESIA. Java. On
Capsicum annuum
,
Feb 24 1939,
J.T. Beauchamp
(
BPI 395383
). NEW ZEA-
LAND. Bay of Plenty. On
Capsicum annuum
(sweet
pepper), fruit rot, Mar 1990,
P.R. Johnston
,
CBS H-21033
,
culture
CBS 128507
5
ICMP 12927
5
C1147-2
; Bay of
Plenty. On
Solanum lycopersicum
, fruit rot, Mar 1990,
P.R.
Johnston
, culture
CBS 128553
5
ICMP 12929
5
C1167-1
.
SERBIA AND MONTENEGRO. Novi Sad, Institute of Field
and Vegetable Crops. On
Helianthus tuberosus
, Feb 1981,
T.
Verbalov
, culture
CBS 288.81
. UNKNOWN LOCATION. On
Capsicum annuum
fruit, Autumn 1903, unknown collector
(
BPI 395380
); Unknown substrate, Autumn 1903, unknown
collector (
BPI 395374
); Probably on
Solanum lycopersicum
,
unknown collection date and collector, culture
CBS 175.59
;
Probably on
Solanum lycopersicum
, unknown collection date
and collector, culture
CBS 174.59
5
ATCC 12521
5
SC
2145
. USA. NEW JERSEY: probably Swedesboro, Gloucester
Co. On fruit of
Capsicum annuum
, fruit anthracnose,
probably Aug 1890, probably
B.D. Halsted
(NY, Ellis
collection, no. 158 [not seen] lectotype designated here,
BPI 797145
isolectotype); NEW JERSEY: New Brunswick.
On
Capsicum annuum
fruits, Aug 1896,
B.D. Halsted
(
BPI
399228
); NEW JERSEY: Vineland Market. On
Capsicum
annuum
, Oct 1889,
J.B. Ellis
(
BPI 395379
, possible type of
Gloeosporium piperatum
); NEW JERSEY: Vineland Market.
On
Capsicum annuum
, Oct 1889,
J.B. Ellis
(
BPI 797997
);
NEW JERSEY: New Brunswick. On cultivated
Capsicum
annuum
, May 1896,
B.D. Halsted
(
BPI 395373
); CONNEC-
TICUT: Hamden. On
Capsicum annuum
, Oct 1903,
G.P.
Clinton
(
BPI 395382
); DELAWARE: Newark. On
Capsicum
annuum
, Nov 1903,
C.O. Smith
(
BPI 395381
); DELAWARE:
Newark. On
Lycopersicon esculentum
, Oct 1891,
F.D. Chester
(
BPI 399511
, possible type of
Colletotrichum lycopersici
);
DELAWARE: Newark. On
Lycopersicon esculentum
fruit, Oct
1891,
F.D. Chester
(
BPI 399513
); DELAWARE: Newark. On
Lycopersicon esculentum
fruit, Oct 1891,
F.D. Chester
(
BPI
398647
); DELAWARE: Newark. On
Lycopersicon esculentum
fruit, Oct 1891,
F.D. Chester
(
BPI 398648
); OHIO. On
Solanum lycopersicum
, unknown collection date and collec-
tor, culture
CBS 132451
5
Sa3
; OHIO. On
Solanum
lycopersicum
, unknown collection date and collector, culture
CBS 132450
5
Hu20
; WASHINGTON. On
Solanum
lycopersicum
, 1921,
F.T. Brooks
, culture
IMI 17310
5
CPC
20244
5
NCTC 1130
.
Note: Anthracnose of peppers was reported from
New Jersey, USA by Halsted (1891a, b), who described
the causal agents as
C. nigrum
and
Gm. piperatum
.
Halsted (1891b) observed that the two species caused
different symptoms on peppers.
Colletotrichum nigrum
LIU ET AL.: EPITYPIFICATION OF TWO
C
OLLETOTRICHUM
SPECIES 855
formed decayed patches that turned black due to the
setae of the fungus, while the color of decayed
patches caused by
Gm. piperatum
remained un-
changed. The conidia of
C. nigrum
are cylindrical,
nearly straight, hyaline, and measure 20–22 34mm
(Halsted 1891a). Later, Halsted (1896) reported
C.
nigrum
as the main cause of anthracnose of peppers
in the USA.
