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119
Mycologia,
96(1), 2004, pp. 119–134.
q2004 by The Mycological Society of America, Lawrence, KS 66044-8897
Molecular systematics of citrus-associated
Alternaria
species
T.L. Peever
1
G. Su
L. Carpenter-Boggs
Department of Plant Pathology, Washington State
University, P.O. Box 646430, Pullman, Washington
99164-6430
L.W. Timmer
Citrus Research and Education Center, University of
Florida, 700 Experiment Station Road, Lake Alfred,
Florida 33850
Abstract:
The causal agents of Alternaria brown
spot of tangerines and tangerine hybrids, Alternaria
leaf spot of rough lemon and Alternaria black rot of
citrus historically have been referred to as
Alternaria
citri
or
A. alternata
. Ten species of
Alternaria
recently
were described among a set of isolates from leaf le-
sions on rough lemon (
Citrus jambhiri
) and tangelo
(
C. paradisi
3
C. reticulata
), and none of these iso-
lates was considered representative of
A. alternata
or
A. citri
. To test the hypothesis that these newly de-
scribed morphological species are congruent with
phylogenetic species, selected Alternaria brown spot
and leaf spot isolates, citrus black rot isolates (post-
harvest pathogens), isolates associated with healthy
citrus tissue and reference species of
Alternaria
from
noncitrus hosts were scored for sequence variation at
five genomic regions and used to estimate phyloge-
nies. These data included 432 bp from the 5
9
end of
the mitochondrial ribosomal large subunit (mtLSU),
365 bp from the 5
9
end of the beta-tubulin gene, 464
bp of an endopolygalacturonase gene (endoPG) and
559 and 571 bp, respectively, of two anonymous ge-
nomic regions (OPA1–3 and OPA2–1). The mtLSU
and beta-tubulin phylogenies clearly differentiated
A.
limicola
, a large-spored species causing leaf spot of
Mexican lime, from the small-spored isolates associ-
ated with citrus but were insufficiently variable to re-
solve evolutionary relationships among the small-
spored isolates from citrus and other hosts. Sequence
analysis of translation elongation factor alpha, cal-
modulin, actin, chitin synthase and 1, 3, 8-trihydrox-
ynaphthalene reductase genes similarly failed to un-
cover significant variation among the small-spored
Accepted for publication June 2, 2003.
1
Corresponding author. E-mail: tpeever@wsu.edu
isolates. Phylogenies estimated independently from
endoPG, OPA1–3 and OPA2–1 data were congruent,
and analysis of the combined data from these regions
revealed nine clades, eight of which contained small-
spored, citrus-associated isolates. Lineages inferred
from analysis of the combined dataset were in gen-
eral agreement with described morphospecies, how-
ever, three clades contained more than one morpho-
logical species and one morphospecies (
A. citrima-
cularis
) was polyphyletic. Citrus black rot isolates also
were found to be members of more than a single
lineage. The number of morphospecies associated
with citrus exceeded that which could be supported
under a phylogenetic species concept, and isolates in
only five of nine phylogenetic lineages consistently
were correlated with a specific host, disease or eco-
logical niche on citrus. We advocate collapsing all
small-spored, citrus-associated isolates of
Alternaria
into a single phylogenetic species,
A. alternata
.
Key words:
anonymous region, beta-tubulin,
black rot, endopolygalacturonase, mitochondrial
LSU rDNA, phylogeny, phytopathogen
INTRODUCTION
Alternaria
Nees is a cosmopolitan fungal genus that
includes saprophytic, endophytic and pathogenic
species. Plant pathogenic species of
Alternaria
infect
a number of economically important plants such as
tangerine (
Citrus reticulata
Blanco), apple (
Malus do-
mestica
Borkh.), pear (
Pyrus pyrifolia
(Burm. f.) Na-
kai), tomato (
Lycopersicon esculentum
Mill.) and po-
tato (
Solanum tuberosum
L.). Many of the pathogenic
species produce host-specific toxins (Nishimura et al
1983, Otani et al 1995) that are demonstrated path-
ogenicity factors in disease (Hatta et al 2002, Johnson
et al 2000, 2001, Tanaka et al 1999). The four de-
scribed
Alternaria
diseases of citrus include: (i) Al-
ternaria brown spot of mandarins, tangerines and
various tangerine hybrids; (ii) Alternaria leaf spot of
rough lemon (
C. jambhiri
Lush.); (iii) black rot of
citrus, a post harvest disease; and (iv) citrus leaf spot
(
mancha foliar de los citricos
) of Mexican (Key) lime
(
C. aurantiifolia
[Christm.] Swingle).
Alternaria brown spot of mandarin and tangerines,
caused by
A. citri
Ellis & Pierce, first was described
on emperor mandarin in Australia in 1903 (Cobb
120 M
YCOLOGIA
1903). Alternaria leaf spot of rough lemon, also at-
tributed to
A. citri
, originally was described from
South Africa in 1929 (Doidge 1929) and Florida in
1937 (Ruehle 1937). The former pathogen has been
referred to as
A. alternata
(Fr. : Fr.) Keissl. ‘‘tangerine
pathotype’’ and the latter as
A. alternata
‘‘rough lem-
on pathotype’’ because each form is host-specific and
produces a chemically distinct host-specific toxin
(Pegg 1966, Kohmoto et al 1979, Whiteside 1976).
Black rot of citrus also has been attributed to
A. citri
,
and symptoms include a stem-end decay of mature
fruit in storage, which can occur on all commercial
citrus cultivars (Bliss and Fawcett 1944). Mancha fo-
liar de los citricos is caused by
A. limicola
Simmons
& Palm (Palm and Civerolo 1994, Simmons 1990)
and is a weak pathogen of other species of
Citrus
L.
The taxonomic status of the fungi that cause Al-
ternaria brown spot and Alternaria leaf spot is un-
clear. These pathogens originally were identified as
A. citri
based on morphological similarities to isolates
causing black rot (Doidge 1929, Kiely 1964, Pegg
1966, Ruehle 1937) and later were considered to rep-
resent a distinct strain based on their ability to infect
leaves and young fruit and produce host-specific tox-
ins (Kiely 1964, Whiteside 1976). Nishimura and
Kohmoto (1983) treated these pathogens as
A. alter-
nata
based on a published description of the mor-
phology and size of the conidia of this species. The
fungi causing Alternaria brown spot also have been
called
A. alternata
pv. citri (Solel 1991) to differen-
tiate them from saprophytic isolates of
A. alternata
.
The taxonomic status of isolates causing black rot
similarly is uncertain. Isolates causing black rot are
considered biologically and taxonomically distinct
from the brown spot pathogens because they are un-
able to cause disease on leaves or young fruit and do
not produce host-specific toxins (Kiely 1964, Pegg
1966, Ruehle 1937). However, they are also small-
spored and morphologically similar to the brown
spot pathogens and it is not clear if they represent a
distinct taxon.
