For. Path. 44 (2014) 1–20 doi: 10.1111/efp.

12064
© 2013 Blackwell Verlag GmbH

A taxonomic re-evaluation reveals that Phytophthora cinnamomi and P. cinnamomi

var. parvispora are separate species

By B. Scanu1,5, G. C. Hunter2, B. T. Linaldeddu1, A. Franceschini1, L. Maddau1, T. Jung3,4 and S. Denman2

1
Dipartimento di Agraria, Sezione di Patologia vegetale ed Entomologia (SPaVE), Universit
a degli Studi di Sassari, Viale Italia 39, 07100,
Sassari, Italy; 2Forest Research, Alice Holt Lodge, Farnham, Surrey, UK; 3Phytophthora Research and Consultancy, Brannenburg, Germany;
4
Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology (IBB/CGB), Plant and Animal Genomic Group,
University of Algarve, Faro, Portugal; 5E-mail: bscanu@uniss.it (for correspondence)

Summary
Between 2008 and 2011, severe dieback associated with root and collar rot was reported on Arbutus unedo in several sites in Sardinia,
Italy. Isolations from infected tissues and rhizosphere soil samples consistently yielded a Phytophthora species. It was initially identified as
Phytophthora cinnamomi var. parvispora Kr€ ober and Marwitz by comparing morphological features with the original description and the
internal transcribed spacer (ITS) sequences with those present in GenBank. A multigene phylogeny based on DNA sequence data from two
nuclear (ITS and b-tubulin) and two mitochondrial (cox1 and cox2) gene regions combined with extensive morphological and physiological
properties of these isolates, including the ex-type culture of P. cinnamomi var. parvispora, demonstrates that this taxon is unique and it is
redesignated here as Phytophthora parvispora sp. nov. Although morphologically similar to P. cinnamomi, P. parvispora differs by its smal-
ler-sized sporangia, chlamydospores, oogonia and oospores, higher oospore wall index, single-celled antheridia, higher minimum and maxi-
mum temperatures for growth and faster growth at optimum temperature. In the phylogeny, P. parvispora falls within Phytophthora ITS
clade 7a, grouped in a well-supported clade sister to P. cinnamomi. In pathogenicity tests, P. parvispora and P. cinnamomi were equally
aggressive towards A. unedo seedlings. The possible geographic origin of P. parvispora is also discussed.

1 Introduction
Arbutus unedo L., commonly known as the strawberry tree, is an evergreen shrub in the family Ericaceae that can grow to
a height of about 8 m (Camarda and Valsecchi 2008). Native to parts of Southern Europe, it is a typical component of the
Mediterranean sclerophyllous and laurel-like vegetation mainly in coastal and inland areas with mild climates, where nei-
ther frost nor summer dryness is very intense. In Sardinia (Italy), A. unedo is one of the main components of the Mediterra-
nean ‘maquis’ biome and the understorey in natural stands dominated by oak species. Due to its drought hardiness,
tolerance of calcaric soils and attractive red fruit and pinkish-white flowers, A. unedo is a valuable ornamental plant for
gardening and landscaping that is increasingly cultivated in commercial nurseries (Dalla Serra et al. 1999).
In December 2008, severe dieback of potted A. unedo (Fig. 1a) associated with root and collar rot was found in a forest
nursery in western Sardinia. Two years later, similar symptoms were observed in two locations along the north-east coast
of Sardinia on free-standing A. unedo plants in a private garden (Fig. 1b,c) and in a protected natural area (Fig. 1d,e). Isola-
tions from infected tissues and rhizosphere soil samples of symptomatic plants consistently yielded a Phytophthora species.
Initial characterization of the isolates using morphological and sequence analysis of the internal transcribed spacer (ITS)
region of the rDNA operon indicated that they belonged to a single taxon, Phytophthora cinnamomi var. parvispora Kr€ ober
and Marwitz.
Phytophthora cinnamomi var. parvispora was originally described as a variety of Phytophthora cinnamomi Rands based
on isolates obtained from diseased Beaucarnea sp. grown in a nursery greenhouse in Germany (Kr€ ober and Marwitz 1993).
Despite different morphological and adaptive characteristics, Kr€ ober and Marwitz (1993) considered this taxon a variety of
P. cinnamomi rather than a separate species. Shortly thereafter and against a backdrop of developing molecular technolo-
gies, Erwin and Ribeiro (1996) commented that ‘it would be of interest to determine how P. cinnamomi var. parvispora
compares with isolates of P. cinnamomi’.
With the advent of DNA-based identification methods and the development of phylogenetic inference, the systematics of
the genus Phytophthora has advanced resulting in a natural phylogeny of the genus (Cooke et al. 2000; Martin and Tooley
2003; Kroon et al. 2004; Blair et al. 2008). Combining new molecular methods with extensive morphological, adaptive and
pathogenicity analysis, the taxonomy of several complex morphospecies of Phytophthora could be elucidated, and multiple
novel Phytophthora species have been described (Hansen and Maxwell 1991; Jung et al. 2002, 2011; Brasier et al. 2003;
Hong et al. 2009, 2011; Jung and Burgess 2009; Scott et al. 2009; Bezuidenhout et al. 2010; Rea et al. 2010; Martin et al.
2012; Nechwatal et al. 2012). In several studies, considerable genetic distance between P. cinnamomi var. parvispora and
P. cinnamomi was shown, suggesting long-term separation and little or no gene flow between these two lineages
(Oudemans and Coffey 1991; Blair et al. 2008; Robideau et al. 2011). Consequently, it has been proposed that P. cinnamomi
var. parvispora could be raised to species level (Blair et al. 2008; Gallegy and Hong 2008). Also Bezuidenhout et al. (2010)
demonstrated that P. cinnamomi var. parvispora differed markedly from P. cinnamomi, based on ITS phylogenetic analysis
and pathogenicity experiments.

Received: 7.2.2013; accepted: 21.5.2013; editor: V. Andrea

http://wileyonlinelibrary.com/
2 B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

(a) (b)

(c) (d) (e)

Fig. 1. Symptoms caused by Phytophthora parvispora on Arbutus unedo in Sardinia; (a) Two-year-old seedling in a nursery with shoot
dieback and acute wilting caused by root rot; (b) Dieback and wilting of a five-year-old plant in an ornamental planting due to root rot; (c)
Death of a five-year-old plant in an ornamental planting caused by root and collar rot; (d, e) Dieback and wilting of A. unedo in a natural
woodland due to root rot.

The objective of this study was to clarify the taxonomic status of P. cinnamomi var. parvispora. Based on its unique com-
bination of morphological characters, temperature–growth relations and phylogenetic position in a multilocus analysis,
P. cinnamomi var. parvispora is described as Phytophthora parvispora sp. nov.

2 Material and methods

2.1 Sample collection and Phytophthora isolation

In December 2008, five 2- to 3-year-old seedlings of A. unedo with symptoms of shoot tip dieback and root and collar rot
were harvested from a forest nursery in western Sardinia (Oristano, 39°57′N, 9°13′E; Fig. 1a). In addition, in 2010 and
2011, rhizosphere soil samples and necrotic collar tissues were collected from symptomatic mature A. unedo plants grow-
ing in an ornamental planting (San Teodoro, 40°49′N, 9°40′E; Fig. 1b,c). In addition, rhizosphere soil was sampled from
both young and mature plants growing in a natural woodland area (National Park of La Maddalena Archipelago, 41°12′N,
9°27′E; Fig. 1d,e). Both sites are located on the north-east coast of Sardinia.
Symptomatic plants and rhizosphere soils were transported in cool boxes to the laboratory and processed within
24 hours. Symptomatic shoots and twigs and stem bases with collar necrosis were detached from the rest of the plant.
A representative number of necrotic roots were sampled from each plant. Symptomatic tissue was washed and surface-
sterilized with 70% ethanol for 60 s, followed by a 60-s rinse in sterile distilled water and left to air-dry on a clean paper
towel. Small pieces of plant tissue were aseptically cut from the transition zone between dead and living tissue and plated
onto synthetic Mucor agar (SMA) medium (Elliott et al. 1966) supplemented with 50 ml carrot juice and 0.5 ml of a 4%
MBC (benomyl hydrochloride) solution (Brasier et al. 2005). The pH was adjusted to 6.5 with 1 M NaOH. After autoclaving
at 121°C for 15 min, the agar was cooled to ca 45°C and amended with 0.4 ml of a 2.5% (wt/v) aqueous suspension of
pimaricin and 3 ml of a 1% (w/v) aqueous solution of rifamycin sv sodium salt (all from Sigma-Aldrich, Milan, Italy)
 Phytophthora parvispora sp. nov. 3

(Brasier et al. 2005). For isolations from rhizosphere, soil samples of approximately 100 g of fresh soil were flooded in
10 9 10 9 15 cm plastic trays with 500 ml of distilled water (Themann et al. 2002). Then, young leaflets taken from 2- to
3-month-old Quercus suber and Quercus ilex seedlings were used as baits floated on the surface of the water (Scott et al.
2009). After 3–5 days, leaflets with necrotic lesions were blotted dry on filter paper, plated on SMA and incubated at 25°C
in the dark. The plates were checked daily under the stereomicroscope, and any developing colonies were subcultured on
carrot agar (CA; 12 g agar technical no. 3, Oxoid Ltd, Basingstoke, UK; 2.4 g CaCO3, 160 g carrot and 800 ml distilled
water) (Brasier 1967). Pure cultures were incubated at 25°C and examined within 5 days under a compound microscope
for preliminary species identification.

