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A unique species in Phytophthora clade 10,
Phytophthora intercalaris sp. nov., recovered from
stream and irrigation water in the eastern USA
X. Yang,
1
Y. Balci,
2
N. J. Brazee,
3
A. L. Loyd
4
3 and C. X. Hong
1
Correspondence
X. Yang
yxiao9@vt.edu
1
Hampton Roads Agricultural Research and Extension Center, Virginia Tech, Virginia Beach, VA,
USA
2
Department of Plant Sciences and Landscape Architecture, University of Maryland, College Park,
MD, USA
3
UMass Extension, Center for Food, Agriculture and the Environment, University of Massachusetts,
Amherst, MA, USA
4
Department of Plant Pathology, North Carolina State University, Raleigh, NC, USA
A novel species of the genus Phytophthora was recovered during surveys of stream and nursery
irrigation water in Maryland, Massachusetts, North Carolina, Virginia and West Virginia in the
USA. The novel species is heterothallic, and all examined isolates were A
1
mating type. It
produced rare ornamented oogonia and amphigynous antheridia when paired with A
2
mating
type testers of Phytophthora cinnamomi and Phytophthora cryptogea. Sporangia of this novel
species were non-papillate and non-caducous. Thin-walled intercalary chlamydospores were
abundant in hemp seed agar and carrot agar, while they were produced only rarely in aged
cultures grown in clarified V8 juice agar. Phylogenetic analyses based on sequences of the
internal transcribed spacer region and the b-tubulin and mitochondrial cytochrome-c oxidase 1
(cox1) genes indicated that the novel species is phylogenetically close to Phytophthora gallica
in Phytophthora clade 10. The novel species has morphological and molecular features that are
distinct from those of other species in Phytophthora clade 10. It is formally described here as
Phytophthora intercalaris sp. nov. Description of this unique clade-10 species is important for
understanding the phylogeny and evolution of Phytophthora clade 10.
INTRODUCTION
The genus Phytophthora currently includes more than 130
species (Kroon et al., 2012; Martin et al., 2012, 2014).
Although species of Phytophthora resemble filamentous
fungi morphologically, they actually belong to the kin gdom
Chromalveolata, which is more closely related to plants and
algae (Adl et al., 2005). Most species of Phytophthora are
ecologically and economically important plant pathogens
in forest and agricultur al settings (Erwin & Ribeiro,
1996). For example, Phytophthora ramorum causes the
notorious sudden oak death (SOD) on many oak species
and has wiped out millions of forest trees in California
and Oregon (Rizzo et al., 2002, 2005). It also causes
ramorum blight on hundreds of ornamental plants
(Werres et al., 2001). Excluding species that are well-estab-
lished plant pathogens (e.g. Phytophthora cinnamomi and
P. ramorum), many species of Phytophthora have unre-
solved host ranges, such as the recently described Phy-
tophthora amnicola (Crous et al., 2012), Phytophthora
borealis, Phytophthora riparia (Hansen et al., 2012), Phy-
tophthora fluvialis (Crous et al., 2011), Phytophthora hydro-
gena (Yang et al., 2014b), Phytophthora macilentosa,
Phytophthora stricta (Yang et al., 2014a), Phytophthora mis-
sissippiae (Yang et al., 2013), Phytophthora moyootj (Crous
et al., 2014) and Phytophthora virginiana (Yang & Hong,
2013). While limited information on host specificity does
exist, it is assumed that some of these newly described
species may be saprophytes of dead organic matter (Brasier
et al., 2003; Jung et al., 2011; Yang et al., 2013).
The genus Phytophthora has been expanding quickly over
the past 15 years, and many novel species in the genus
3Present address: Bartlett Tree Research Laboratories, Charlotte, NC,
USA.
Abbreviations: ITS, internal transcribed spacer; ML, maximum-
likelihood; SOD, sudden oak death.
The GenBank/EMBL/DDBJ accession numbers for the ITS and cox1
and b-tubulin gene sequences of isolate 45B7
T
are KT163268,
KT163315 and KT163336, respectively.
A supplementary table and two supplementary figures are available
with the online Supplementary Material.
International Journal of Systematic and Evolutionary Microbiology (2016), 66, 845–855 DOI 10.1099/ijsem.0.000800
000800
G
2015 IUMS Printed in Great Britain 845
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were initially recovered from aquatic ecosystems. Since the
2000s, the SOD epidemic has reminded the scientific and
public communities of the importance of species of Phy-
tophthora and catalysed research on this important plant-
pathogen group. Extensive surveys have been conducted
to study species of Phytophthora in many previously unex-
plored ecosystems such as forests, streams, irrigation water
and riparian ecosystems. Since then, there has been a
‘golden era’ for discovering novel species of Phytophthora.