Phylogenetic analyses of species of
Alternaria
based
on DNA sequence data from several regions of the
genome have revealed that small-spored species such
as
A. alternata
,
A. longipes
(Ellis & Everh.) Mason and
A. tenuissima
(Nees : Fr.) Wiltshire are readily distin-
guished from large-spored species such as
A. solani
Sorauer (Kusaba and Tsuge 1995, McKay et al 1999,
Pryor and Gilbertson 2000, Pr yor and Michailides
2002). In contrast, differentiation of the small-spored
species has been difficult due to lack of variation in
nuclear ribosomal internal transcribed spacer (ITS)
and beta-tubulin sequences, two genomic regions typ-
ically used in fungal systematics. Analysis of nuclear
rDNA and mitochondrial RFLP also failed to differ-
entiate several small-spored species including
A. al-
ternata
,
A. citri
,
A. gaisen
Nagano,
A. longipes
and
A.
mali
Roberts (Kusaba and Tsuge 1994, 1997). Anal-
ysis of ITS sequences revealed that small-spored, tox-
in-producing taxa such as
A. alternata
tangerine path-
otype,
A. alternata
rough lemon pathotype,
A. alter-
nata
strawberry pathotype,
A. alternata
tomato path-
otype,
A. gaisen
and
A. mali
could not be
differentiated from each other or from several sap-
rophytic isolates of
A. alternata
(Kusaba and Tsuge
1995). Pryor and Gilbertson (2000), Pryor and Mi-
chailides (2002), Kang et al (2002) and McKay et al
(1999) also failed to differentiate small-spored
Alter-
naria
species using ITS sequence data.
Recent research has attempted to clarify the sys-
tematics of the species of
Alternaria
associated with
Alternaria brown spot and leaf spot of citrus (Sim-
mons 1999a). One hundred thirty-five isolates from
a worldwide collection, including isolates from leaf
lesions on rough lemon, tangerine and tangerine
3
grapefruit hybrids, were examined (Simmons 1999a).
Ten morphological species of
Alternaria
were de-
scribed from this material, none of which was consid-
ered representative of
A. alternata
or
A. citri
. Because
this research was based primarily on isolates from the
culture collections of T.L. Peever and L.W. Timmer
(Peever et al 1999, 2002), it represented an ideal op-
portunity to test the hypothesis that
Alternaria
species
defined under a morphological species concept were
concordant with taxa defined under a phylogenetic
species concept using sequence data from multiple
regions of the genome. To test this hypothesis, we
used sequence data from the mitochondrial ribosom-
al large ribosomal subunit (mtLSU), the 5
9
end of
the beta-tubulin gene, an endopolygalacturonase
gene (endoPG) and two anonymous regions of the
genome (OPA1–3, OPA2–1) that together provided
the necessary variation to resolve a phylogeny among
all known citrus-associated isolates of
Alternaria
.A
secondary objective was to test the hypothesis that
isolates associated with citrus black rot are monophy-
letic and phylogenetically distinct from citrus brown
spot isolates. A preliminary report of this research
has been published (Su et al 2001).
MATERIALS AND METHODS
Cultivation of fungi and extraction of DNA.
Sixty-eight sin-
gle-conidial isolates were selected to represent described
morphospecies of
Alternaria
from citrus (T
ABLE
I). Addi-
tional morphologically well-characterized members of the
genus from other hosts were obtained from E.G. Simmons,
Crawfordsville, Indiana, and other cooperators and includ-
ed as reference species. Citrus isolates included: (i) isolates
causing brown spot of tangerine and tangerine hybrids, and
121P
EEVER ET AL
:
A
LTERNARIA SPECIES SYSTEMATICS
Alternaria leaf spot of rough lemon collected for previous
population genetics studies (Peever et al 1999, 2002), and
examined by Simmons (1999a); (ii) isolates causing leaf
spot of Mexican lime (Simmons 1990, Palm and Civerolo
1994); (iii) isolates from healthy citrus tissue; and (iv) iso-
lates sampled from mature citrus fruit with symptoms of
black rot. When available, ex-type strains or strains consid-
ered representative of each described morphospecies were
used (T
ABLE
I). All isolates were stored on sterile filter pa-
per as previously described (Peever et al 1999). For pro-
duction of mycelium, isolates were grown 4 d in 2-YEG me-
dium (10 g dextrose, 2 g yeast extract per L) on a rotary
shaker, and mycelium was collected and lyophilized as pre-
viously described (Peever et al 1999). Genomic DNA was
extracted from 50 mg lyophilized mycelium as described by
Peever et al (1999) with modifications. One phenol/chlo-
roform/isoamyl alcohol (25:24:1) extraction and one chlo-
roform/isoamyl alcohol (24:1) extraction were used. DNA
concentrations were estimated visually in 0.7% agarose gels
containing 5 mg/mL ethidium bromide by comparing band
intensity with known quantities of lDNA/
Hind
III markers.
Cloning and sequencing of mtLSU.
Primers ML1 and ML6
(White et al 1990) were used to amplify 1700 bp of the
mtLSU from
A. alternata
(EGS 34–016),
A. gaisen
(EGS 90–
0512),
A. tangelonis
(SH-MIL-4s),
A. limicola
(Colima A90),
A. longipes
(isolate EGS 30–033) and
A. solani
(49ss) (T
A
-
BLE
I). This 1700 bp amplicon corresponded to nucleotide
positions 587–2436 of the coding region of the
Penicillium
chrysogenum
Thom mtLSU rDNA (GenBank accession
D13859). Twenty five mL PCR contained 13reaction buffer,
0.2 mM of each primer, 200 mM dNTP, 2.5 mM MgCl
2
,25
ng of DNA and 1 unit of
Taq
polymerase. PCR was carried
out in a Hybaid Omn-E thermocycler (Hybaid, Ashford,
Middlesex, U.K.), and cycling conditions consisted of 95 C
for 1 min followed by 35 cycles of 95 C for 1 min, 60 C for
1 min and 72 C for 1 min. Amplicons from all six isolates
were cloned into the pCR4-TOPO vector (Invitrogen, Carls-
bad, California) and used to chemically transform compe-
tent TOP 10
Escherichia coli
cells (Invitrogen) following the
manufacturer’s directions. Transformed cells were grown
overnight in Luria-Bertani broth amended with 100 mg/mL
ampicillin, and plasmid DNA was extracted using QIAprep
Spin Mini Prep columns (Qiagen, Valencia, California) or
BIO 101 RPM spin columns (Q-BIOgene, Carlsbad, Califor-
nia). Concentrations of plasmid DNA were determined us-
ing a fluorometer (Dynatech Laboratories, Chantilly, Vir-
ginia). Inserts were sequenced using T3 and T7 primers
with each sequence reaction containing 40–90 ng DNA, 320
nM primer, 4 mL BigDye Terminator Cycle Sequencing
Ready Reaction Mix (Applied Biosystems, Foster City, Cali-
fornia) and sterile distilled water in 10 mL total volumes.
Cycle sequence reactions were performed in a Hybaid
Omn-E thermal cycler and cycling conditions consisted of
25 cycles of 96 C for 15 s, 50 C for 15 s, and 60 C for 4
min. Products were purified using Centriflex Gel Filtration
Cartridges (Edge BioSystems, Gaithersburg, Maryland),
dried in a rotary evaporator and sequenced on a PE Bio-
systems Model 377 Automated DNA Sequencer (Applera
Corp., Norwalk, Connecticut). All sequencing was per-
formed in the Laboratory for Biotechnology and Bioanaly-
sis, School of Molecular Biosciences, Washington State Uni-
versity. Based on alignment of mtLSU sequences from the
six species of
Alternaria
, a new reverse primer ML-R1 (59-
GCCCTTCCGAGAGCAAATAC-39) was designed using
Primer3 software to amplify a product of convenient size
for direct sequencing each strand in a single sequencing
reaction. Primers ML1 and ML-R1 amplified a 460 bp frag-
ment from all tested isolates using the same PCR conditions
as described above. This amplicon corresponded to nucle-
otide positions 611–1081 of the
P. chrysogenum
mtLSU
rDNA. Amplicons were purified through QiaQuick PCR pu-
rification columns (Qiagen) and direct sequenced on both
strands using the same conditions described above with
primers ML-1 and ML-R1. Cycle sequence reactions and au-
tomated sequencing were carried out as described above.