2.2 Phytophthora isolates

Four of the nine P. cinnamomi var. parvispora isolates from the three sites in Sardinia were included in this study. Five con-
firmed isolates of this taxon were obtained from several sources and used for intraspecific comparisons of morphological and
physiological characters and phylogenetic analysis (Table 1). These isolates included the ex-type culture (CBS 413.96) used in
the original description of P. cinnamomi var. parvispora (Kr€
ober and Marwitz 1993) and three isolates collected during Phytoph-
thora surveys in South Africa, Sicily and Portugal (Bezuidenhout et al. 2010; Pane et al. 2010; Horta et al. 2012). Five isolates of
P. cinnamomi including the ex-type culture (CBS 144.22) (Rands 1922) were included for interspecific comparisons (Table 1).

2.3 Phenotypic characterization

Colony morphology was characterized from 5-day-old cultures incubated at 20°C in the dark on CA, V8 juice agar (V8A;
Erwin and Ribeiro 1996), potato dextrose agar (PDA), malt extract agar (MEA) and corn meal agar (CMA) (all from Oxoid
Ltd, UK). Colony morphologies were visually assessed, grouped and described according to descriptions in the literature
(Erwin and Ribeiro 1996; Brasier et al. 2004; Jung et al. 2011).
For temperature–growth rate studies, agar plugs (5 mm diameter) from actively growing cultures of four isolates of
P. cinnamomi var. parvispora and four isolates of P. cinnamomi (Table 1) were placed in the centre of individual 90-mm
Petri dishes containing CA. Three replicates of each isolate were incubated at 5, 10, 15, 20, 25, 30, 35 and 40°C. After
4 days, plates were removed from the incubator and radial growth scored at the base of the Petri dish along two lines
intersecting at right angles in the centre of the inoculum plug. Means were calculated based on six diameter readings per
isolate per temperature. Optimum, minimum and maximum temperatures were determined by growing the isolates at one-
degree intervals in the temperature ranges 25–30, 5–10 and 35–40°C, respectively (Vettraino et al. 2011).
Eight isolates of P. cinnamomi var. parvispora and five isolates of P. cinnamomi were included in the morphological stud-
ies. Measurements and photographs of morphological structures were made at 2009 and 4009 magnification and recorded
using a digital camera connected to an Olympus BX51 compound microscope (Olympus, Japan) and CellD imaging software
(Olympus, Japan). All measured structures were in a mature stage and selected at random. To induce the production of
sporangia, four mycelial plugs (10 mm diameter) were cut from the edges of actively growing CA colonies, placed in sterile
60-mm Petri dishes and flooded with unsterile pond water. Water cultures were incubated at 20–25°C in natural daylight
until sporangia were observed. Chlamydospores and hyphal swellings were assessed directly on CA plates if present. Spo-
rangial length (l), breadth (b), l/b ratio and characteristic features of 25 sporangia as well as shape and diameters of 25
chlamydospores and hyphal swellings (two perpendicular measurements per structure) were recorded for each isolate.
Sexual compatibility type was determined in paired cultures on 90-mm plates containing 10 ml CA. A preliminary test
was carried out by pairing each isolate of P. cinnamomi var. parvispora and P. cinnamomi with known A1 and A2 tester
strains of P. cinnamomi (P904, P1889) to determine the mating type of each isolate. Subsequently, in a second trial, isolates
of P. cinnamomi var. parvispora were paired with each other to produce gametangia and oospores of P. cinnamomi var. par-
vispora. As a comparison, four A2 isolates of P. cinnamomi were paired with the A1 isolate P904. Both tests plates were
incubated at 20°C in darkness and scored for gametangial formation after 15–20 days. Twenty-five gametangia were chosen
at random, and dimensions and characteristic features of antheridia, oogonia and oospores were measured and recorded at
2009 and 4009 magnification. The oospore aplerotic index and wall index were calculated according to Dick (1990).

2.4 DNA isolation, amplification and sequencing

DNA was extracted from nine and five isolates each of P. cinnamomi var. parvispora and P. cinnamomi, respectively
(Table 1). The isolates were grown for 7 days on cellophane membranes placed over CA in 90-mm plates. Mycelium from
actively growing cultures was scraped from the surface of the cellophane and stored in 1.5-ml sterile microtubes at 80°C
prior to DNA extraction. DNA was extracted from mycelium following the CTAB method of Doyle and Doyle (1987).
Extracted DNA was visualized in 1% agarose gels stained with GelRed (Biotium, Hayward, CA, USA) and viewed under
ultraviolet light in the gel documentation system (Gel Doc 1000, Bio-Rad, Hercules, CA, USA).
Four gene regions were targeted for PCR and DNA sequencing, namely the ITS region of the rDNA operon, b-tubulin and
part of the mitochondrial cox1 and cox2 gene regions. The ITS region was amplified using primers ITS-6 (Cooke et al.
2000) and ITS-4 (White et al. 1990); b-tubulin using primers Btub F1 (Blair et al. 2008) and Btub R1 (Kroon et al. 2004);
cox1 using primers FM 84 and FM 83; and cox2 using primers FM 75 and FM 78 (Martin and Tooley 2003). PCR amplifica-
tions were performed using a GeneAmpâ PCR System 9700 (Applied Biosystems, Life Technologies, Carlsbad, CA, USA), and
the PCR conditions and reaction mixture followed Bezuidenhout et al. (2010). PCR amplification products were purified
 4
Table 1. Identity, host, location, isolation data and GenBank accession numbers for Phytophthora isolates used in this study.

GenBank accession no.2

Collection no.1 Identification Host Location, year Isolated by, reference ITS b-tubulin cox1 cox2