Approximately 80 novel species of Phytophthora have
been described since 2000, with 16 of these novel species
(plus many provisional species) first recovered from
aquatic ecosystems. Specifically, these include P. amnicola
(Crous et al., 2012), P. borealis (Hansen et al., 2012), Phy-
tophthora chlamydospora (5Phytophthora taxon Pgchla-
mydo) (Brasier et al., 2003; Hansen et al., 2015),
P. fluvialis (Crous et al., 2011), P. lacustris (Nechwatal
et al., 2013), P. moyootj (Cro us et al., 2014) and Phy-
tophthora taxon aquatilis (Hong et al., 2012) from stream
water, Phytophthora aquimorbida (Hong et al., 2012), P.
hydrogena (Yang et al., 2014b), Phytophthora hydropathica
(Hong et al., 2010), P. macilentosa (Yang et al., 2014a), P.
mississippiae (Yang et al., 2013), P. stricta (Yang et al.,
2014a) and P. virginiana (Yang & Hong, 2013) from irriga-
tion water and Phytophthora pluvialis (Reeser et al.,2013)
from canopy drip. These species, along with Phytophthora
gonapodyides, exemplify endemic and dominant species in
aquatic environments worldwide, while their less-frequent
presence in terrestrial environments indicates the potential
variation in composition of species of Phytophthora between
aquatic and terrestrial habitats.
Species concepts for t he genus Phytophthora have evolved
from being based on morphology to phylogeny. Tradition-
ally, classification of species of Phytophthora was accom-
plished using morphological characters (Waterhouse,
1963). Now, by sequence analyses, species of Phytophthora
have generally been classified into 10 phylogenetic clades
(Blair et al., 2008; Cooke et al., 2000; Kroon et al., 2004;
Martin et al., 2014).
Phytophthora clade 10 currently contains four formal
species: Phytophth ora boehmeriae, Phytophthora gallica,
Phytophthora kernoviae and Phytophthora morindae.
Except for P. boehmeriae, all these species were described
after 2005. Most of these species pose threats to agricultural
production and natural plantations (Brasier et al., 2005;
Erwin & Ribeiro, 1996; Jung & Nechwatal, 2008 ; Nelson
& Abad, 2010). P. kernoviae is an invasive pathogen to
many forest and ornamental plants in the UK and presents
a significant threat to global biosecurity (Brasier et al.,
2005). P. boehmeriae causes a variety of diseases on a
number of host plants such as brown rot of citrus fruits
(Erwin & Ribeiro, 1996), foliar blight of hot pepper (Cap-
sicum annuum) (Chowdappa et al., 2014) and boll rot of
cotton (Gossypium sp.) (Erwi n & Ribeiro, 1996). P. morin-
dae causes black flag disease of noni (Morinda citrifolia)
(Nelson & Abad, 2010), an evergreen fruit tree in Hawaii.
The pathogenicity of P. gallica has not been fully
determined. This species was originally isolated from rhizo-
sphere soil of declining oak and reed stands in France and
Germany (Jung & Nechwatal, 2008) and later detected
from streams in Spain (Catala
`
et al., 2015) and Oregon,
USA (Sims et al., 2015). It has been shown to be weakly
to moderately aggressive to pedunculate oak (Quercus
robur), white willow (Salix alba), common alder (Alnus
glutinosa) and European beech (Fagus sylvatica ) in patho-
genicity tests (Jung & Nechwatal, 2008). Also, P. gallica is
morphologically distinct in producing non-papillate and
persistent sporangia and being self-sterile (Jung & Nechwa-
tal, 2008), while the remaining three clade-10 species are
homothallic and produce papillate and caducous sporangia
(Brasier et al., 2005; Erwin & Ribeiro, 1996; Nelson &
Abad, 2010). Additionally, P. boehmeriae, P. kernoviae
and P. morindae are phylogenetically closely related and
form a distinct cluster within clade 10, while P. gallica
was placed basal to them according to previous phyloge-
netic analyses (Jung & Nechwatal, 2008; Martin et al.,
2014). In addition to the formal species, a provisional
species in clade 10, Phytophthora gondwanense prov.
nom., has recently been recovered from rivers in the Gond-
wana Rainforests of Australia World Heritage Area (Scar-
lett et al., 2015).
Isolates of a previously unknown species of Phytophthora
have been recovered frequently from stream water in the
eastern USA. Preliminary sequence analyses found that
this novel species is phylogenetically close to species in
clade 10. In this study, it is described based on its distinct
morphological, physiological and molecular characters and
formally named as Phytophthora intercalaris sp. nov.
METHODS
Isolate collection and maintenance. Aside from one isolate that
was recovered from irrigation water at an ornamental plant nursery in
North Carolina, all isolates of Phytophthora intercalaris sp. nov. were
recovered from streams in five eastern states: Maryland, Massachu-
setts, North Carolina, Virginia and West Virginia. Information on
these isolates is given in Table 1.
In Virginia, P. intercalaris isolates were recovered by the Virginia
Department of Forestry from a wide range of distinct stream locations
using rhododendron leaf baits during stream surveys for P. ramorum
from 2007 to 2012. The baits were deployed in the surveyed streams
for 7 to 14 days and then transferred to the Ornamental Plant Path-
ology Lab in Virginia Beach, VA. They were cut into 10
|10 mm
sections and plated onto PARP selective medium (Jeffers & Martin,
1986). Pure cultures were obtained from hyphal tips of emerging
colonies from the edge of leaf pieces. Cultures were maintained and
routinely subcultured onto 20% clarified V8 juice agar (cV8A) during
morphological examination in this study. Blocks of fresh cultures
growing in cV8A were transferred into microtubes containing sterile
distilled water and autoclaved hemp seeds for long-term storage at
15
u
C. In Maryland and West Virginia, P. intercalaris was isolated as
part of SOD stream surveys using the same protocol described above.