MtLSU sequences have been deposited in GenBank (acces-
sion numbers AY293857–64).
Cloning and sequencing of the beta-tubulin gene.
One thou-
sand one hundred twenty-four bp of the beta-tubulin gene
was amplified from
A. alternata
(EGS 34–016),
A. limicola
(Colima A90),
A. solani
(49ss) and
A. tangelonis
(SH-MIL-
4s) (T
ABLE
I) using primers T1 (O’ Donnell and Cigelnik
1997) and beta-tub-2 (59-ATCATGTTCTTGGGGTCGAA-
39). Primer beta-tub-2 was designed to prime at nucleotide
positions 806–787 relative to a partial
A. alternata
beta-tu-
bulin sequence (GenBank accession Y17073) and nucleo-
tide positions 1641–1661 in exon 6 of the
Venturia inae-
qualis
Aderhold beta-tubulin gene (GenBank accession
M97951). These 1100 bp amplicons corresponded to nucle-
otide positions 447–1662 of the
V. inaequalis
beta-tubulin
gene and included ;900 bp of putative exon sequence and
;200 bp of putative intron sequence. Amplification of par-
tial beta-tubulin sequences was carried out as described
above, and amplicons were cloned and sequenced as de-
scribed above. Sequences were aligned to design degener-
ate primers beta-3 (59-GAGATTGYAAGTATCGCCTGSM-39)
and beta-4 (59-GCACGAACTTGTTGTTGGAS-39) that am-
plified a 400 bp fragment corresponding to nucleotide po-
sitions 456–943 of the
V. inaequalis
beta-tubulin gene. This
amplicon included ;300 bp of putative exon sequence and
;100 bp of putative intron sequence. Amplicons were pu-
rified and direct sequenced with primers beta-3 and beta-4
as described above. Cycle sequence reactions and sequenc-
ing were carried out as described above. Beta-tubulin se-
quences have been deposited in GenBank (accession num-
bers AY293867–82).
Sequencing of additional gene regions.
Primers PG3 and
PG2 were used to amplify a portion of an endopolygalctu-
ronase gene (endoPG, GenBank accession ABO47682)
characterized from the rough lemon pathotype of
A. alter-
nata
(Isshiki et al 2001). These primers amplified a 500 bp
amplicon consisting entirely of exon sequence. Amplifica-
tions were carried out as described for the mtLSU and beta-
tubulin gene regions. Cycling conditions consisted of 95 C
for 2 min followed by 35 cycles of 95 C for 1 min, 50 C for
1 min, and 72 C for 1 min. These cycles were followed by
a final 5 min elongation cycle at 72 C. Amplicons were pu-
rified and sequenced as described above. EndoPG sequenc-
122 M
YCOLOGIA
T
ABLE
I. Citrus-associated and reference isolates of
Alternaria
used in this study
Species
a
Isolate Code
b
Host Disease
c
Accession No.
d
Source
e
Reference(s)
A. citrimacularis
Simmons BC2-RLR-17s
Citrus jambhiri
non-pathogen — T.L. Peever Simmons 1999a; Peever
et al 1999
A. citrimacularis
Simmons BC2-RLR-32s T
Citrus jambhiri
non-pathogen BPI 746366 T.L. Peever Simmons 1999a; Peever
et al 1999
A. limoniasperae
Simmons BC2-RLR-1s
Citrus jambhiri
leaf spot — T.L. Peever Simmons 1999a; Peever
et al 1999
A. tangelonis
Simmons SH-MIL-4s
Citrus reticulata
3
C.
paradisi
Macfady
brown spot — T.L. Peever Simmons 1999a; Peever
et al 1999
A. tangelonis
Simmons EV-MIL-2s T
Citrus reticulata
3
C.
paradisi
brown spot BPI 746364 T.L. Peever Simmons 1999a; Peever
et al 1999
A. citriarbusti
Simmons SH-MIL-15s T
Citrus reticulata
3
C.
paradisi
brown spot BPI 746369 T.L. Peever Simmons 1999a; Peever
et al 1999
A. citriarbusti
Simmons SH-MIL-8s
Citrus reticulata
3
C.
paradisi
brown spot — T.L. Peever Simmons 1999a; Peever
et al 1999
A. toxicogenica
Simmons PR320 T
Citrus reticulata
cv. ‘‘Dan-
cy’’
brown spot BPI 746370 L.W. Timmer Simmons 1999b
A. limoniasperae
Simmons PR325
Citrus jambhiri
leaf spot — L.W. Timmer Simmons 1999a, 1999b
nd Acitri-1
Citrus sinensis
(L.) Os-
beck cv. ‘‘Valencia’’
black rot — L.W. Timmer —
nd Australia D
Citrus reticulata
cv. ‘‘Em-
peror’’
black rot — L.W. Timmer —
nd 39-189
Citrus sinensis
cv. ‘‘Valen-
cia’’
black rot — L.W. Timmer —
nd 39-190
Citrus sinensis
cv. ‘‘Valen-
cia’’
black rot — L.W. Timmer —
nd 39-191
Citrus sinensis
cv. ‘‘Valen-
cia’’
black rot — L.W. Timmer —
nd 39-192
Citrus paradisi
cv. ‘‘Dun-
can’’
black rot — L.W. Timmer —
nd Acitri-2
Citrus sinensis
cv. ‘‘Fall-
glo’’
black rot — L.W. Timmer —
A. turkisafria
Simmons EGS 44-159
Citrus reticulata
3
C.
paradisi
brown spot — L.W. Timmer Simmons 1999a; Peever
et al 2002
A. perangusta
Simmons EGS 44-160 T
Citrus reticulata
3
C.
paradisi
brown spot BPI 746361 L.W. Timmer Simmons 1999a; Peever
et al 2002
A. turkisafria
Simmons EGS 45-003
Citrus reticulata
3
C.
paradisi
brown spot — L.W. Timmer Simmons 1999a; Peever
et al 2002
A. dumosa
Simmons EGS 45-007 T
Citrus reticulata
3
C.
paradisi
non-pathogen BPI 746365 L.W. Timmer Simmons 1999a; Peever
et al 2002
A. interrupta
Simmons EGS 45-011 T
Citrus reticulata
3
C.
paradisi
brown spot BPI 746362 L.W. Timmer Simmons 1999a; Peever
et al 2002
123P
EEVER ET AL
:
A
LTERNARIA SPECIES SYSTEMATICS
T
ABLE
I. Continued
Species
a
Isolate Code
b
Host Disease
c
Accession No.
d
Source
e
Reference(s)
A. colombiana
Simmons EGS 45-017 T
Citrus reticulata
3
C.
paradisi
brown spot BPI 746368 L.W. Timmer Simmons 1999a; Peever
et al 2002
A limicola
Colima A90
Citrus aurantiifolia
leaf spot — M.E. Palm Palm and Civerolo 1994;
Simmons 1990
A. limicola
Guerrero 8
Citrus aurantiifolia
leaf spot — M.E. Palm Palm and Civerolo 1994;
Simmons 1990
A. limicola
Colima E
Citrus paradisi
leaf spot — M.E. Palm Palm and Civerolo 1994
A. limicola
Jalisco G
Citrus aurantium
L. leaf spot — M.E. Palm —
nd UC-1s
Citrus reticulata
unknown — J. Menge —
nd UC-2s
Citrus sinensis
unknown — J. Menge —
nd UC-4s
Citrus sinensis
unknown — J. Menge —
nd UC-5s
Citrus sinensis
unknown — J. Menge —
nd UC-6s
Citrus sinensis
unknown — J. Menge —
nd UC-7s
Citrus limon
(L.) N. L.