CBS 132771, P. parvispora Arbutus unedo, rotted roots Forest nursery, Sardinia, Italy, 2008 B. Scanu GU460376 KC609401 KC609412 KC559760
PH0283,4,5
CBS 132772, P. parvispora A. unedo, collar rot Ornamental planting, Sardinia, Italy, 2011 B. Scanu KC478667 KC609402 KC609413 KC559763
PH0723,4,5,6
PH073 P. parvispora A. unedo, rotted roots Ornamental planting, Sardinia, Italy, 2011 B. Scanu n.a. n.a. n.a. n.a.
PH080 P. parvispora A. unedo, rhizosphere Forest, Sardinia, Italy, 2011 B. Scanu KC478666 KC609403 KC609414 KC559764
PH0884 P. parvispora Pinus pinea, rhizosphere Forest nursery, Algarve, Portugal, 2011 T. Jung & M. Horta-Jung n.a. n.a. n.a. n.a.
IMI39761863,4 P. parvispora Mandevilla sp., rotted roots Nursery, Sicily, Italy, 2004 Pane et al. 2010 KC478669 KC609404 KC609415 KC559761
STEU 62655 P. parvispora Agathosma betulina, Plantation, Western Cape Province, Bezuidenhout et al. 2010 KC478671 KC609405 KC609416 KC559762
rhizosphere South Africa, 2005
CBS 413.96, P. parvispora Beaucarnea sp., stem base Greenhouse, Germany, 1990 Kr€
ober and Marwitz 1993 KC478668 KC609406 KC609417 KC559758
P84943,4,5
CBS 411.96, P. parvispora Beaucarnea sp., stem base Greenhouse, Germany, 1990 Kr€
ober and Marwitz 1993 KC478672 KC609407 KC609418 KC559759
P84954,5
P2404 P. parvispora Citrus sp. Taiwan, 1985 Ann and Ko 1985 FJ801815 n.a. n.a. n.a.
1335 P. parvispora Vigna unguiculata Planting, Itabuna, Brazil, 2008 Dos Santos 2010 JF917304 n.a. n.a. n.a.
1337 P. parvispora Vigna unguiculata Planting, Itabuna, Brazil, 2008 Dos Santos 2010 JF917305 n.a. n.a. n.a.
CBS 144.223,6 P. cinnamomi Cinnamomum sp., stripe Plantation, Sumatra, 1922 Rands 1922 KC478663 KC609408 KC609419 KC559768
canker
PH060 P. cinnamomi Quercus ilex, rhizosphere Forest, Sardinia, Italy, 2010 B. Scanu n.a. n.a. n.a. n.a.
PH0673,4 P. cinnamomi Quercus suber, stem base Garden, Sardinia, Italy, 2011 B. Scanu KC478664 KC609409 KC609420 KC559767
P9043,4,7 P. cinnamomi n.a. Australia n.a. KC478662 KC609410 KC609421 KC559765
P18893,4,7 P. cinnamomi Fagus sylvatica Devon, UK, 2005 C.M. Brasier KC478665 KC609411 KC609422 KC559766
P6305 P. cinnamomi Persea americana Sulawesi, Indonesia, 1989 M.D.Coffey HQ261525 EU079894 HQ261272 GU221969
P3232 P. cinnamomi Rhododendron sp. China, 1986 P. Tsao HQ261526 EU079797 HQ261273 GU221968
STE-U 6255 P. cinnamomi Agathosma crenulata Nursery, Western Cape Province, Bezuidenhout et al. 2010 GU191204 GU191304 GU191273 n.a.
South Africa, 2005
STE-U 6258 P. cinnamomi A. betulina Plantation, Western Cape Province, Bezuidenhout et al. 2010 GU191208 GU191306 GU191289 n.a.
South Africa, 2005
STE-U 6259 P. cinnamomi A. betulina Plantation, Western Cape Province, Bezuidenhout et al. 2010 GU191209 GU191303 GU191283 n.a.
South Africa, 2005
P0592 P. cambivora Abies procera Oregon, USA n.a. HQ261516 EU080551 HQ261263 JF771263
P3105 P. cajani Cajanus cajani India Kannaiyan et al. 1980 HQ261515 EU080101 HQ261262 GU221954
P10331 P. drechsleri n.a. USA, 2003 n.a PD_00086_ITS8 EU079507 HQ261300 PD_00086_cox28
B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

P10324 P. europaea Quercus sp., rhizosphere France Jung et al. 2002 HQ261556 EU079482 HQ261303 GU222005
P3820 P. fragariae Fragaria 9 ananassa California, USA Converse 1970 HQ261563 EU079835 HQ261310 GU222013
P3609 P. melonis Cucumis melo Japan n a. HQ261617 EU080472 HQ261364 GU222072
P6197 P. pistaciae Pistacia vera Kerman, Iran Mirabolfathy et al. 2001 HQ261644 EU080326 HQ261391 GU222109
P1475 P. sinensis Cucumis sativus n.a. n.a. HQ261671 EU079750 HQ261418 GU222135
P3114 P. sojae Glycine max Wisconsin, USA n.a. HQ261677 EU079790 HQ261424 GU222142
P10413 P. uliginosa Quercus robur, rhizosphere Poland Jung et al. 2002 HQ261721 EU080012 HQ261468 GU222156
P3019 P. vignae Vigna unguiculata Australia n.a. HQ261724 EU079783 HQ261471 GU222157

n.a., not available.

1
Abbreviations of isolates and culture collections: CBS = CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; IMI = CABI Bioscience (Imperial Mycological Institute), UK;
PH = culture collection of the University of Sassari; P = Worldwide Phytophthora Collection, University of California, Riverside, USA; STEU = University of Stellenbosch, South Africa.
2
Sequence numbers in italics were retrieved from GenBank. All others were determined in this study.
3
Isolate used in the temperature–growth test.
4
Isolate used in the fungicide sensitivity assay.
5
Isolate included in the pathogenicity test.
6
Ex-type isolate.
7
Forest Research Phytophthora culture collection, Farnham, UK.
8
Phytophthora database (www.phytophthoradb.org)
 Phytophthora parvispora sp. nov. 5
TM
using the Zymo-Spin IC_XL Column extraction kit (ZymoResearch, Irvine, CA, USA) according to the manufacturer’s
instructions. For DNA sequencing, purified PCR amplicons (5–20 ng ll 1) and sequencing primer (3.5 pmol ll 1) were sent
to GenePool (DNA sequencing service, University of Edinburgh, UK) for the sequence reaction using the BigDye Terminator
Cycle Sequencing ready reaction kit version 3.1. Purified reactions were run on an ABI3730 capillary sequencer (Applied
Biosystems, Life Technologies). DNA sequence chromatograms were viewed and edited using BioEdit version 5.0.6 software
(Hall 2001).

2.5 Phylogenetic analysis

DNA sequences representing species from Phytophthora subclades 7a and 7b (Blair et al. 2008) were downloaded from
GenBank and the Phytophthora Database (www.phytophthoradb.org) for the four genetic loci sequenced during this study
(ITS, b-tubulin, cox1 and cox2) and combined with the sequences derived during this study (Table 1). Sequences were
aligned for the individual data sets using MAFFT version 6 (http://mafft.cbrc.jp/alignment/software/) (Katoh and Toh
2008) employing the FFT-NS-I and 200PAM/K = 2 alignment strategy. Alignments were visualized in MacClade version
4.08 (Maddison and Maddison 2003) and manually adjusted where necessary. DNA sequence alignments of the four gene
regions were uploaded to CIPRES (Cyberinfrastructure for Phylogenetic Research) Science Gateway version 3.1 (http://
www.phylo.org/index.php/portal) (Miller et al. 2010) in Phylip format. Individual sequence data sets were subject to maxi-
mum-likelihood (ML) phylogeny reconstruction using RAxML (Stamatakis et al. 2005, 2008) as implemented through the
RAxML-HPC2 run on XSEDE. Maximum-likelihood runs employed the GTRMIX model using the following partitions: ITS and
the first, second and third codon positions of b-tubulin, cox1 and cox2. Node support was evaluated through 1000 boot-
strap pseudoreplicates. Maximum-likelihood results were visualized in MEGA (Molecular Evolutionary Genetics Analysis)
version 5.05 (Tamura et al. 2011), and all phylograms were rooted to Phytophthora drechsleri (P10331). The congruence
between the four data sets was tested using a 70% reciprocal bootstrap criterion (Mason-Gamer and Kellogg 1996). Phylo-
gram topology was evaluated for each genetic locus individually following which the DNA sequence data from the two
nuclear loci (ITS and b-tubulin) and the two mitochondrial loci (cox1 and cox2) were combined. Final ML analyses were
determined for the two combined data sets as described for the individual data sets. New sequences were deposited in
GenBank (Table 1) and the DNA sequence alignment files and phylogenetic trees in TreeBASE (http://www.treebase.org/).