In North Carolina, P. intercalaris isolates were recovered from rivers,
streams and, in one incidence, a reclamation reservoir. Samples were
taken using 500 ml sterile polypropylene bottles. At each sampling,
500 ml water was collected at a depth of approximately 20–25 cm
below the surface and the nearest point of the intake pump. A water
X. Yang and others
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Table 1. Isolates of P. intercalaris sp. nov. examined in this study
GenBank accession numbers are listed in Table S1. Culture collections: ATCC, American Type Culture Collection, Manassas, VA, USA; CBS,
CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. Substrates: F, filters; GPB, green pepper baits; PB, pear baits; RLB, rhododen-
dron leaf baits.
Isolate Location State Substrate Date
45B7
T
(5 ATCC TSD-7
T
5 CBS 140632
T
) Rapidan River Virginia Stream water, RLB July 2007
47E2 Ramsey’s Draft Virginia Stream water, RLB September 2008
48A1 East Hawksbill Virginia Stream water, RLB May 2008
48E5 McKittricks Branch Virginia Stream water, RLB October 2008
48H8 Robinson River Virginia Stream water, RLB October 2008
49A7 (5 CBS 140631) Rush River Virginia Stream water, RLB May 2009
49C1 South River Virginia Stream water, RLB May 2009
50B7 North Fork Thornton River Virginia Stream water, RLB September 2009
50F9 Thornton River Virginia Stream water, RLB September 2009
51B6 Hazel River Virginia Stream water, RLB June 2010
52C9 Jordan River Virginia Stream water, RLB October 2010
56J7 Lickinghole Creek Virginia Stream water, RLB September 2011
57B2 Flint Run Virginia Stream water, RLB October 2011
57G3 South Fork Rockfish River Virginia Stream water, RLB October 2011
59J4 Rappahannock River Virginia Stream water, RLB September 2012
59J5 Rappahannock River Virginia Stream water, RLB September 2012
42P2-2 Duncan Creek North Carolina Stream water, F 2009
47P4-2 Green River North Carolina Stream water, F 2009
66P2-1 Irrigation reservoir North Carolina Irrigation water, F 2009
1Pax2 P4-1 French Broad River North Carolina Stream water, F 2012
2Pax2 P4-2 French Broad River North Carolina Stream water, F 2011
AF-15 Connecticut River Massachusetts Stream water, RLB July 2013
AV-W5-1 Connecticut River Massachusetts Stream water, F July 2014
MV-W12-3 Connecticut River Massachusetts Stream water, F August 2014
MV-W1-3 Connecticut River Massachusetts Stream water, F June 2014
SS-W13-1 Connecticut River Massachusetts Stream water, F August 2014
SS-W15-3 Connecticut River Massachusetts Stream water, F September 2014
UMF-32 Connecticut River Massachusetts Stream water, RLB September 2013
UMF-39 Connecticut River Massachusetts Stream water, GPB October 2013
HR-07 Fort River Massachusetts Stream water, RLB July 2013
HR-24 Fort River Massachusetts Stream water, PB August 2013
HR-30A Fort River Massachusetts Stream water, RLB September 2013
HR-35 Fort River Massachusetts Stream water, PB September 2013
MR-10 Mill River Massachusetts Stream water, RLB July 2013
MR-17 Mill River Massachusetts Stream water, PB July 2013
MR-39 Mill River Massachusetts Stream water, RLB September 2013
MR-46 Mill River Massachusetts Stream water, RLB September 2013
MR-48 Mill River Massachusetts Stream water, PB October 2013
MR-52 Mill River Massachusetts Stream water, PB October 2013
KG-W12-2 Mohawk Brook Massachusetts Stream water, F August 2014
RC_6_3A Rock Creek Maryland Stream water, RLB August 2009
BAL_7_1B Ballenger Creek Maryland Stream water, RLB August 2009
GUN_7_1C Gunpowder Falls Maryland Stream water, RLB August 2009
1475_FLA_7_3B Flat Creek Maryland Stream water, RLB July 2009
1365_RC_2_1_g Rock Creek Park Maryland Stream water, RLB June 2009
MIL_1_2C Mill Creek Maryland Stream water, RLB May 2009
1490_SMT2_8_1_c Muddy Creek (Smithsonian) Maryland Stream water, RLB July 2009
1191_RC_4_4_b2 Rock Creek Maryland Stream water, RLB July 2009
262_ROB_1_3c Robinson Creek Maryland Stream water, RLB May 2009
104_BFA Beech Fork West Virginia Stream water, RLB June 2007
117_MC135 Muddlety Creek West Virginia Stream water, RLB October 2007
Phytophthora intercalaris sp. nov., in clade 10
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filtration assay was used to recover isolates of Phytophthora
(MacDonald et al., 1994). Aliquots of each water sample were passed
through Fisher Scientific brand filter papers (1–5 mm pore size). After
the filtrate had been vacuumed, filters were removed and placed top-
side down on PARPH selective medium (Jeffers & Martin, 1986).
Plates with filter papers were incubated in the dark at 21
u
C for 24 h.