Burm.
black rot — J. Menge —
nd UC-10s
Citrus sinensis
unknown — J. Menge —
nd UC-12s
Citrus sinensis
unknown — J. Menge —
A. longipes
EGS 30-033 T
Nicotiana tabacum
L. brown spot — E.G. Simmons Simmons 1981
A. tenuissima
EGS 34-015 T
Dianthus
sp. unknown — E.G. Simmons Simmons 1995, 1999b
A. alternata
EGS 34-016 T
Arachis hypogaea
L. unknown — E.G. Simmons Simmons 1967, 1981,
1995, 1999b
A. alternata
EGS 34-039
Datura metel
L. unknown — E.G. Simmons Simmons 1967, 1981,
1995
A. alternata
EGS 35-193
Musa sapientum
L. unknown — E.G. Simmons Simmons 1967, 1981,
1995, 1999b
A. mali
EGS 38-029 T
Malus domestica
Alternaria blotch CBS 106.24 E.G. Simmons Simmons 1999b
A. gaisen
EGS 90-0512 T
Pyrus pyrifolia
black spot BPI 802725 E.G. Simmons Simmons 1993; Simmons
and Roberts, 1993
A. gaisen
EGS 37-1321
Pyrus pyrifolia
black spot — E.G. Simmons Simmons and Roberts,
1993
A. arborescens
Simmons EGS 39-128 T
Lycopersicon esculentum
stem canker BPI 746367 E.G. Simmons Simmons 1999b
A. arbusti
Simmons EGS 91-129
Pyrus pyrifolia
non-pathogen BPI 802727 E.G. Simmons Simmons 1993
A. conjuncta
Simmons EGS 37-139 T
Pastinaca sativa
L. unknown BPI 445975 E.G. Simmons Simmons 1986
A. infectoria
Simmons EGS 27-193 T
Triticum
sp. unknown BPI 446385 E.G. Simmons Simmons 1986
A. solani
EGS 44-098 T
Solanum tuberosum
early blight — E.G. Simmons Simmons 2000
A. alternata
DAOM 166633
Lycopersicon esculentum
unknown — L. Hutchison —
A. solani
LK 99-02
Solanum tuberosum
early blight — L. Hutchison —
A. solani
Idaho A
Solanum tuberosum
early blight — L. Hutchison —
A. solani
49ss
Solanum tuberosum
early blight — B. Christ Weir et al 1998
A. solani
39ss
Solanum tuberosum
early blight — B. Christ Weir et al 1998
A. solani
22ss
Solanum tuberosum
early blight — B. Christ Weir et al 1998
124 M
YCOLOGIA
T
ABLE
I. Continued
Species
a
Isolate Code
b
Host Disease
c
Accession No.
d
Source
e
Reference(s)
A. alternata
alternata 1A
Linum usitatissimum
L. unknown — G.J. McKay Mckay et al 1999
A. linicola
Groves and
Skolko
linicola 3B
Linum usitatissimum
unknown — G.J. McKay Mckay et al 1999
A. brassicicola
(Schwein.)
Wiltshire
brassicicola 6B
Brassica oleracea
L. unknown — G.J. McKay Mckay et al 1999
A. solani
solani 7G
Ageratum
sp. unknown — G.J. McKay Mckay et al 1999
A. solani
solani 7H
Lycopersicon esculentum
unknown — G.J. McKay Mckay et al 1999
a
Morphological species designations. Underlined species indicate isolates identified by E.G. Simmons and considered representative of the species.nd5morpho-
logical species not determined.
b
Original isolate number. Isolates followed by a ‘‘T’’ represent ex-types of E.G. Simmons.
c
Disease associated with isolate if known. ‘‘Unknown’’ indicates that it was not known if the isolate was obtained from diseased tissue or if it was capable of inducing
disease if artificially inoculated. ‘‘non-pathogen’’ indicates isolates sampled from disease lesions that were non-pathogenic when artificially inoculated on the host of
isolation.
d
BPI or CBS accession numbers given for accessioned isolates. All other isolates stored in duplicate in the laboratories of T.L. Peever and L.W. Timmer.
e
Isolates obtained from E.G. Simmons Crawfordsville, IN, USA; L. Hutchison, Lakehead University, Thunder Bay, Canada; B. Christ, Pennsylvania State University,
University Park, PA, USA; M.E. Palm, USDA-APHIS, Beltsville, MD, USA; G. McKay, University of Belfast, Belfast, UK; J. Menge, University of California, Riverside, CA,
USA.
es have been deposited in GenBank (accession numbers
AY295020–33). Five additional regions of the genome were
amplified from selected isolates with variable endoPG se-
quences. These included actin (ACT), calmodulin (CAL),
chitin synthase (CHS), translation elongation factor alpha
(EF-1a) and 1, 3, 8-trihydroxynaphthalene (THN) reduca-
tase. Primers EF1–728F and EF1–986R, CAL-228F and CAL-
737R, ACT-512F and ACT-783R, CHS-79F and CHS-354R
(Carbone and Kohn 1999) were used to amplify a 300 bp
fragment of EF-1aincluding ;230 bp of intron sequence,
a 550 bp fragment of CAL including ;350 bp of intron
sequence, a 240 bp fragment of ACT including ;130 bp of
intron sequence and a 270 bp fragment of CHS including
no intron sequences, respectively. Amplification of EF-1a
was carried out as described for mtLSU, beta-tubulin and
endoPG. Cycling conditions consisted of 95 C for 2 min
followed by 35 cycles of 95 C for 1 min, 60 C for 1 min,
and 72 C for 1 min. These cycles were followed by a final
5 min elongation cycle at 72 C. Amplifications of CAL, ACT
and CHS were carried out in a PE Applied Biosystems Gene
Amp 9700 thermocycler (Applied Biosystems, Norwalk,
Connecticut), and reagents and cycling conditions were
similar to those used for EF-1aexcept that a 55 C annealing
temperature was used. Primers melanin-3 (59-TCAATCGA-
GCAGACATGGAG-39) and melanin-4 (59-CAACGCAGTT-
GACGGTGAT-39) were designed to amplify a 660 bp frag-
ment corresponding to nucleotide positions 903–1570 of
the
A. alternata
THN reductase gene, BRM2 (GenBank ac-
cession AB015743) involved in melanin biosynthesis. The
amplified sequence included ;570 bp of exon sequence
and ;90 bp of intron sequence. PCR were carried out in a
Hybaid Omn-E thermocycler as described above. Cycling
conditions consisted of 95 C for 2 min followed by 35 cycles
of 95 C for 1 min, 62 C for 1 min, and 72 C for 1 min.