2.6 Pathogenicity test

Pathogenicity of five isolates of P. cinnamomi var. parvispora (Table 1) to seedlings of A. unedo was tested using the soil
infestation method of Jung et al. (1996) with minor modifications. As the large host range of P. cinnamomi also includes
A. unedo, two isolates of P. cinnamomi were included as positive controls (Table 1). Two-month-old A. unedo seedlings
(purchased from Ente Foreste della Sardegna, Italy) were grown in 500-ml pots for two years in a greenhouse and then
used for the pathogenicity experiment. Inoculum was prepared by growing individual isolates in 500-ml Erlenmeyer flasks
containing a mixture of 250 ml vermiculite and 150 ml of Lolium italicum seeds thoroughly moistened with 100 ml of fil-
tered carrot juice (200 ml l 1 carrot, 3 g l 1 CaCO3 and 800 ml l 1 distilled water), which was autoclaved for 20 min at
121°C before use. After cooling to room temperature, the mixture was inoculated with eight 5-mm-diameter plugs cut from
the margins of actively growing CA cultures. Following 4-week incubation at 25°C, the inoculum was rinsed in running dis-
tilled water to remove excess nutrients (Matheron and Mircetich 1985), and 20 ml of washed inoculum per isolate was col-
lected and added to the soil of each pot. Each control plant received 20 ml of the uninoculated mixture. To stimulate the
production of sporangia, pots were flooded immediately after inoculation for 48 h and then at three-week intervals by
immersing pots in 10-litre buckets so that the water level was ca. 1 cm above the soil surface. To confirm viability of the
inoculum and production of zoospores, Q. suber leaves were used as baits during all flooding treatments. Between the
flooding treatments, pots were kept at temperatures ranging from 20 to 28°C and watered as necessary. During the experi-
ment, seedlings were visually assessed for the presence of symptoms on a weekly basis, and symptoms and the number of
dead plants were noted at the end of the experiment. Each plant was removed from the pot after 4 months and the root
system gently washed under tap water. Single roots were cut off at the collar, and after scanning, root length was measured
using the APS Assess 2.0 software (The American Phytopathological Society, USA). Data were analysed as described by
J€
onsson et al. (2003). The soil was baited following the methods described above to determine whether the pathogen was
still viable in the soil. Re-isolations were also made from necrotic roots and collar tissues using SMA selective medium.

2.7 Fungicide sensitivity assays

Sensitivity to mefenoxam (Ridomil Gold SL, 43.88% wt/vol; Syngenta, Monthey, Switzerland) was determined for six iso-
lates of P. cinnamomi var. parvispora and three isolates of P. cinnamomi (Table 1) using the method of Perez-Sierra et al.
(2010) with slight modifications. Mycelial discs (6 mm diameter) were cut from the margin of actively growing CA cultures
and transferred to 90-mm Petri dishes containing CA amended with the following concentrations of mefenoxam: 0, 0.01,
0.05, 0.1, 0.5, 1, 5, 10 and 100 lg ml 1. Three replicates for each isolate–fungicide concentration combination were used,
and the experiment was repeated twice. After incubation at 20°C in darkness for 3 days, radial growth was measured tak-
ing two measurements per plate. Measurements were averaged, and growth was expressed as percentage of the growth
rate in the unamended control. According to Parra and Ristaino (2001) and Taylor et al. (2002), an isolate was considered
sensitive to mefenoxam, if colony growth at 5 lg ml 1 of mefenoxam was <40% compared with the control. Isolates
6 B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

showing, at 5 lg ml of mefenoxam, >40% of the growth on control plates, but <40% on media amended with 100 lg
1

ml 1 of mefenoxam, were considered to have an intermediate sensitivity. If colony growth on media with 100 lg ml 1 was
>40% compared with control plates, isolates were considered resistant. Probit analysis was used to calculate the effective
concentrations that inhibited mycelial growth by 50% (EC50) and by 90% (EC90).

2.8 Statistical analysis

Morphometric and pathogenicity data were analysed by a one-way analysis of variance (ANOVA) using Tukey’s HSD test
(honestly significant difference) as post-test (XLSTAT 2008 software; Addinsoft, Paris, France). Differences with p < 0.05
were considered significant.

3 Results

3.1 Taxonomy
Phytophthora parvispora Scanu and Denman, sp. nov. (Fig. 2).
MycoBank: MB 803239;
Etymology: Name refers to the small-sized reproductive structures.
Phytophthora cinnamomi similis, sed sporangia (36.9 9 22.8 lm), chlamydosporae (21.5 lm), oogonia (26.7–37 lm),
oosporae (20.8–31.1 lm) et inflationes hypharum (18–30 lm) in medio minores, chlamydosporae rarae aut non observa-
tae, antheridia unicellularia, temperaturae minimae (>10°C) et maximae (37°C) altiores et incrementum radiatum ad
optime (27°C) altiores (14.4 mm day 1). Regiones ‘rDNA ITS’, ‘b-tubulin’, ‘cox1’ et ‘cox2’ cum unica sequentia (GenBank
KC478667, KC609402, KC609413, KC559763).
Phytophthora parvispora is heterothallic. Oogonia golden brown, smooth-walled and globose, mean diameter in different
pairings 30.9–32.9 lm. Oospores aplerotic to slightly aplerotic, averaging in different pairings 24.7–25.7 lm, thick-walled
with a mean wall diameter of 1.9  0.2 lm. Antheridia amphigynous, one-celled and globose. Sporangia abundant in liquid
cultures, persistent, terminal and occasionally intercalary, ovoid, ellipsoid or obpyriform, non-papillate with a flat apex,
mean dimensions 36.9  5.8 9 22.8  3.2 lm and length/breadth ratio 1.6. Sporangial proliferation internal, both in a
nested and extended way, and external. Hyphal swellings irregular or globose, often forming dense aggregations. Chlamy-
dospores not abundant, mostly lateral and occasionally terminal, mean diameter 21.5  2.8 lm. Colony morphology on CA
and V8A chrysanthemum to stellate with limited aerial mycelium. Minimum, optimum and maximum temperatures on CA
>12, 27 and 37°C, respectively; radial growth rate on CA at optimum 14.4 mm day 1.

3.1.1 Material examined

Italy, Sardinia. Isolated from collar rot of a declining planted Arbutus unedo. Collected: B. Scanu, 2011; CBS H-21132
(holotype, dried culture on CA, Herbarium CBS-KNAW Fungal Biodiversity Centre), CBS 132772 = PH072 (culture ex-type).

3.1.2 Additional specimens

Italy, Sardinia. Isolated from rotted root of a declining nursery plant of A. unedo. Collected: B. Scanu, 2008; CBS
132771 = PH028. Italy, Sardinia. Isolated from rhizosphere soil of a declining A. unedo in a forest. Collected: B. Scanu,
2011; PH080. Italy, Sicily. Isolated from rotted root of a nursery plant of Mandevilla sp. Collected: A. Pane, 2004;
IMI3976186. Portugal, Algarve. Isolated from rhizosphere soil of a chlorotic declining nursery plant of Pinus pinea.
Collected: T. Jung and M. Horta-Jung, 2011; PH088. Germany. Isolated from stem base of Beaucarnea sp. in a greenhouse.
Collected: H. Kr€ober and R. Marwitz, 1990; CBS 413.96 = P8494. Germany. Isolated from stem base of Beaucamea sp. in a
greenhouse. Collected: H. Kr€ober and R. Marwitz, 1990; CBS 411.96 = P8495. South Africa, Western Cape Province. Isolated
from rhizosphere soil of Agathosma betulina in a plantation. Collected: C.M. Bezuidenhout, 2005; STEU 6265.

3.1.3 Distribution
Germany, Italy, Portugal, South Africa, Taiwan, Australia and Brazil.

3.2 Morphological and phenotypic characterization

3.2.1 Colony morphology

After 5 days growth at 20°C on five different culture media, P. parvispora showed variation of colony patterns across media
types and also amongst isolates on the same medium (Fig. 3). On the nutrient-rich media, V8A and CA colonies generally
developed limited aerial mycelium. On V8A, isolates CBS 413.96 and IMI3976186 had a rosaceous colony morphology,
whereas CBS 411.96 and CBS 132772 formed a loose chrysanthemum pattern. Isolate CBS 132771 had a striate pattern
with coarse fluffy mycelium in the centre, but loose chrysanthemum tuffs near the edge of the colony. Isolate STEU 6265
formed a distinctive colony pattern on V8A and developed only sparse aerial mycelium in all media. On MEA and PDA,
 Phytophthora parvispora sp. nov. 7

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j) (k) (l)

(m) (n) (o) (p) (q)

(r) (s) (t) (u)