After initial incubation, filter papers were removed and plates were
incubated in the dark at 21
u
C for an additional 48 h. Isolations were
made from colonies with visually distinctive morphological characters
and hyphal branching typical of members of Phytophthora. Sub-
cultures were obtained from each colony by transferring a hyphal tip
to PARPH. Pure cultures were maintained on cornmeal agar for
further characterization (Loyd et al., 2014). In Massachusetts,
P. intercalaris isolates were recovered in 2013 and 2014 from the
Connecticut River and various tributaries during a survey of distri-
bution of species of Phytophthora. Isolates were recovered using
various baits (rhododendron leaves, non-organic pear and pepper) in
2013 and by water filtration in 2014. Pure cultures were obtained and
prepared for long-term storage in a similar manner to that described
above.
DNA extraction. An approximately 5|5 mm culture plug of each
isolate was taken from the actively growing area of a fresh culture. It
was then grown in 20% clarified V8 broth at room temperature
(approx. 23
u
C) for 7–10 days to produce mycelium. Virginia isolates
were lysed using a FastPrep-24 Instrument (MP Biomedicals).
Genomic DNA was extracted using a Maxwell Rapid Sample Con-
centrator instrument (Promega). Maryland and West Virginia isolates
were processed at the Department of Plant Science and Landscape
Architecture, University of Maryland. For initial sequencing of the
internal transcribed spacer (ITS) region, genomic DNA was extracted
by harvesting toothpick-tip-sized samples of mycelium grown in
1.5 ml tubes containing potato dextrose broth (PDB; Difco) and
transferring into a 0.2 ml PCR tube containing 10 ml Lyse and Go
PCR Reagent (Pierce Biotechnology). For sequencing of the b-tubulin
gene of a select isolate 104_BFA, genomic DNA was extracted from
homogenized mycelium using an AccuPrep Genomic DNA Extraction
kit (Bioneer) according to the manufacturer’s protocol. A Gentra
Puregene Tissue Extraction kit (Qiagen) was used to extract genomic
DNA from lyophilized mycelium of isolates collected in North Car-
olina. In Massachusetts, genomic DNA was extracted from lyophilized
mycelium using a chloroform/isoamyl alcohol-based protocol and the
Qiagen DNeasy kit (Qiagen).
Sequencing and phylogenetic analyses. All isolates of the novel
species were subjected to sequencing of the ITS region. Also, selected
isolates were sequenced for the protein-coding b-tubulin (Btub) gene
and the maternally inherited cytochrome-c oxidase 1 (cox1) gene.
Sequencing of the ITS region was done using the forward primer ITS6
and reverse primer ITS4 (Cooke et al., 2000). Primers Btub_F1 and
Btub_R1 were used for sequencing the Btub gene (Blair et al., 2008;
Kroon et al., 2004). Sequencing of the cox1 gene was done using the
primers COXF4N and COXR4N (Kroon et al., 2004). PCR conditions
for amplification of ITS, Btub and cox1 were described previously
(Blair et al., 2008; Cooke et al., 2000; Kroon et al., 2004). Sequences in
both directions were visualized with Finch TV version 1.4.0 (Geospiza
Inc.), aligned using
CLUSTAL W and edited manually to correct
obvious errors.
For phylogenetic analyses, sequences of isolates of P. intercalaris were
aligned with those of representative species of Phytophthora using
MAFFT online version 7 (Katoh & Standley, 2013) and the Q-INS-i
algorithm for alignment of ITS sequences (Katoh & Toh, 2008) and
G-INS-i for alignments of Btub and cox1 sequences (Katoh et al.,
2005). Maximum-likelihood (ML) inference was carried out
with
MEGA 6.06 (Tamura et al., 2013) using the Tamura–Nei
model (Tamura & Nei, 1993) with 1000 bootstrap replicates.
Pythium aphanidermatum was used as an outgroup for the ITS and
cox1 phylogenies and Halophytophthora fluviatilis and Phytopythium
vexans (Pythium vexans) were used as the outgroup for the Btub
phylogeny.
Colony morphology. Colony pattern was determined by growing
three representative isolates (45B7
T
, 48A1 and 49A7) on carrot agar
(CA), cV8A, hemp seed agar (HSA), malt extract agar (MEA) and
potato dextrose agar (PDA) in the dark at 20
u
C. Colony patterns
were photographed after 10 and 30 days.
Cardinal temperatures. The same three representative isolates were
examined for their cardinal temperatures on CA and cV8A. Agar
blocks (5 mm in diameter) taken from actively growing areas of
7-day-old cultures were placed at the centre of 10 cm Petri dishes
containing 12 ml freshly made medium. Triplicate dishes per isolate
per temperature were placed in the dark at 5, 10, 15, 20, 25, 30 and
35
u
C. Two perpendicular measurements of each colony were taken
after 8 days. The entire experiment was repeated twice. Means and
standard deviations of radial growth were plotted against temperature
with the gplot package 2.16.0 (Warnes et al., 2015) in R statistical
software 3.1.3 (R Core Team, 2015). Analysis of variance was also
conducted with R to determine whether there were any differences in
radial growth measurements between experiments and/or among
representative isolates.