These cycles were followed by a final 5 min elongation cycle
at 72 C. Amplicons were purified and sequenced as de-
scribed above.
Sequencing of anonymous regions.
Two anonymous regions
of the
Alternaria
genome were amplified using RAPD-PCR
with commercially available random decamer primers. Poly-
morphic amplicons were cloned and sequenced to design
specific primers that would amplify regions of the genome
useful for phylogenetic analysis among the small-spored cit-
rus-associated species of
Alternaria
. Four isolates of
Alter-
naria
from citrus (SH-MIL-1s, 4s, 5s, 37s) previously shown
to have divergent RAPD haplotypes (Peever et al 1999) were
used to screen for polymorphisms with primers OPA1 and
OPA2 (‘‘Kit OPA’’, Operon). Twenty five mL PCR reactions
contained 13reaction/loading buffer, 0.2 mM of either
primer, 200 mM dNTP, 2.0 mM MgCl
2
, 25 ng of DNA and
1 unit of
Taq
polymerase. PCR was carried out in a Hybaid
Omn-E thermocycler (Hybaid) with cycling conditions of 97
C for 1 min followed by 35 cycles of 96 C for 15 s, 45 C for
15 s, and 72 C for 15 s. A polymorphic 922 bp amplicon
produced by primer OPA1 from isolate SH-MIL-37s and a
polymorphic 614 bp amplicon produced by primer OPA2
from isolate SH-MIL-4s were purified from agarose gels us-
ing QiaQuick Gel Extraction columns (Qiagen) and cloned
into pCR4-TOPO (Invitrogen) as described above. Plasmid
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LTERNARIA SPECIES SYSTEMATICS
DNA was prepared as described above, and insert size was
verified by
Eco
R1 digestion and electrophoresis. Plasmid in-
serts were sequenced on both strands using T3 and T7
primers as described above. Primers were designed to the
ends of the inserts using Primer 3 so that the new primers
included the decamer priming site. Primers OPA1–3-L (59-
CAGGCCCTTCCAATCCAT-39) and OPA1–3-R (59-AGGCC
CTTCAAGCTCTCTTC-39) or OPA2–1-L (59-TGCCGAGCT
GTCAGATAATTG-39) and OPA2–1-R (59-GCCGAGCTGG
TGGAGAGAGT-39) were used to amplify a 900 bp or 600
bp fragment, respectively, from selected small-spored spe-
cies of
Alternaria
with different endoPG haplotypes (ap-
proximately two isolates representing each endoPG clade)
to facilitate comparison among all three datasets and min-
imize sequencing effort. BLAST searches revealed no sig-
nificant matches to these sequences in the databases. Ten
mL PCR reactions contained 13reaction/loading buffer,
0.3 mM of each primer, 200 mM dNTP, 2 mM MgCl
2
,10ng
DNA, 0.8 M betaine and 0.5 units
Taq
polymerase. PCR was
carried out in a GeneAmp PCR System 9700 thermocycler
(Applied Biosystems), and cycling conditions consisted of
94 C for 1 min followed by 35 cycles of 94 C for 20 s, 56 C
for 20 s and 72 C for 40 s followed by a final extension at
72 C for 7 min. Amplicons were purified and sequenced as
described above. OPA1–3 and OPA 2–1 sequences have
been deposited in GenBank (accession numbers AY295034–
53 and AY295054–72, respectively).
DNA sequence alignment and phylogenetic analysis.
MtLSU
and beta-tubulin sequences were aligned using ClustalX 1.8
(Thompson et al 1997) and a rooted, maximum likelihood
phylogeny was estimated for each gene using the DNAML
program in PHYLIP (Felsenstein 1993) with
A. infectoria
as
the outgroup. Previous phylogenetic studies of
Alternaria
have revealed that
A. infectoria
consistently forms the basal
clade of the genus (McKay et al 1999, Pryor and Gilbertson
2000, Pryor and Michailides 2001). Models of sequence evo-
lution were tested and model parameter estimates obtained
for each alignment using MODELTEST 3.06 (Posada and
Crandall 1998) as implemented in PAUP* 4.0b10 (Swofford
2002). For the mtLSU data, MODELTEST selected the F81
1G model with unequal base frequencies, a gamma shape
parameter of 0.0083 and a transition:transversion ratio of
1.0. For the beta-tubulin data, MODELTEST selected the
HKY 1G model with unequal base frequencies, a gamma
shape parameter of 0.462, and a transition:transversion ra-
tio of 1.571. Statistical support for phylogram topologies was
estimated using 100 bootstrapped datasets generated in the
SEQBOOT program of PHYLIP. One hundred phylograms
were estimated using DNAML with transition:transversion
ratios, gamma shape parameters, base frequencies estimat-
ed using MODELTEST and random input order of taxa
with three jumbling steps. Insertions and deletions in the
alignments are ignored by DNAML. A majority-rule consen-
sus tree was produced by the program CONSENSE and the
consensus phylograms were visualized in TREEVIEW (Page
1996). Maximum-likelihood branch lengths for the consen-
sus trees were estimated by removing the branch lengths
from the consensus tree and using this tree as a user tree
in DNAML. Clades with bootstrap values greater than 80%
were considered significantly supported. EndoPG, OPA1–3
and OPA2–1 sequences were aligned using Clustal-X and
rooted, maximum-likelihood phylogenies were estimated in-
dependently for each sequence using DNAML with
Alter-
naria gaisen
as outgroup.
Alternaria gaisen
has been dem-
onstrated to be phylogenetically distinct from the small-
spored citrus isolates and forms the basal clade in phylo-
genetic analyses of the small-spored species of
Alternaria
(Peever et al 2002). For the endoPG data, MODELTEST
selected the K80 model with equal base frequencies, equal
rates among sites and a transition:transversion ratio of 3.31.
For the OPA1–3 data, MODELTEST selected the K80 1G
model with equal base frequencies, a gamma shape param-
eter of 0.329, and a transition:transversion ratio of 2.08. For
the OPA2–1 data, MODELTEST selected the K80 model
with equal base frequencies, and a transition:transversion
ratio of 4.686. For the combined endoPG, OPA1–3 and
OPA2–1 data, MODELTEST selected the K80 1G model
with equal base frequencies, a gamma shape parameter of
0.013, and a transition:transversion ratio of 2.589. These pa-
rameter estimates were used in DNAML for phylogenetic
analyses. Topologies of the resulting phylograms were com-
pared using incongruence length difference (ILD) tests
(Farris et al 1994) to determine the suitability of combining
the endoPG, OPA1–3 and OPA2–1 data. ILD tests were im-
plemented in PAUP* (referred to as ‘‘partition homoge-
neity tests’’ in PAUP*) with invariant characters removed
and 500 randomized partitions. Tested data partitions in-
cluded: (i) endoPG, OPA1 and OPA2; (ii) endoPG and
OPA1; (iii) endoPG and OPA2; and (iv) OPA1 and OPA2.