Fig. 2. Morphological structures of Phytophthora parvispora formed on V8 agar; (a–l) Sporangia produced in non-sterile pond water; (a–e)
Mature non-papillate sporangia; (a, b) Ellipsoid; (c) Obpyriform; (d) Ovoid, with swollen apex before release of the already differentiated
zoospores; (e) Ellipsoid mature sporangium and empty internally proliferating sporangium; (f) Internal nested proliferation; (g) Internal
nested and extended proliferation; (h) Internal extended proliferation; (i) External proliferation close to sporangial base; (j) Release of
individual zoospores; (k) Differentiation of zoospores outside the sporangium; (l) Released zoospores temporarily sticking together; (m, n)
Clusters of subglobose to globose hyphal swellings forming dense aggregations; (o) Appressoria-like hyphal swellings; (p, q) Laterally
borne globose chlamydospores; (r–u) Oogonia formed in pairings of A1 and A2 isolates; (r) Mature golden brown oogonia with plerotic
(left) and slightly aplerotic (right) oospores and single-celled amphigynous antheridia with finger-like projections; (s, t) Mature golden
brown oogonia with slightly aplerotic oospores and single-celled amphigynous antheridia; (u) Aborted oogonia. Scale bars = 25 lm.

most isolates produced chrysanthemum patterns with limited aerial mycelium, often with irregular margins. All isolates
formed submerged colonies with faintly stellate patterns on CMA. Colony morphology of different P. cinnamomi isolates
showed no variation on the same culture media (Fig. 3). Colonies of P. cinnamomi were faintly stellate to chrysanthemum
on CA and faintly stellate on V8A with sparse aerial mycelium and petaloid to rosaceous patterns with wooly mycelium on
MEA and PDA. On CMA, colonies were faintly stellate and submerged. Both P. parvispora and P. cinnamomi showed slow
growth on MEA and PDA (Fig. 3). Phytophthora cinnamomi grew faster than P. parvispora on PDA and CMA (Fig. 3).

3.2.2 Cardinal temperatures and growth rates

Cardinal temperatures for growth on CA for P. parvispora and P. cinnamomi isolates are summarized in Table 2, and the
temperature–growth rate curves of both species are shown in Fig. 4. There was little variation in the temperature–growth
8 B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

16
P. parvispora
14 P. cinnamomi * *
12

Growth rate mm day–1

NS

10

6
*
ND
4
ND
2

0
10 15 20 25 30 35
Temperature °C

Fig. 3. Radial growth rates of Phytophthora parvispora (continuous line) and Phytophthora cinnamomi (dashed line) on carrot agar at six
different temperature. The data are plotted as average  SD. Statistical analysis was performed using the Student’s t-test. (*) p < 0.01, NS,
not significant; ND, not determined.

rate amongst the six isolates of P. parvispora tested, and with the exception of STEU 6265, all P. parvispora isolates had
identical cardinal temperatures, with a minimum temperature above 10°C, maximum temperature at 37°C and optimum
growth at 27°C (Table 2; Fig. 4). Isolate STEU 6265 showed one-degree lower maximum (36°C) and optimum temperature
(26°C) of growth compared with other P. parvispora isolates. All four P. cinnamomi isolates showed similar growth trends
but had different cardinal temperatures and slower growth rates above 17°C compared with P. parvispora. With 27–28°C,
the optimum temperature was similar to P. parvispora, while both maximum (32–33°C) and minimum (below 10°C) tem-
peratures were lower than in P. parvispora (Table 2; Fig. 4). Phytophthora parvispora showed radial growth rates of 12.8 to
14.4 mm day 1 at optimum temperature (27°C) and 9.3 to 10 mm day 1 at 20°C with isolate STEU 6265 being the excep-
tion showing much slower growth at optimum temperature and at 20°C (Table 2). Radial growth rates of P. cinnamomi
ranged from 10.5 to 11.9 mm day 1 at optimum temperature (27°C) and from 7.9 to 9.2 mm day 1 at 20°C.

3.2.3 Sporangia
After 24 h, in liquid culture, sporangia were abundantly produced by almost all P. parvispora isolates (Fig. 2a–l). In con-
trast, P. cinnamomi isolates formed sporangia less abundantly and required 48 h to form mature sporangia (Fig. 5a–d).
Both species formed non-papillate, persistent sporangia, which were mostly ovoid, obpyriform, ellipsoid- or tuber-shaped
(Figs 2a–f and 5a–d). In P. parvispora, the sporangial apex was always flat (Fig. 2a–e, h–i), whereas in some isolates of
P. cinnamomi (PH067, PH060, P1889), sporangia appeared almost semipapillate. In addition, in one of the P. cinnamomi
isolates (PH067), some sporangia had a long pedicel. Sporangia of P. parvispora were borne terminal or occasionally inter-
calary, sometimes with a laterally displaced attachment of the sporangiophore. Widening of the sporangiophores towards
the base of the sporangium was observed infrequently in P. parvispora. Internal nested and extended proliferation of spo-
rangia occurred in both species after zoospore release (Figs 2f–h and 5c,d). In P. parvispora, chains of proliferating sporan-
gia along the same sporangiophore were frequently observed (Fig. 2g). Nested proliferation was more frequent in
P. parvispora than in P. cinnamomi. External proliferation was also common in both species, with the hypha originating
close to the sporangial base (Fig. 2i). In both species, zoospores were usually released individually (Fig. 2j), but in P. parvis-
pora, they could also be discharged as a group (Fig. 2k) or string held together outside the sporangium (Fig. 2l). In both
species, zoospore cysts often germinated indirectly by releasing a secondary zoospore (diplanetism). Sporangial dimensions
of eight isolates of P. parvispora and five isolates of P. cinnamomi are given in Table 2. Sporangial length 9 breadth dimen-
sions of P. parvispora were 35.6  0.4 9 23.7  0.3 lm (mean  SE), while in P. cinnamomi, sporangia were significantly
larger averaging 61.1  1.2 9 38.4  0.6 lm. There were no significant differences of sporangial dimensions between
different P. parvispora isolates (Table 2).

3.2.4 Hyphal swellings

Diameters of primary hyphae ranged from 2.1 to 5.5 lm in P. parvispora and from 2.6 to 6.3 lm in P. cinnamomi (Figs 2m,
n and 5e,f). In both species, hyphal swellings often in clusters were abundantly produced in both solid agar and liquid cul-
ture. Phytophthora cinnamomi almost exclusively produced coralloid irregular swellings (Fig. 5e,f), while swellings of
P. parvispora were predominantly globose (Fig. 2m,n) with a diameter of 18–30 lm or slightly irregular resembling
appressoria (Fig. 2o) produced by some Phytophthora species, for example P. plurivora (Jung and Burgess 2009), and by
species belonging to the genus Pythium. Aggregations of hyphae and swellings were frequently observed in both P. parvis-
pora (Fig. 2m) and P. cinnamomi (Fig. 5e).
 Table 2. Dimensions of sporangia and chlamydospores (lm) and temperature–growth relationships of Phytophthora parvispora and Phytophthora cinnamomi.

Sporangia Chlamydospores

Length Breadth Length/Breadth Diameter Cardinal temperatures (°C)2 Growth rate Growth rate
at optimum at 20 °C
Mean1 Range Mean1 Range Mean1 Range Mean1 Range Min Max Opt (mm day 1) (mm day 1)

P. parvispora
P8494 39.9 a 29.0–49.6 26.4 cd 19.8–32.0 1.5 abcd 1.3–1.9 20.5 a 15.9–29.9 >10 37 27 14.1 8.9
P8495 37.0 a 27.0–49.6 25.5 bc 19.9–36.4 1.5 bcd 1.2–2.1 20.3 a 13.0–24.7 >10 37 27 14.4 9.8
PH028 33.3 a 16.1–38.8 23.1 abc 19.7–26.6 1.4 bcd 0.8–2.0 19.3 a 14.2–26.7 >10 37 27 12.8 9.4
PH072 36.9 a 20.5–52.0 22.8 ab 17.3–30.1 1.6 ab 0.7–2.0 21.5 a 15.9–27.6 >10 37 27 14.4 10.0
PH080 34.1 a 25.0–42.5 24.6 bc 19.0–36.0 1.4 d 1.1–1.7 21.1 a 13.0–27.6 >10 37 27 14.1 9.8
PH088 33.1 a 18.6–40.2 22.9 ab 17.9–30.3 1.5 bcd 0.9–2.0 20.3 a 15.0–28.1 >10 37 27 13.9 9.7
IMI397618 33.4 a 26.8–40.3 23.3 abc 19.5–25.8 1.4 cd 1.2–1.7 20.5 a 15.9–29.7 >10 37 27 13.9 9.3
STEU 6265 34.3 a 23.5–44.3 20.3 a 14.7–24.3 1.7 a 1.1–2.4 Not observed >10 36 26 8.6 6.5
Over all isolates 35.6 16.1–52 23.7 14.7–36.4 1.5 0.7–2.4 20.4 13–32.9 >10 37 27 12.5 8.4
P. cinnamomi
CBS 144.22 62.8 c 48.4–81.0 39.9 e 34.0–43.1 1.6 abc 1.3–2.0 48.6 c 35.5–82.7 <10 33 27 10.6 8.1
Phytophthora parvispora sp. nov.