Morphology. Sporangia were produced by transferring agar plugs
(10
|10 mm) from actively growing cultures on cV8A to Petri dishes
containing non-sterile, 15 % soil water extract (SWE; 15 g sandy loam
soil per litre water) and incubating at room temperature under cool-
white fluorescent light. Caducity of sporangia was determined by
vigorously agitating agar plugs bearing sporangia on a microscope
slide in a drop of water (Gallegly & Hong, 2008).
Production of chlamydospores was determined in various growth
media including CA, cV8A and HSA after 10–90 days at room tem-
perature in the dark.
The mating type of isolates of P. intercalaris was determined in dual
culture with an A
1
or A
2
tester of P. cinnamomi on cV8A. Selfed
gametangia of the three representative isolates were induced in
polycarbonate membrane tests with opposite mating-type testers of P.
cinnamomi, Phytophthora cryptogea, Phytophthora meadii and Phy-
tophthora nicotianae using HSA (Gallegly & Hong, 2008; Ko, 1978).
Eight replicates per isolate per tester were used in each polycarbonate
membrane test. The polycarbonate membrane test with the mating-
type testers of P. cinnamomi and P. cryptogea was repeated twice.
Asexual and sexual bodies were photographed with an Olympus IX71
inverted microscope. Fifty randomly selected mature sporangia and
chlamydospores per representative isolate and all observed game-
tangia were measured using Image-Pro Plus version 5.1.2.53.
RESULTS
Sequence analysis
The three representative isolates of P. intercalaris produced
848 bp ITS sequences, which differed from each other in
one or two positions. These sequences were also 99% iden-
tical to those of other isolates of P. intercalaris in homolo-
gous regions. The ITS sequence of the proposed type isolate
45B7
T
was distinct from that of any known species. BLAST
searches indicated P. gallica (GenBank accession no.
DQ286726) as the clos est species. However, there were
X. Yang and others
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136 nucleotide differences between the ITS sequences of the
type isolates of these two species (87% identity). The ML
inference based on the ITS sequences of 127 species of Phy-
tophthora placed isolates of P. intercalaris in a distinct clus-
ter (Fig. 1), which was close to P. gallica with moderate
bootstrap clustering support (54%).
All isolates of P intercalaris except 59J4 (1 nucleotide differ-
ence from the other isolates) produced an identical cox1
sequence. The closest matches to P. intercalar is in the
867 bp homologous sequence were Phytophthora
macrochlamydospora (56 nucleotide differences; GenBank
accession no. KC733454) and P. gallica (58 nucleotide
differences; KF317112). In the ML phylogeny based on
cox1 sequen ces of 39 species of Phytophthora (Fig. S1, avail-
able in the online Supplementary Material), isolates of
P. intercalaris were grouped in a distinct cluster that was
close to P. gallica but with weak bootstrap support
(v50%). The other three clade-10 species, P. boehmeriae,
P. kernoviae and P. morindae, were scattered in the phylo-
genetic tree and were more closely related to species in the
main Phytophthora clades (Fig. S1).
0.05
Clade 2
Clade 1
Clade 8
Clade 7
Clade 6
Clade 4
Clade 3
Clade 5
99
55
74
98
99
97
94
87
P. quercina 30A5 (KF358229)
P. hydrogena 46A3 (KC249959)
P. hydropathica 5D1 (EU583793)
P. virginiana 46A2 (KC295544)
P. parsiana 47C3 (KC733446)
P. irrigata 23J7 (EU334634)
P. macilentosa 58A7 (KF192700)
P. chrysanthemi 61F1 (KT183038)
P. aquimorbida 40A6 (FJ666127)
P. insolita PMC5-1 (GU111612)
P. polonica 49J9 (KF358225)
P. macrochlamydospora 33E1 (KC733445)
P. richardiae IMI340618 (AF271221)
P. quininea CBS 406.48 (DQ275189)
P. constricta CBS 125801 (HQ013225)
P. captiosa 310C (DQ297402)
P. fallax 310L (DQ297391)
Phytophthora intercalaris 48A1 (KT163270)
Phytophthora intercalaris 49A7 (KT163273)
Phytophthora intercalaris 45B7
T
(KT163268)
P. gallica 50A1 (KF317090)
P. kernoviae P1571 (AY940661)
P. morindae Ph697 (FJ469147)
P. boehmeriae P6950 (FJ801955)
P. boehmeriae 45F9 (KT183036)
P. boehmeriae 22G7 (KT183035)
P. boehmeriae 45F8 (KT317089)
Pythium aphanidermatum P1779 (GU983641)
94
90 57
92
99
99
99
99
99
99
99
98
95
69
54
69
91
97
94
Fig. 1. ML phylogenetic tree based on ITS sequences of 127 taxa of Phytophthora . Alignment was done with MAFFT version
7. The phylogenetic tree was reconstructed and compressed in
MEGA6. Bootstrap support values (percentages of 1000
replicates) are shown on branches (values ,50 % are not shown). The alignment and an uncompressed tree are available
in TreeBASE (S17827).
Phytophthora intercalaris sp. nov., in clade 10
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Isolates of P. intercalaris produced almost identical Btub
sequences, with a maximum of 3 nucleotide differences.