Data partitions were considered significantly different at
P
,0.01 (Sullivan 1996). Sequence alignments have been de-
posited in TreeBASE (study accession number S891, data
matrices accession numbers M1461–64).
RESULTS
MtLSU and beta-tubulin phylogenies.
Amplification
of the mtLSU yielded amplicons that varied in length
from 418 to 432 bp. Data was not obtained for
A.
colombiana
,
A. limicola
,
A. linicola
, and
A. solani
had
5 and 9 bp deletions separated by bases AAA at the
5
9
end of the sequence. Forty isolates had identical
mtLSU sequence including
A. alternata
,
A. arbores-
cens
,
A. gaisen
,
A. infectoria
,
A. longipes
,
A. mali
,
A.
tenuissima
and nine morphospecies from citrus (F
IG
.
1). The mtLSU phylogeny revealed two well-support-
ed clades (bootstrap values
.
80%) with Clade 1 con-
taining
A. alternata
,
A. arborescens
,
A. brassicicola
,
A.
gaisen
,
A. infectoria
,
A. longipes
,
A. mali
,
A. tenuissima
,
and nine citrus-associated morphospecies and Clade
2 containing
A. limicola
,
A. linicola
and
A. solani
(F
IG
.
1). Amplification of the beta-tubulin gene yielded
amplicons that varied in length from 342 to 365 bp.
The beta-tubulin phylogeny revealed five well-sup-
ported clades (bootstrap support
.
80%) with Clade
1 containing
A. arbusti
,
A. conjuncta
and
A. infectoria
126 M
YCOLOGIA
F
IG
. 1. Rooted, consensus phylogeny estimated among species of
Alternaria
sampled from citrus (bold type) plus reference
Alternaria
species using partial mtLSU sequence data. Phylogeny was rooted by
A. infectoria
and was estimated using maximum
likelihood with the DNAML program in PHY LIP. Numbers at the major branches indicate the percentage occurrence of the
clade to the right of the branch in 100 bootstrapped datasets. Only bootstrap values greater than 50% are shown. Branch
lengths are proportional to the inferred amount of evolutionary change, and the scale bar represents 0.01 nucleotide sub-
stitutions per site. Clades 1 and 2 inferred based on bootstrap values greater than 80% are shown. Isolates shaded in gray
have identical sequence.
and Clade 2 containing
A. limicola
and Clade 3 con-
taining
A. solani
(F
IG
. 2). Clade 4 contained isolates
of
A. alternata
,
A. gaisen
,
A. toxicogenica
,
A. turkisaf-
ria
and 39–191. Clade 5 included four isolates from
healthy citrus tissue (UC-4, 6, 10, 12) and
A. longipes
.
Twenty-nine isolates, including 23 from citrus, had
beta-tubulin sequences identical to
A. toxicogenica
(F
IG
. 2, Clade 4).
EndoPG phylogeny.
The predicted 489 bp endoPG
fragment was amplified from all isolates except
A.
arbusti
,
A. brassicicola
,
A. conjuncta
,
A. infectoria
,
A.
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LTERNARIA SPECIES SYSTEMATICS
F
IG
. 2. Rooted, consensus phylogeny estimated among species of
Alternaria
sampled from citrus (bold type) plus reference
Alternaria
species using partial beta-tubulin sequence data. Phylogeny was rooted by
A. infectoria
and was estimated using
maximum likelihood with the DNAML program in PHYLIP. Numbers at the major branches indicate the percentage occur-
rence of the clade to the right of the branch in 100 bootstrapped datasets. Only bootstrap values greater than 50% are
shown. Branch lengths are proportional to the inferred amount of evolutionar y change and the scale bar represents 0.1
nucleotide substitutions per site. Clades 1–5 inferred based on bootstrap values greater than 80% are shown. Isolates shaded
in gray have identical sequence. The beta-tubulin sequence of
A. toxicogenica
is identical to those of 28 other citrus-associated
and reference species including:
A. alternata
,
A. arborescens
,
A. citriarbusti
,
A. citrimacularis
,
A. dumosa
,
A. interrupta
,
A.
limoniasperae
,
A. mali
,
A. perangusta
,
A. tangelonis
,
A. tenuissima
,
A. turkisafria
, Acitri-1,-2, Australia D, UC-1, 2, 5, 7, 39–189,
39–190 and 39–192.
linicola
and
A. solani
. All attempts to obtain ampli-
cons from these isolates by modifying PCR conditions
were unsuccessful. Amplicons of isolates of
A. limicola
were not included in the phylogenetic analysis due
to extensive sequence divergence relative to the
small-spored species. The endoPG phylogeny re-
vealed significantly more variation among the citrus-
associated isolates than either mtLSU or beta-tubulin
128 M
YCOLOGIA
F
IG
. 3. Rooted, consensus phylogeny estimated among small-spored species of
Alternaria
sampled from citrus (boldface)
plus reference
Alternaria
species using partial endoPG sequence data. Phylogeny was rooted by
A. gaisen
and was estimated
using maximum likelihood with the DNAML program in PHYLIP. Numbers at the major branches indicate the percentage
occurrence of the clade to the right of the branch in 100 bootstrapped datasets. Only bootstrap values greater than 50% are
shown. Branch lengths are proportional to the inferred amount of evolutionary change and the scale bar represents 0.01
nucleotide substitutions per site. Clades 1–5 inferred based on bootstrap values greater than 80% are shown. Isolates shaded
in gray have identical sequence.
phylogenies and defined five clades with bootstrap
values greater than 80% (F
IG
. 3). Clade 1 contained
A. gaisen
and Clade 2 contained
A. alternata
,
A. ar-
borescens
, Acitri-1, and UC-1. Clade 3 contained
A.
citrimacularis
,
A. dumosa
,
A. interrupta
,
A. limonias-
perae
,
A. mali
,
A. perangusta
,
A. tenuissima
,
A. turki-
safria
, UC-2, 4, 5, 6, 7, 10, 12, and Clade 4 contained
A. alternata
and 39–192 while Clade 5 contained
A.
colombiana
,
A. longipes
, and
A. tangelonis
. Morpho-
species
A. citrimacularis
was polyphyletic with isolate
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LTERNARIA SPECIES SYSTEMATICS
BC2-RLR-32s occurring in Clade 3 and isolate BC2-
RLR-17s in the poorly supported clade that also in-
cluded
A. citriarbusti
and
A. toxicogenica
(F
IG
. 3).
Combined endoPG, OPA1 and OPA2 phylogeny.
Spe-
cific primers designed to the cloned OPA1–3 and
OPA2–1 fragments produced amplicons of the ex-
pected size from all isolates including those which
did not amplify with the original decamer primer
(i.e., null alleles). This indicated that the RAPD poly-
morphisms (i.e., presence or absence of a band) like-
ly were due to mutations in the priming sites. Incon-
gruence length difference tests performed on all
three datasets (endoPG, OPA1 and OPA2) or on
endoPG plus OPA1 were significant (
P
5
0.002), but
tree lengths of the randomized datasets were only a
few steps longer than the sum of lengths for the orig-
inal partitions. All other pairwise comparisons were
not significantly different (
P
.
0.01). MtLSU and
beta-tubulin sequences were not included in the
combined dataset because they provided no phylo-
genetic signal in analyses of the small-spored species.