PH060 48.1 b 34.8–71.2 29.6 d 22.1–42.3 1.6 ab 1.3–2.1 51.5 c 42.6–83.5 <10 33 28 10.8 8.9
PH067 77.9 d 62.0–98.4 45.7 f 39.0–54.0 1.7 a 1.4–2.1 53.9 c 42.6–82.7 <10 32 27 11.9 9.2
P904 59.0 c 45.8–74.5 39.3 e 33.7–45.6 1.5 bcd 1.2–1.8 41.3 b 32.0–55.1 <10 33 28 10.9 8.3
P1889 57.5 c 49.8–77.3 38.6 e 34.2–45.0 1.5 bcd 1.3–1.8 40.3 b 31.0–55.1 <10 32 28 10.5 7.9
Over all isolates 61.1 34.8–98.4 38.4 22.1–54 1.6 1.2–2.1 47.1 31–83.5 <10 32.5 27.5 10.2 8.4
1
Different letters indicate significant differences according to Tukey’s (HSD) test (p < 0.05).
2
Min, minimum for growth; Max, maximum for growth; Opt, optimum for growth.
9
10 B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

Fig. 4. Colony morphology of Phytophthora parvispora isolates CBS 132772 (ex-type), CBS 132771, CBS 413.96 and STEU 6265 and
Phytophthora cinnamomi isolates CBS 14422 (ex-type) and P904 (from top to bottom) after 5 day growth at 20°C on carrot agar, V8 agar,
malt extract agar, potato dextrose agar and corn meal agar (from left to right).

Chlamydospores (Figs 2p,q and 5g,h) were infrequently produced by all isolates of P. parvispora except by STEU 6265,
which did not form chlamydospores. Phytophthora parvispora chlamydospores were mostly solitary in lateral and less fre-
quently in terminal positions (Fig. 2p,q). In contrast, P. cinnamomi chlamydospores were produced abundantly, often in
grape-like clusters (Fig. 5g,h). With diameters of 13–29.9 lm, chlamydospores of P. parvispora chlamydospores were signif-
icantly smaller than those produced by P. cinnamomi (31–83.5 lm) with no overlap between both species (Table 2).

3.2.5 Oogonia, oospores and antheridia

All P. parvispora isolates were heterothallic and produced gametangia readily when A1 and A2 isolates were paired against
each other or against tester strains of P. cinnamomi with opposite mating type (Figs 2r–u and 5i–m). As expected, self-
crossed isolates and A1 9 A1 as well as A2 9 A2 combinations did not result in gametangia production. Both mating types
of P. parvispora occurred in Sardinia and Germany, while in South Africa and Portugal, only the A1 and A2, respectively,
were found (Table 3). Oogonium production was observed 20–30 days after onset of the mating test. The abundance of
oospore production varied between the different A1 9 A2 combinations. Almost all A1 9 A2 pairings of P. parvispora iso-
lates mated successfully, except of the pairing STEU 6265 9 IMI397618, where no oogonia were observed (Table 3).
However, both isolates behaved as self-sterile silent A1 (STEU 6265) or A2 (IMI397618) mating types capable of
stimulating the formation of oogonia when paired with other isolates of opposite mating types from both P. parvispora and
P. cinnamomi (Table 3). Interestingly, two A2 isolates of P. parvispora (CBS 132771 and CBS 411.96) formed a low number
 Phytophthora parvispora sp. nov. 11

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l) (m)

Fig. 5. Morphological structures of Phytophthora cinnamomi formed on V8 agar; (a–d). Sporangia produced in non-sterile pond water; a
mature, ovoid non-papillate sporangium; (b) Mature, ellipsoid non-papillate to semipapillate sporangium; (c, d) Internal nested
proliferation; (e) Cluster of irregular coralloid hyphal swellings, some forming a dense aggregation; (f) Irregular coralloid hyphal swellings;
(g) Botryose cluster of chlamydospores; (h) Chlamydospore; (i–m) Oogonia with typical tapering bases, slightly aplerotic to nearly plerotic
oospores and amphigynous, characteristically two-celled antheridia. Scale bars = 25 lm.

Table 3. Abundance of oogonium production and dimensions of oogonia; oospores; and antheridia (lm) of Phytophthora parvispora and
Phytophthora cinnamomi.

Oogonia Oospores Antheridia

Oogonial Wall Length/

production1 Mean2 Range Mean2 Range thickness2 Length2 Breadth2 Breadth2

P. parvispora
P8495 (A2) 9 P8494 (A1) +++ 30.9 ab 28.4–33.2 25.6 a 22.4–28.8 2.0 a 14.3 a 14.1 a 0.9 a
PH028 (A2) 9 PH072 (A1) +++ 32.9 b 29.8–37.0 25.7 a 20.8–31.1 1.9 a 15.7 ab 16.7 b 0.9 ab
P8495 (A2) 9 PH072 (A1) +++ 30.9 ab 26.7–35.3 24.7 a 21.1–30.0 1.9 a 17.5 b 16.1 ab 1.1 bc
PH028 (A2) 9 P8494 (A1) ++ 31.0 ab 27.3–34.3 24.9 a 21.4–28.0 1.9 a 15.3 ab 15.6 ab 1.0 abc
PH028 (A2) 9 STEU 6265 (A1) + 31.2 ab 27.4–34.8 24.5 a 22.3–30.0 1.9 a 17.0 b 15.8 ab 1.1 bc
PH033 (A2) 9 PH072 (A1) + 31.8 ab 29.3–34.0 25.6 a 23.0–28.0 2.0 a 17.2 b 16.5 ab 1.0 abc
PH033 (A2) 9 P8494 (A1) + 30.3 ab 27.3–33.4 24.8 a 21.7–28.0 1.9 a 16.3 ab 15.5 ab 1.1 abc
P8495 (A2) 9 STEU 6265 (A1) + 30.4 a 26.7–35.3 24.8 a 21.7–28.9 1.9 a 16.8 b 15.5 ab 1.1 c
PH033 (A2) 9 STEU 6265 (A1) – – – – – – – –
Over all isolates 31.1 26.7–37 25 20.8–31.1 1.9 16.3 15.7 1
P. cinnamomi
CBS 144.22 (A2) 9 P904 (A1) +++ 43.5 cd 39.2–49.7 37.8 b 33.3–42.5 1.9 a 23.6 c 16.3 ab 1.4 d
PH060 (A2) 9 P904 (A1) +++ 41.5 c 38.0–44.8 36.5 b 31.7–39.6 2.0 a 24.9 cd 16.6 ab 1.5 de
PH067 (A2) 9 P904 (A1) +++ 41.5 c 35.0–47.7 36.7 b 33.0–41.4 2.0 a 26.7 d 16.3 ab 1.6 ef
P1889 (A2) 9 P904 (A1) +++ 44.5 d 37.7–48.9 38.4 b 33.0–43.0 2.1 a 27.3 d 16.5 ab 1.7 f
Over all isolates 42.8 35–49.7 37.4 31.7–43 2 25.6 16.4 1.6
1
Abundance of oogonium production: –, none; +, occasional; ++, frequent; +++, abundant.
2
Different letters indicate significant differences according to Tukey’s (HSD) test (p < 0.05).
12 B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