The Btub sequence of isolate 45B7
T
differed in 56 positions
from that of the P. gallica type isolate (GAL1) in the
1136 bp homologous region (TreeBASE submission ID
17827). In the ML phylogenetic tree based on Btub
sequences of 113 taxa of Phytophthora (Fig. S2), isolates
of P. intercalaris formed a distinct cluster that was basal
to the other clade-10 species.
The GenBank/EMBL/DDBJ accession numbers for the ITS,
cox1 and b-tubulin sequences of isolates of P. intercalaris
examined in this study are included in Table S1. All
sequences, alignments and uncompressed phylogenetic
trees derived in this study are available in TreeBASE (S17827).
Colony morphology
The three representative isolates produced very similar
colony patterns after 10 and 30 days. Thus, only colonies
of isolate 45B7
T
are shown in Fig. 2. Isolates of P. interca-
laris produced stellate patterns on CA, cV8A and HSA
(Fig. 2). Abundant aerial mycelium was produced at the
colony centre around the seeded plugs on CA and cV8A
after 10 days of incubation in the dark at 20
u
C (Fig. 2).
While aerial mycelium eventually covered the CA plate, it
remained limited to the colony centre on cV8A after
30 days (Fig. 2). Aerial mycelium was produced at both
the centre and edges of colonies on HSA after 30 days.
The colony patterns on MEA and PDA were sparsely petal-
late and rosaceous, respectively.
Cardinal temperatures for vegetative growth
The three representative isolates had almost identical daily
radial growth rates (P50.95) in two cardinal temperature
tests (P50.94). Thus, data from the three isolates and the
repeated tests were pooled and means were plotted agai nst
temperature (Fig. 3). The optimum growth temperature
was 25–30
u
C in CA and 25
u
C in cV8A. Limited growth
occurred at 5
u
C. No growth was observed at 35
u
C
during cardinal tem perature tests (Fig. 3). The isolates
did not resume growth when returned to room tempera-
ture after exposure to 35
u
C for 8 days.
Taxonomy
Phytophthora intercalaris X. Yang, Y. Balci, N. J.
Brazee, A. L. Loyd & C. X. Hong, sp. nov. (Figs 4
and 5)
Phytophthora intercalaris (in.ter.ca.la9ris. N.L. fem. adj.
intercalaris referring to the abundant intercalary chlamy-
dospores produced in hemp seed agar and carrot agar).
MycoBank: MB812881
Abundant sporangia were produced from fresh mycelial
plugs grown in cV8A after they were submerged in 1.5%
SWE for approximately 20 h. Sporangia were mostly
ovoid (Fig. 4a–d), and occasionally ellipsoid (Fig . 4e),
limoniform (Fig. 4f), pyriform (Fig. 4g) or obpyriform
(Fig. 4h). Sporangia were terminal, sometimes formed on
a simple sympodial sporangiophore (Fig. 4i). Sporangia
ranged from 25.2 to 52.5 mm in length (mean
38.7
+
5.0 mm) and 19.2 to 37.0 mm in breadth (mean
27.0
+
2.9 mm) for the three representative isolates. They
were non-papillate and non-caducous. However, sporangia
were occasionally dislodged, with pedicels ranging from
18.9 to 63.9 mm (mean 37.5
+
13.3 mm) (Fig. 4j–l).
Nested or extended internal proliferations were frequently
Fig. 2. Colony morphology of P. intercalaris isolate 45B7
T
after 10 (top) and 30 (bottom) days at 20 8C on (left to right) CA,
cV8A, HSA, MEA and PDA.
X. Yang and others
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produced (Fig. 4m). Hyphal swellings were rarely produced
in cV8A, while they were abundant in both young and aged
cultures grown in HSA and CA (Fig. 4p). Abundant thin-
walled chlamydospores were produced in HSA and CA
after 10 days (Fig. 4q–u), but were only rarely produced
by aged cultures (w30 days) in cV8A. They were mostly
intercalary (85%) (Fig. 4q, r), occasionally terminal
(10%) (Fig. 4s, t) and rarely lateral (4%) (Fig. 4u). The
chlamydospores ranged from 24.8 to 63.1 mm in diameter
(mean 49.8
+
7.0 mm).
P. intercalaris is heterothallic, and all tested isolates were of
A
1
mating type. Abundant oogonia typical of P. cinnamomi
were produced in dual culture when each isolate of
(i)
(j)
(k)
(l)
(m)(n)
(o)
(p)
(q)
(r)(s)(t)(u)
(a) (b)(c) (d) (e)(f)(g)(h)
Fig. 4. Morphology of asexual structures of P. intercalaris. (a–o) Sporangia produced on cV8A flooded with 15 % SWE,
showing ovoid non-papillate sporangia (a, b), ovoid to globose sporangia (c, d), an ellipsoid sporangium (e), a limoniform
sporangium (f), an obovoid sporangium (g), an obpyriform sporangium (h), sporangia formed on a simple sympodial sporan-
giophore (i), dislodged sporangia with short (j), medium-length (k) and long (l) pedicels, an ovoid sporangium with internal
proliferation (m), a mature sporangium about to release zoospores (n) and a sporangium releasing zoospores (o). (p–u) Struc-
tures produced on HSA, showing hyphal swellings (p), an intercalary thin-walled chlamydospore (q), an off-centre intercalary
chlamydospore (r), a terminal thin-walled chlamydospore on a long pedicel (s), a terminal thin-walled chlamydospore on a
short pedicel (t) and a lateral thin-walled chlamydospore (u). The bar (10 mm) in (u) applies to all subfigures.