The phylogeny estimated from the combined data
defined nine clades with bootstrap values greater
than 80% (F
IG
. 4). Clade 1 contained
A. gaisen
,
Clade 2 contained only
A. citrimacularis
, Clade 3 con-
tained
A. perangusta
and
A. turkisafria
and Clade 4
contained
A. alternata
and 39–192. Clade 5 con-
tained
A. citriarbusti
,
A. citrimacularis
and 39–190.
Clade 6 contained only
A. toxicogenica
, Clade 7 con-
tained
A. limoniasperae
, Clade 8 contained
A. arbores-
cens
and Acitri-1, and Clade 9 contained
A. colombi-
ana
,
A. longipes
and
A. tangelonis
. Clades 5 and 6 of
the combined phylogeny contained isolates that were
found in a poorly supported clade in the endoPG
phylogeny (
A. citriarbusti
,
A. citrimacularis
,
A. toxi-
cogenica
and 39–190). Clades 3, 5 and 9 contained
more than one morphological species.
Alternaria ci-
trimacularis
was polyphyletic with isolate BC2-RLR-
32s in Clade 2 and isolate BC2-RLR-17s in Clade 5
(F
IG
. 4).
Analysis of ACT, CAL, CHS, EF-1
a
and THN.
Align-
ment of sequences from five additional regions of the
Alternaria
genome revealed little polymorphism
among selected small-spored
Alternaria
isolates. Iso-
lates selected from clades 3, 4, 5, 6, 7, 8 and 9 of the
combined analysis were invariant for ACT and THN
(results not shown). Alignment of EF-1
a
from nine
small-spored isolates representing different clades in
the combined analysis revealed four polymorphic
sites, one of which separated
A. gaisen
from all other
isolates and three sites that separated
A. tangelonis
(EV-MIL-2s) from all other isolates. The same set of
isolates revealed three polymorphic CAL sites and 2
polymorphic CHS sites.
DISCUSSION
Analysis of mtLSU and beta-tubulin sequences re-
vealed two monophyletic
Alternaria
lineages associ-
ated with citrus. The first lineage consisted of isolates
of large-spored
Alternaria limicola
causing leaf spot
of Mexican lime.
Alternaria limicola
appears to rep-
resent a valid phylogenetic as well as morphological
taxon. This species has constricted, moniliform co-
nidia, which often are longer than 100
m
m (Simmons
1990) and are easily distinguished morphologically
from the small-spored citrus isolates, which generally
are 30–50
m
m in length (Simmons 1999). MtLSU and
beta-tubulin sequence data indicated that
A. limicola
is most closely related to other large-spored species
such as
A. solani
and
A. linicola
. Symptoms of man-
cha foliar de los citricos on Mexican lime include the
production of water-soaked pustules that more close-
ly resemble a bacterial disease; the disease originally
was mistaken for citrus canker (Palm and Civerolo
1994). These symptoms are very different from those
associated with Alternaria brown spot and leaf spot,
and
A. limicola
has never been associated with black
rot of citrus. The second well-supported clade in-
ferred from mtLSU and beta-tubulin sequences in-
cluded small-spored species of
Alternaria
associated
with Alternaria brown spot of tangerines and hybrids,
Alternaria leaf spot of rough lemon and citrus black
rot. These gene regions were insufficiently variable
to estimate a phylogeny among the small-spored iso-
lates as were EF-1
a
, CAL, CHS and THN sequences.
This result is similar to that reported previously for
phylogenetic analyses of beta-tubulin (McKay et al
1999) and ITS sequences (Kusaba and Tsuge 1995,
McKay et al 1999, Pryor and Gilbertson 2000, Pryor
and Michailides 2002).
MtLSU sequence data were sufficiently variable to
differentiate the large-spored species of
Alternaria
(
A. solani
,
A. linicola
and
A. limicola
) from the small-
spored species (
A. alternata
and
A. tenuissima
). How-
ever, analysis of this gene region did not allow us to
differentiate
A. brassicicola
or
A. infectoria
from any
of the small-spored citrus isolates or reference iso-
lates such as
A. alternata
and
A. tenuissima
. The
mtLSU appears to be considerably less variable
among species of
Alternaria
than the mtSSU (Pryor
and Gilbertson 2000), a gene region used to differ-
entiate
A. alternata
,
A. brassicicola
and
A. infectoria
.
Sequence data from the 5
9
end of the beta-tubulin
gene revealed four clades in the current study and
appears to be slightly more variable than either ITS,
mtSSU, or exon 6 of the beta-tubulin gene (Kusaba
and Tsuge 1995, Pryor and Gilbertson 2000, Pr yor
and Michailides 2002, McKay et al 1999). This por-
tion of the beta-tubulin gene has been successful in
130 M
YCOLOGIA
F
IG
. 4. Rooted, consensus phylogeny estimated among small-spored species of
Alternaria
sampled from citrus (bold type)
plus reference
Alternaria
species using combined endoPG, OPA1–3, and OPA2–1 sequence data. Phylogeny was rooted by
A. gaisen
and was estimated using maximum likelihood with the DNAML program in PHYLIP. Numbers at the major branches
indicate the percentage occurrence of the clade to the right of the branch in 100 bootstrapped datasets. Only bootstrap
values greater than 50% are shown. Branch lengths are proportional to the inferred amount of evolutionary change, and
the scale bar represents 0.01 nucleotide substitutions per site. Clades 1–9 inferred based on bootstrap values greater than
80% are shown. Isolates shaded in gray have identical sequence.
delimiting phylogenetic species of
Cylindrocladium
Morgan (Crous et al 1999),
Fusarium
Link : Fr.
(O’Donnell et al 1998, 2000) and basidiomycetes
(Thon and Royse 1999).
Phylogenetic analysis of the combined endoPG,
OPA1–3 and OPA2–1 data revealed substantially
more variation than the beta-tubulin phylogeny for
the small-spored isolates from citrus as well as several
reference species. The analysis revealed nine clades
that generally were congruent with described mor-
phospecies (F
IG
. 4). These nine clades could be in-
terpreted as defining nine species under a phyloge-
netic species concept with genealogical concordance
(Taylor et al 2000). Other regions of the genome,
including EF-1
a
, CAL, ACT and CHS, have been use-
ful in estimating phylogenies at the inter- and intra-
131P
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:
A
LTERNARIA SPECIES SYSTEMATICS
specific levels in other fungi (Carbone and Kohn
1999, 2001,Crous et al 1999, O’Donnell et al 1998,
2000), but had little variation among the small-
spored
Alternaria
isolates associated with citrus. We
consider this further evidence of the close evolution-
ary relationship among these fungi. EndoPG has
proven useful for the elucidation of phylogeograph-
ical patterns among Alternaria brown spot and leaf
spot isolates (Peever et al 2002), and several isolates
from that study were included here. EndoPG also
might prove useful for phylogenetic or phylogeo-
graphic studies of other closely related fungi. To our
knowledge, this is the first reported use of this gene
for molecular systematics. The results of Peever et al
(2002) revealed three endoPG clades among a world-
wide sample of citrus brown spot isolates that corre-
spond to clades 2, 4 and 6 of the combined analysis
in the present study. The anonymous regions that
were generated from RAPD for this study also appear
to be very useful for phylogenetic studies of closely
related
Alternaria
species.