of viable oogonia in single culture when submerged in unsterile pond water suggesting a sexual stimulus in the pond water.
These selfed oogonia were mainly hyaline in contrast to the golden brown oogonia produced in the A1 9 A2 pairings. Sel-
fing was not observed in any of the P. cinnamomi isolates used in this study. The abundance of oogonia formation and the
dimensions of oogonia, oospores and antheridia in all tested pairing combinations of P. parvispora and P. cinnamomi are
given in Table 3. In P. parvispora, oogonia were borne terminally and laterally (Fig. 2r); had smooth walls, globose to
subglobose shape; turned golden brown to bronze brown soon after pairing (Fig. 2r–u); and averaged 31.1  0.2 lm
(26.7–37 lm). Oogonia of P. cinnamomi also had smooth walls, but were significantly larger averaging 42.8  0.4 lm (35–
49.7 lm), less frequently golden brown and usually had a tapering base (Fig. 5i–m). Phytophthora parvispora oospores
were often slightly aplerotic (plerotic index = 52.4%) with a mean diameter of 25.0  0.2 lm (20.8–31.1 lm) and thick
walls of 1.9 lm diameter (Table 3). Conversely, P. cinnamomi oospores were more frequently almost plerotic (plerotic
index = 67.2%) and had a mean diameter of 37.4  0.3 (31.7–43 lm) and a mean wall thickness of 2.0 lm (Table 3). Hav-
ing similar wall thickness but significantly different oospore sizes, the oospore wall index of both species was significantly
different, averaging 0.4 and 0.29 in P. parvispora and P. cinnamomi, respectively. Aborted oospores were observed in both
P. parvispora (Fig. 2u) and P. cinnamomi, and the abortion rates were averaged 43.2 and 23.6% in P. parvispora and P. cin-
namomi, respectively. Interestingly, in P. parvispora, abortion rates in pairings of A1 9 A2 isolates from the same local pop-
ulation (Germany, Sardinia) were significantly lower (34.6–35.8%) than in pairings of isolates from different geographic
origin (50.1–52.0%), suggesting reproductive barriers as a result of long-term separation. Antheridia of P. parvispora were
usually amphigynous, occasionally paragynous and exclusively one-celled (Fig. 2r–u). Digitate projections were sometimes
observed at the base of the antheridia. In contrast, antheridia of P. cinnamomi were amphigynous, predominantly two-
celled and significantly longer than that of P. parvispora (Fig. 5i–m; Table 3).

3.3 Phylogenetic analysis

All phylogenetic analyses of the individual nuclear (ITS, b-tubulin) and mitochondrial (cox1, cox2) data sets resulted in sim-
ilar overall tree topologies (data not shown). The ITS and b-tubulin data sets were combined and so too were the cox1 and
cox2 data sets. Maximum-likelihood analyses were conducted on these two combined data sets, and the resultant ML phylo-
grams are presented in Figures 6 and 7 (TreeBASE: http://purl.org/phylo/treebase/phylows/study/TB2:S14021).
The combined nuclear data set (ITS, b-tubulin) contained 27 taxa and a total of 1646 characters (ITS = 805,
b-tubulin = 841). The resultant phylogeny was rooted to P. drechsleri from ITS major clade 8 and resolved several terminal
clades of Phytophthora species from subclades 7a and 7b (Fig. 6). Isolates of P. parvispora grouped in a well-supported
clade (100%) sister to isolates of P. cinnamomi. Thirteen ITS and 26 b-tubulin-fixed polymorphisms differentiated
sequences of P. cinnamomi from P. parvispora (Table 4). The phylogenetic relationship between P. parvispora and
P. cinnamomi was well-supported (100%), and these two clades were sister to the remaining ingroup taxa representing
species from subclades 7a and 7b (Fig. 6). Genetic resolution was good at terminal clades and allowed for the differentia-
tion of P. europaea, P. uliginosa, P. fragariae and P. cambivora into a moderately supported clade (84%) sister to a well-
supported clade (100%) accommodating P. sojae, P. pistaciae, P. sinensis, P. melonis, P. vignae and P. cajani. However, this
phylogeny did not recover support for the node joining these two clades.
The combined mitochondrial DNA sequence data set (cox1 and cox2) contained 24 taxa and a total of 1150 characters
(cox1 = 581 and cox2 = 569). The overall topology of the phylogram was similar to the nuclear phylogeny with some
important differences (Fig. 7). Isolates of P. cinnamomi and P. parvispora were also accommodated in well-supported sister
clades (100% and 99%, respectively). Thirty-two cox1 and 14 cox2 fixed polymorphisms distinguished DNA sequences of
P. parvispora from those of P. cinnamomi (Table 4). In contrast to the nuclear phylogeny, P. parvispora and P. cinnamomi
were sister to a single well-supported clade accommodating P. europaea, P. cambivora, P. fragariae and P. uliginosa (Fig. 7).
A separate unsupported clade containing P. pistaciae, P. cajani, P. vignae, P. sinensis, P. melonis and P. sojae was basal to
these three other clades.

3.4 Pathogenicity test

Arbutus unedo seedlings inoculated with P. parvispora started to show symptoms of leaf chlorosis and shoot dieback
50 days post-inoculation, except for those inoculated with isolate STEU 6265, which remained asymptomatic throughout
the experiment. Death of seedlings inoculated with P. parvispora occurred between the third and fourth month after inoc-
ulation. The experiment was terminated after four months because all A. unedo seedlings were dead, with the exception
of those inoculated with isolate STEU 6265. Phytophthora cinnamomi caused similar leaf and dieback symptoms, but they
appeared three weeks earlier compared with plants inoculated with P. parvispora. All seedlings inoculated with P. cinnam-
omi died after three months. Control plants did not show any aboveground symptoms. Collar necrosis was only observed
in plants inoculated with the two isolates of P. cinnamomi (PH067, P1889). Both Phytophthora species caused fine root
decay and necroses of mother roots, and a significant reduction in root length compared with control seedlings. The only
exception was P. parvispora isolate STEU 6265 where root length did not differ significantly from the control (Fig. 8).
The two P. cinnamomi isolates were the most aggressive pathogens, but the difference to the four P. parvispora isolates
was not significant. P. parvispora and P. cinnamomi were equally aggressive towards A. unedo seedlings. All Phytophthora
isolates were re-isolated from both necrotic roots and soil. No Phytophthora isolates were recovered from control
seedlings.
 Phytophthora parvispora sp. nov. 13

Fig. 6. Maximum-likelihood phylogram of combined ITS and b-tubulin DNA sequences indicating the phylogenetic relationships for species
from Phytophthora clades 7a and 7b. The phylogram is rooted to Phytophthora drechsleri P10331.

3.5 Fungicide sensitivity assays

Mefenoxam significantly inhibited growth of both P. cinnamomi and P. parvispora isolates compared with the controls.
Based on proportional growth rates on media with 5 lg ml 1 of mefenoxam, all P. parvispora and P. cinnamomi isolates
studied were classified as sensitive. At this concentration, the mean growth rates of P. parvispora isolates ranged from
7.6% for isolate IMI397618 to 8.6% for isolate CBS 413.96, while for P. cinnamomi, they ranged from 16.1% for isolate
CBS 14422 to 24% in PH067. Although all isolates were considered to be sensitive to the fungicide, analysis of variance
showed that P. parvispora was more sensitive than P. cinnamomi to mefenoxam at 5 lg ml 1 (p < 0.001). The estimated
EC50 and EC90 values for P. parvispora ranged from 0.002 to 0.84 lg ml 1 and from 1.45 to 3.10 lg ml 1, respectively. Phy-
tophthora cinnamomi isolates showed EC50 around 0.1 lg ml 1 except for the isolate PH067, which had an EC50 value of
0.8 lg ml 1. The estimated EC90 value for P. cinnamomi ranged from 7.2 to 31.5 lg ml 1, with the latter value coming from
isolate PH067.