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Daily radial growth rate (mm)
5 10152025303540
Temperature (ºC)
CA
cV8A
Fig. 3. Mean daily radial growth of isolates 45B7
T
, 48A1 and
49A7 in CA and cV8A over an 8-day period.
Phytophthora intercalaris sp. nov., in clade 10
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P. intercalaris was paired with an A
2
tester of P. cinnamomi
in HSA for approximately 15 days. In polycarbonate
membrane tests, gametangia were rarely produced. Only
10 gametangia of P. intercalaris were recorded after
30 days when three representative isolates of P. intercalaris
were paired with A
2
tester strains of P. cinnamomi (Fig. 5a–c)
and P. cryptogea (Fig. 5d). No gametangia were produced
when isolates of P. intercalaris were paired with mating-type
testers of P. meadii and P. nicotianae. Oogonia of
P. intercalaris were 40.8
+
6.5 mm in diameter. Oogonial wall
was ornamented with abundant (Fig. 5a, d) to a few (Fig.
5b, c) protuberances. All observed oospores were aborted
(Fig. 5a–d). The mean
+
SD diameter was 35.8
+
5.2 mm.
Antheridia were amphigynous (Fig. 5a–c) and showed mean
dimensions of 20.3 mminlengthand15.5mminwidth.
The type isolate 45B7
T
, recovered from Rapidan Stream,
VA, USA, in July 2007, has been deposited in the American
Type Culture Collection, Manassas, VA, USA, as ATCC
TSD-7
T
(holotype), and the Centraalbureau voor Schim-
melcultures (CBS) Fungal Biodiversity Centre, Utrecht,
The Netherlands, as CBS 140632
T
(ex-type). Other
examined isolates are listed in Table 1.
DISCUSSION
A novel species in Phytophthora clade 10 from streams and
irrigation water in the eastern USA is formally named here
as Phytophthora intercalaris sp. nov. This novel species
has unique morphological, physiological and molecular
features. Furthermore, unli ke other quickly expanding
(a) (b)
(c) (d)
Fig. 5. Morphology of sexual structures of P. intercalaris. (a–c) Selfed gametangia of P. intercalaris induced by an A
2
mating-
type tester of P. cinnamomi, showing an aborted ornamented oogonium with abundant protuberances and a distorted amphi-
gynous antheridum (a), an aborted ornamented oogonium with a cylindrical amphigynous antheridum (b) and an aborted orna-
mented oogonium with anamphigynous antheridum (c). (d) An aborted ornamented oogonium induced by an A
2
mating-type
tester of P. cryptogea. The bar (10 mm) in (d) applies to all subfigures.
X. Yang and others
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phylogenetic groups such as Phytophthora clades 2, 6, 7, 8
and 9, no other novel clade-10 species have been described
in the past 5 years. However, the current assemblage of five
species in clade 10 is di verse in their morphological and
genetic characters. These interesting differences are import-
ant in understanding the phylogeny and evolution of Phy-
tophthora clade 10 and are discussed below.
P. intercalaris can be readily separated from all known
species of Phytophthora . Firstly, all examined isolates of
P. intercalaris produced abundant thin-walled, intercalary
chlamydospores in HSA and CA, while they rarely pro-
duced chlamydospores in cV8A. This is a distinguishing
characteristic that is not seen in other species of Phy-
tophthora. Secondly, among the four heterothallic species
that produce ornamented oogonia, P. intercalaris can be dis-
tinguished from Phytophthora cambivora by producing
abundant hyphal swellings and chlamydospores in HSA
and CA, from P. mississippiae (Yang et al., 2013) by its
lower optimum growth temperature in cV8A (25 vs
30
u
C) and fro m Phytophth ora | stagnum (Yang et al.,
2014c) by not producing abundant hyphal swellings and
chlamydospores in cV8A. Thirdly, P. intercalaris can also
be easily identified by its distinct molecular profile, as illus-
trated through the sequences of the ITS region and cox1 and
Btub genes. These features are important in identifying this
novel species and diagnosing potential diseases in the future
that it may cause. While P. intercalaris has never been found
associated with a diseased plant or disease epidemic, its
formal description is also important to reduce the risk of
misidentifying high-impact species of Phytophthora,
especially those also found in aquatic environments, such
as the quarantine pathogens P. ramorum, Phytophthora
alni and P. kernoviae (also in Phytophthora clade 10).