In almost all cases where more than one isolate of
a morphospecies was analyzed, both isolates clustered
together in the same clade. However, the number of
morphospecies exceeded that which could be sup-
ported phylogenetically and at least one morphospe-
cies appeared to be polyphyletic (
A. citrimacularis
).
Only two morphospecies,
A. limoniasperae
and
A. tox-
icogenica
, were associated uniquely with a single clade
in the combined analysis. The eight remaining mor-
phospecies were not associated uniquely with a spe-
cific clade, host, disease or ecological niche. The
small-spored citrus isolates examined in this study
were sampled from different ecological niches (leaf
spots, fruit rots, healthy tissue) and have been shown
to differ in host specificity and virulence (Peever et
al 1999, 2002). However, these ecological differences
did not map uniquely to specific clades defined
through phylogenetic analysis of the combined
endoPG, OPA1 and OPA2 data. Among the brown
spot isolates, there were more morphospecies than
could be supported by the phylogenetic analysis. This
pattern contrasts with that revealed in many phylo-
genetic studies of morphologically well-characterized
fungi. In these cases, morphospecies have been
found to be polytypic and new phylogenetic species
are typically uncovered (Geiser et al 1998, Giraud et
al 1997, O’Donnell et al 1998). One definition of a
fungal species consists of monophyletic groups that
have unique, diagnosable phenotypic characters
(Harrington and Rizzo 1999), and we advocate this
species concept for the small-spored
Alternaria
iso-
lates associated with citrus. The lack of strict corre-
lation between each phylogenetic lineage and unique
phenotypic, ecological or biological characters
among the small-spored citrus-associated
Alternaria
and the occurrence of multiple morphospecies in
several clades calls into question the practical utility
of both the morphospecies and species defined by
strictly phylogenetic criteria. Until such time as
unique phenotypic, biological or ecological differ-
ences can be consistently and uniquely associated
with isolates in a given clade, we advocate collapsing
the small-spored species of
Alternaria
from citrus into
a single phylogenetic species,
A. alternata
.
Isolates sampled from citrus fruit with symptoms of
black rot were polyphyletic. It is not known if the
seven black rot isolates studied here fit the morpho-
logical description of
A. citri
(Simmons 1990) but our
data clearly indicate that black rot can be caused by
several phylogenetically distinct small-spored
Alter-
naria
isolates. Black rot isolates and brown spot iso-
lates sampled from different citrus hosts in several
parts of the world had identical beta-tubulin sequenc-
es, and the former were distributed throughout the
combined analysis in three of nine clades with the
brown spot isolates. This might indicate that phylo-
genetically distinct isolates associated with citrus (in-
cluding brown spot isolates) can induce citrus black
rot. A similar result was obtained by Kang et al
(2002), who found that phylogenetically diverse
small-spored
Alternaria
isolates were associated with
black rot of citrus in South Africa. In the present
study, one of the black rot isolates (Acitri-1) was
found in a clade with
A. arborescens
, a host specific
toxin-producing tomato pathogen. We speculate that
most or all small-spored species of
Alternaria
associ-
ated with citrus or other hosts can cause black rot.
The morphological similarity of citrus black rot iso-
lates and brown spot isolates in culture has been ob-
served previously, and brown spot isolates were ini-
tially described as a distinct strain of
A. citri
(Doidge
1929, Kiely 1964, Pegg 1966, Ruehle 1937, Whiteside
1976). Based on our data, there appears to be no
basis for considering black rot isolates as a distinct
taxon and we also advocate the use of
A. alternata
for these isolates.
Alternaria brown spot and leaf spot pathogens
were found in two of five clades in the endoPG phy-
logeny and five of nine clades in the combined
endoPG, OPA1–3 and OPA2–1 phylogeny. Rooting
the endoPG phylogeny by
A. limicola
(data not
shown) revealed that
A. colombiana
,
A. gaisen
,
A. lon-
gipes
and
A. tangelonis
occupy a basal position in this
phylogeny. Citrus morphospecies in this basal clade
(
A. colombiana
,
A. tangelonis
) are pathogenic on tan-
gerine and tangerine hybrids and produce host-spe-
cific ACT-toxin (Masunaka et al 2000).
Alternaria
gaisen
, which causes black spot of Japanese pear, is
known to produce host-specific AK-toxins (Nishimura
132 M
YCOLOGIA
and Kohmoto 1983, Simmons and Roberts 1993, Ta-
naka et al 1999) that are structurally similar to ACT-
toxins produced by citrus brown spot isolates (Otani
et al 1995). Homologs of genes controlling AK-toxin
production have been identified in brown spot iso-
lates, including isolates in this basal clade (Masunaka
et al 2000). The occurrence of toxin-producing citrus
brown spot isolates in several clades derived from the
A. gaisen
clade indicates that toxin production may
be an ancestral character in this group of organisms.
In addition, it appears that rough lemon leaf spot
pathogens (
A. limoniasperae
) also have evolved from
an ACT-toxin producing ancestor, even though they
produce an unrelated toxin (ACRL-toxin) and are
specific to rough lemon (Peever et al 1999).
A. ar-
borescens
,
A. longipes
, and
A. mali
similarly appear to
have evolved from an AK-toxin-producing ancestor
yet produce host-specific toxins that are unrelated to
AK- and ACT-toxin (Nishimura and Kohmoto 1983,
Otani et al 1995). The recent availability of sequence
data from the entire AK- and AF-toxin gene clusters
(Hatta et al 2002, Tanaka et al 1999, 2000) and
knowledge of their chromosomal location (Hatta et
al 2002, Johnson et al 2001) will allow tests of specific
hypotheses about the evolution of these toxin genes,
the evolution of host specificity, correlations between
toxin production and virulence and the molecular
mechanisms conferring loss of toxin production in
specific phylogenetic lineages of these fungi.
The basal position of
A. gaisen
in the combined
phylogeny also suggests that citrus black rot isolates
have evolved from a toxin-producing ancestor. Black
rot isolates are nonpathogenic on leaves and imma-
ture fruit (Kiely 1964, Ruehle 1937, Masunaka et al
2000), and the few isolates that have been tested do
not produce host-specific toxins (Pegg 1966, Masu-
naka et al 2000). Although the black rot isolates em-
ployed in the present study have not been tested for
ACT-toxin production, they were found in three of
nine clades with known toxin-producing strains. It is
possible that black rot isolates have lost the ability to
produce toxins but have retained some or all of the
genes controlling their synthesis. On the other hand,
toxin sequences might have been lost due to the loss
of entire chromosomes carr ying a cluster of toxin
gene sequences (Hatta et al 2002, Johnson et al
2001). Our working hypothesis is that all small-
spored isolates of
Alternaria
associated with citrus are
potential black rot pathogens, and this hypothesis is
being tested using host inoculations and a larger sam-
ple of isolates from diverse geographic regions.
ACKNOWLEDGMENTS
PPNS No. 0352, Department of Plant Pathology, College of
Agriculture and Home Economics Research Center, Project
No. 0300, Washington State University, Pullman, Washing-
ton 99164–6430. Research was supported in part by the
Florida Citrus Production Advisory Council project No.
013–16P to L. W. Timmer. The authors would like to thank
Patrick Friel and Tamara Reynolds for technical assistance
and Lori M. Carris and Jack D. Rogers for valuable discus-
sions regarding fungal nomenclature.
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