We located authentic specimens of
C. nigrum
and
Gm. piperatum
from the herbarium BPI. The label of
one of the
C. nigrum
specimens (BPI 797145) states,
‘‘158, New York Botanical Garden,
Colletotrichum
nigrum
E & H, on pepper fruit’’ with a stamp ‘‘CO-
TYPE’’. The morphology of this fungus agrees with the
original description of
C. nigrum
by Halsted (Halsted
1891a). The small size and shape of this specimen as
well as the number ‘‘158’’ identifies it as part of a
specimen located in the New York Botanical Garden
herbarium. There was no type designated either in the
original description of
C. nigrum
(Halsted 1891a) or in
the following reports of this species by Halsted (1891b,
1896). The label of this NY specimen does not state
that it is the holotype, but we are confident that it is
authentic material of
C. nigrum
,andtherefore
designate it here as the lectotype.
We also examined a possible type specimen of
Gm.
piperatum
(BPI 395379), which differed from
C.
nigrum
in forming fusiform conidia, indicating
affinity to the
C. acutatum
species complex.
The ex-epitype culture (CBS 169.49) was collected
in Argentina (South America), which is nearest to the
location where the type specimen was collected
(USA). Unfortunately, strain CBS 169.49 is sterile,
thus we chose a genetically identical strain, CBS
128507 from
Capsicum
in New Zealand, as basis for
the morphological description. Several strains from
other hosts collected in the USA confirm the
occurrence of
C. nigrum
in this country.
Colletotrichum nigrum
forms significantly longer
conidia than
C. coccodes
with a larger L/W ratio both
on SNA medium (average size of conidia of
C.
nigrum
, CBS 128507, 17.5 34mm, L/W 54.4 vs.
C.
coccodes
, CBS 369.75, 12 33.5 mm, L/W 53.4) and on
Anthriscus
stem (average size of conidia of
C. nigrum
,
CBS 128507, 19.5 33.5 mm, L/W 55.5 vs.
C. coccodes
,
CBS 369.75, 14 34mm, L/W 53.5). According to our
results,
C. nigrum
is not host-specific, and can also
infect
Cichorium intybus
,
Fragaria
sp.,
Helianthus
tuberosus, Lens culinare
and
Solanum lycopersicum
.
There are two other names related to this species,
Gm. phomoides
and
C. lycopersici
, both described from
S. lycopersicum
.
Gloeosporium phomoides
, described
from tomato in Italy by Saccardo (Saccardo 1884),
was prior to
C. lycopersici
(Chester 1891) and
C.
nigrum
(Halsted 1891a).
Gloeosporium phomoides
was
described as having conidia with an abruptly attenu-
ate base and a rounded apex, oblong to clavate
(10–12 32.5–3 mm), characters typical of
C. coccodes
.
We were unable to locate the type specimen of
Gm.
phomoides
. In 1884, Arthur reported the occurrence
of the fungus in New York, and in 1891, Chester
reported a fungus in the USA similar to that
described by Saccardo, but lacking setae. He de-
scribed it as a new species,
C. lycopersici
(Chester
1891). In a later publication, Chester (1893) recog-
nized that setae may be present or absent on artificial
medium and regarded
C. lycopersici
as a synonym of
Gm. phomoides
and therefore combined it in
Colleto-
trichum
as
C. phomoides.
Chester (1891) described
C.
lycopersici
as forming oblong conidia, with subacute
ends, measuring 16–22 34mm, similar to the size of
C. nigrum
. Our examination of the possible type
specimen of
C. lycopersici
(BPI 399513) confirmed
this. Two strains from tomato in the USA (CBS
132450 and CBS 132451) are included in this study,
which cluster together with
C. nigrum
(FIG.1).
Therefore we regard
C. lycopersici
as a synonym of
C. nigrum
. While
Glomerella lycopersici
W. Kru¨ ger
(anamorph
Gloeosporium lycopersici
) (Kru¨ ger 1913) is
a synonym of
C. salicis
that belongs to the
C.
acutatum
species complex (Damm et al. 2012a), and
is not closely related to
C. lycopersici
.
There are two species described from
Lens
culinaris
:
C. savulescui
Sandu-Ville from Romania
(Sandu-Ville 1959) and
Glomerella truncata
from
Canada (Armstrong-Cho and Banniza 2006).