Notable variations in morphological characters correlate
with distinct ecological roles of Phytophthora clade-10
species. Phytophthora clade 10 is one of the understudied
groups within the genus Phytophthora. Even with the
addition of P. intercalaris, Phytophthora clade 10 now
includes only five formal species. The two other clades
that contain fewer species are clades 3 and 5 (Kroon
et al., 2012; Martin et al., 2012; Weir et al., 2015). Clade
3 contains four homothallic species; all produce semipapil-
late and caducous sporangia (Kroon et al., 2012; Martin
et al., 2012). All four clade-5 species are homothallic and
produce papillate and non-caducous sporangia (Kroon
et al., 2012; Martin et al., 2012; Weir et al., 2015). Previous
studies have suggested that morphological features,
especially papilla tion, are mostly consistent within individ-
ual clades or subclades (Kroon et al., 2012; Martin et al.,
2012; Yang, 2014). However, clade-10 species are diverse
in morphological features. P. boehmeriae, P. kernoviae
and P. morindae (Brasier et al., 2005; Erwin & Ribeiro,
1996; Nelson & Abad, 2010) are homothallic species that
produce papillate and caducous sporangia, which are mor-
phological advantages for airborne pathogens that affect
foliage and fruits. The self-sterile species P. gallica
(Jung & Nechwatal, 2008) and P. intercalaris produce
non-papillate and non-caducous sporangia. Although
their exact ecological roles remain unknown, these two
species have been discovered exc lusively from soil and
water (Catala
`
et al., 2015; Jung & Nechwatal, 2008),
which indicates their unique soilborne and waterborne
lifestyle.
In addition to the variation in morphological features,
clade-10 species are also highly diverse in their genetic
characters. P. intercalaris demonstrates 136 nucleotide
differences from its closest relative, P. gallica, based on
ITS sequences, and their grouping is supported only mod-
erately or weakly by phylogen etic analyses based on ITS
(Fig. 1) and cox1 (Fig. S1) sequences and is not supported
by the Btub phylogeny (Fig. S2). Additionally, P. gallica
and P. boehmeriae differ in their ITS sequences by 121 bp
(Jung & Nechwatal, 2008). Even between the two geneti-
cally most similar species within clade 10, P. kernoviae
and P. morindae, there are at least 38 nucleotide differences
in the ITS sequence. Generally, variation in the ITS
sequence between closely related species in other Phy-
tophthora clades is much smaller than that illustrated
among clade-10 species. There are even fewer than 10
nucleotide differences in ITS sequence among species in
some phylogenetic groups that have shown recent specia-
tion and radiation, such as subclades 1c and 6b (Cooke
et al., 2000; Jung et al., 2011), as well as the high-tempera-
ture-tolerant cluster of clade 9 (Yang et al., 2014a, b; Yang
& Hong, 2013) and the ‘Phytophthora citricola complex’ of
clade 2 (Ho ng et al., 2011; Jung & Burgess, 2009). The gen-
etic variation implicates an earlier divergence within clade-
10 species than in other Phytophthora clades. It also high-
lights the absence of knowledge of the biology and evol-
utionary pro cesses of Phytophthora clade 10.
Recovery of abundant isolates of P. intercalaris from many
geographically distant locations indicates that this novel
species is well adapted and maybe native to the stream eco-
system in the eastern USA. A query about the presence of
this novel species has been sent to researchers who conduct
Phytophthora surveys in Asia, Australia, Europe and the
western USA (T. Burgess, G. Chastagner, M. Elliott,
E. Hansen, J. Hwang, S. Jeffers, T. Jung and J. Parke, per-
sonal comm unications). To date, no isolates of P. interca-
laris have been recovered from these areas, which supports
the hypothesis that it is endemic to the eastern USA.
The exact ecological role of P. intercalaris remains to be
investigated. Although other clade-10 species have been
detected occasionally from aquatic environments, so far it
is the only member of clade 10 that has been recovered
exclusively from water. One isolate of P. inte rcalaris was
recovered from irrigation water in a reclamation reservoir
in North Carolina. However, a single recovery from irrig a-
tion water does not provide sufficient evidence of its adap-
tation to nursery environments, especially as it has only
been detected from streams in other states of the eastern
USA where irrigation water has also been surveyed for
Phytophthora. Therefore, it is likely that this isolate was
Phytophthora intercalaris sp. nov., in clade 10
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introduced to the irrigatio n system through a flooding
event. Another possibility is that this species is actually
adapted to irrigation systems, but is difficult to detect by
baiting methods because of its slow growth rate, coloniza-
tion of baits and low occupancy within the overall commu-
nity compared with other species of Phytophthora that are
well adapted to irrigation water. This is supported by the
fact that the only irrigation water isolate of P. intercalaris
was recovered using the filtration assay and not by baiting
methods. The closest species to P. intercalaris, P. gallica,is
also a slowly growing species compared with P. gonapo-
dyides, P. lacustris (5Phytophthora taxon Salixsoil) and
P. chlamydo spora (Hansen et al., 2015; Jung & Nechwatal,
2008), three species frequently recovered from aquatic
environments. Isolates of P. gallica were only occasionally
recovered, and its presence in streams of nor thern Spain
was only recently confirmed using pyrosequencing
(Catala
`
et al., 2015). Therefore, whether P. intercalar is is
consistently present in irrigation water warrants further
investigation, perhaps using different detection methods.
ACKNOWLEDGEMENTS
This research was supported in part by the Virginia Agricultural
Experiment Station, the Specialty Crop Research Initiative (agreement
no. 2010-51181-21140), the Hatch Program of the United States
Department of Agriculture/National Institute of Food and Agriculture
and UMass Extension and the Massachusetts Agricultural Experiment
Station (MAS00443). Virginia isolates of Phytophthora intercalaris
were recovered by Patricia Richardson from rhododendron leaf
baits received from Todd Edgerton at the Virginia Department of
Forestry in Charlottesville, Virginia.
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