Glomer-
ella truncata
is more closely related to
C. destructivum
(Gossen et al. 2009, O’Connell et al. 2012). Conidia
of
C. savulescui
are hyaline, cylindrical with both sides
rounded, straight or slightly curved, measuring 7.5–18
33–4.5 mm. The shape of the conidia is often slightly
curved which indicates this species might be a
synonym of
Glomerella truncata
, which however has
conidia that are usually larger (18–24 34–6 mm)
(Armstrong-Cho and Banniza 2006).
DISCUSSION
Colletotrichum
isolates from bean (
Phaseolus
sp.) are
usually identified as
C. lindemuthianum
based on
morphological characteristics (Bigirimana et al. 2000,
Ansari et al. 2004, Gonc¸alves-Vidigal et al. 2008).
However, morphology does not always reflect phylo-
genetic relationships between species in this genus.
For example,
C. petchii
with conidia resembling
C.
gloeosporioides
does not belong to the
C. gloeosporioides
species complex, but to the
C. boninense
species
complex (Damm et al. 2012b). Our results show that
although
C. lindemuthianum
produces conidia and
appressoria with shapes similar to
C. gloeosporioides
s.
856 MYCOLOGIA
str. (Cannon et al. 2008), it does not belong to the
C.
gloeosporioides
species complex. Our analysis reveals
that
C. lindemuthianum
is basal to the other species
included (FIG. 1). The epitypification of other species
in this complex, e.g.
C. orbiculare
,
C. malvarum
and
C.
trifolii
are still needed.
Based on this study,
C. lindemuthianum
seems to be
restricted to
Phaseolus vulgaris
and
P. coccineus
.
Other host plants from which this species has been
reported need to be reconfirmed based on a
comparison to the designated ex-epitype strain.
The infraspecific morphological variation observed
among the 17
C. lindemuthianum
strains studied may
be due to the different age and repeated subculturing
that can result in degeneration. The strains included
in this study were up to 84 years old. Many of the old
strains in this study and even a more recently
collected strain (CBS 569.97, collected in 1997)
produced a yellow pigment and did not sporulate,
while well-sporulating cultures of
C. lindemuthianum
are gray olivaceous to olivaceous black.
In the present study, 33 strains previously identified
as
C. coccodes
, segregated into two clades in the
multigene phylogeny obtained. There were isolates
from peppers and tomato in both clades, but all
isolates from potato (and potato cyst nematodes)
clustered with the ex-neotype strain of
C. coccodes
.
The second clade includes the ex-epitype strain of
C.
nigrum
. Both
C. coccodes
and
C. nigrum
can cause
anthracnose of peppers and tomato, while potato is
only infected by
C. coccodes
.
ACKNOWLEDGMENTS
We thank the curators of the CBS and CABI culture
collections as well as Prof. dr Lisa Vaillancourt (Department
of Plant Pathology, University of Kentucky, USA), Dr Peter
Johnston and Dr Bevan Weir (Landcare Research, Auck-
land, New Zealand), Prof. dr Annemiek C. Schilder
(Department of Plant Pathology, Michigan State University,
USA), Dr Alena Kuba´tova´ (Culture Collection of Fungi
(CCF), Charles University in Prague, Prague, Czech
Republic), Dr Ellis T.M. Meekes (Naktuinbouw, Research
& Development, Roelofarendsveen, the Netherlands), Dr
Lizel Mostert (Department of Plant Pathology, University of
Stellenbosch, South Africa) and Dr Franc¸oise Munot
(Mycothe`que de l’Universite´ catholique de Louvain, Unite´
de Microbiologie, Louvain-la-Neuve, Belgique) for kindly
supplying isolates for this study. We kindly thank the
curators of the fungaria at the Herbarium Hamburgense,
Hamburg, Germany and the US National Fungus Collec-
tions, Beltsville, Maryland, USA for providing us with
important herbarium specimens. This study was financially
supported by CAS KSCX2-YW-Z-1026 and the National
Natural Science Foundation of China (NSFC 31070020).
This research was also supported by the Dutch Ministry of
Agriculture, Nature and Food Quality through an endow-
ment of the FES programme ‘‘Versterking infrastructuur
Plantgezondheid’’.
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