4. Discussion
In the recent history of Phytophthora systematics, several varieties and forma specialis of well-known Phytophthora species
have been elevated to species status, most often using population study approaches that include modern molecular
14 B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

Fig. 7. Maximum-likelihood phylogram of combined cox1 and cox2 DNA sequences generated by RA 9 ML indicating the phylogenetic
relationships of Phytophthora species in clades 7a and 7b. The phylogram was rooted to Phytophthora drechsleri P10331.

techniques, RFLP and isozyme differences, phylogeny, adaptive and morphological features and host range. For example,
P. megasperma var. sojae (also named P. megasperma f. sp. glycinea), P. megasperma f. sp. medicaginis and P. megasperma f.
sp. trifolii were described by Hansen and Maxwell (1991) as P. sojae, P. medicaginis and P. trifolii. In the case of P. fragariae
and P. fragariae var. rubi, which share similar morphology and growth–temperature relations but differ in their host specifici-
ties, the latter was relegated to species status (P. rubi) because analyses of isozyme profiles and cox1 sequences demon-
strated absence of gene flow between both taxa (Man in ‘t Veld 2007). Phytophthora infestans and P. mirabilis share identical
ITS sequences, and the latter was considered a variety of P. infestans before Galindo and Hohl (1985) described it as a
distinct species. This description was based on significant phenotypic and behavioural differences of P. mirabilis compared
with P. infestans: larger sporangia, higher sensitivity to malachite green, faster growth on oat meal – V8 agar than on PDA,
ability to use nitrate as nitrogen source and host specificity to Mirabilis jalapa. Later, Goodwin et al. (1999) reinforced the
species status of P. mirabilis by demonstrating the absence of gene flow between P. mirabilis and P. infestans.
Phytophthora parvispora was first recorded from nursery plants of Beaucarnea sp. in a greenhouse in Germany, and at
that time, it was considered a variety of the polyphagous pathogen P. cinnamomi based on its smaller-sized sporangia and
chlamydospores and its higher maximum temperature for growth (Kr€ ober and Marwitz 1993; Erwin and Ribeiro 1996).
However, P. parvispora was apparently isolated before the work of Kr€ ober and Marwitz (1993) from citrus in Taiwan by
Ann and Ko (1985). Based on extensive isozyme analyses and morphological studies of a wide range of P. cinnamomi iso-
lates including two isolates from citrus in Taiwan, Oudemans and Coffey (1991) already hypothesized that the latter might
belong to a distinct species. Sequences from the ITS and cox2 regions of one of these citrus isolates (P2404) are available
at GenBank (FJ801815 and JF771391, respectively) and show 99% identity to the ex-type isolate CBS 132772 of P. parvis-
pora. Using a multigene phylogeny approach, Blair et al. (2008) demonstrated considerable genetic distance between P. cin-
namomi and P. cinnamomi var. parvispora, indicating absence of gene flow between both populations, and suggested that
P. cinnamomi var. parvispora could be elevated to species status. Similar results were produced by Bezuidenhout et al.
(2010) analysing three nuclear (ITS, b-tubulin and EF-1a) and two mitochondrial gene regions (cox1 and NADH) and by
Robideau et al. (2011) using an ITS and COI barcoding approach. In the present study, the multigene phylogeny (ITS,
b-tubulin, cox1 and cox2) of the ex-type specimen and another original isolate of P. cinnamomi var. parvispora from
Table 4. Positions of polymorphic nucleotides between Phytophthora parvispora and Phytophthora cinnamomi DNA sequences in the internal transcribed spacer (ITS), b-tubulin, cox1 and
cox2 gene regions.

Species Position of polymorphisms in DNA sequence alignment of individual loci

ITS
15 41 42 92 170 175 446 476 483 487 714 745 755
P. parvispora T C C G C A C C G T A G A
P. cinnamomi A A A A T C T T A G T T G

b-tubulin
18 24 36 60 81 108 123 132 174 219 273 327 394 408 423 453 471 549 579 642 657 744 771 780 804 810
P. parvispora C G C T C T T C T T C G T A C C T C C G C C T G C T
P. cinnamomi T C G A/W T C C T C G A C C G T G C T T C T T C T T C

cox1
3 15 46 93 111 144 147 177 224 228 234 237 243 249 258 270 279 291 296 303 315 345 360 391 399 423
P. parvispora A T G A C T A C T C A A T T T A C T A A T A C A T T
P. cinnamomi G C A T T A T A A T G T A A A T T C T T A T T C C A

cox1
450 492 504 546 550 552
Phytophthora parvispora sp. nov.

P. parvispora C C A A T A
P. cinnamomi A A G T G G

cox2
24 51 66 106 133 135 141 174 255 264 267 369 504 513
P. parvispora C C T A A A T A T G T A A A
P. cinnamomi T T G G G G A T A A A T T T
15
16 B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

2000
a
1600 ab

Root lengh (cm)

1200 bc
bc
c c c c
800

400

0
Control STE-U CBS CBS CBS CBS P904 PH067
6265 413.96 132772 132771 411.96
Isolate

Fig. 8. Mean total root length of 2-year-old Arbutus unedo seedlings after 4 months growth in soil infested with Phytophthora parvispora
(isolates STEU 6265, CBS 413.96, CBS 132772, CBS 132771 and CBS 411.96) and Phytophthora cinnamomi (isolates CBS 14422 and
PH067). Letters above columns indicate significant differences according to Tukey’ s HSD test (p ≤ 0.05). Bars represent standard errors.

Germany (Kr€ ober and Marwitz 1993) and a range of P. parvispora and P. cinnamomi isolates from a variety of hosts and
different geographic origins clearly demonstrated that P. parvispora is a distinct monophyletic taxon. All P. parvispora iso-
lates grouped in a highly supported terminal cluster within major ITS clade 7 (Cooke et al. 2000) with P. cinnamomi being
the closest relative. Detailed morphological and physiological analyses reinforced that P. parvispora is a unique species. Key
differences between P. parvispora and P. cinnamomi are the significantly smaller sizes of sporangia, chlamydospores, oogo-
nia and oospores and the higher oospore wall index of P. parvispora with virtually no overlap between the size ranges of
both species; much less abundant production of chlamydospores in P. parvispora; single-celled antheridia in P. parvispora
vs. two-celled antheridia in P. cinnamomi; higher minimum and maximum temperatures for growth and faster growth at
optimum temperature in P. parvispora.
The morphological and physiological attributes of P. parvispora provide insights into the ecology and survival strategy of
this pathogen. Having high cardinal temperatures for growth >12, 27 and 37°C, respectively, P. parvispora is well adapted
to tropical and subtropical climates and greenhouse conditions, which is reflected by all known disease outbreaks. Because
the oospore wall index of P. parvispora is significantly higher than in P. cinnamomi and comparable to species that thrive
in seasonally dry soils like P. quercina, P. psychrophila or P. elongata or dry necrotic leaves like P. ilicis (Scott et al. 2009;
Rea et al. 2010), seasonally dry conditions in its native habitat seem likely. However, in situations where only the A2 mat-
ing type is present, other enduring resting structures are required to survive seasonally dry conditions. Chlamydospores
are only infrequently produced in P. parvispora and have similarly thin walls as in P. cinnamomi, suggesting an analogous
role as short-term survival structures between consecutive periods of rain (Jung et al. 2013a). In P. cinnamomi, stromata-
like hyphal aggregations and selfed oospores have recently been shown to be the main long-term survival propagules (Jung
et al. 2013a). Phytophthora parvispora might have a similar survival strategy as it also forms dense hyphal aggregations in
solid agar and in water. In addition, production of oospores by selfing was induced by non-sterile pond water in several A2
isolates of P. parvispora, a phenomenon previously reported for the self-sterile aquatic P. thermophila enabling survival
when ephemeral streams fall dry (Jung et al. 2011).
Fifteen to twenty years after the first records from nursery plants of Beaucarnea sp. in Germany and citrus orchards in
Taiwan, respectively, P. parvispora has recently been isolated from the following sources: (i) in Australia from potting mixes
of nursery plants sent from the Northern Territory to Western Australia (WA) and from an irrigation channel surrounded
by intensely managed agricultural fields in the north of WA (Davison et al. 2006; http://www.fishingforphytophthora.mur-
doch.edu.au); (ii) from A. betulina in a plantation in South Africa (Bezuidenhout et al. 2010); (iii) from the rhizosphere of
V. unguiculata plantations in Brazil (Dos Santos 2010); (iv) from potted Mandevilla sp. in a nursery in Sicily (Italy) (Pane
et al. 2010); and (v) from container plants of P. pinea in a nursery in Portugal (Horta et al. 2012) (vi) as well as from
A. unedo in Sardinia (this study). It is apparent that almost all findings of P. parvispora are linked to the trade of plants-
for-planting, which is considered as major pathway of Phytophthora species into ornamental, horticultural and natural envi-
ronments (Erwin and Ribeiro 1996; Jung and Blaschke 2004; Brasier 2008; Jung 2009; Perez-Sierra and Jung 2013).
The occurrence of P. parvispora on five continents raises the question of its centre of origin. Because there are no
records from natural undisturbed vegetation, which might be indicative of a native host–pathogen co-evolution, only indi-
rect evidence may hint at possible origins. As P. parvispora and P. cinnamomi are the closest relatives of each other and
share a common ancestor, it could be likely that both species evolved in the same region. Due to the widespread occur-
rence of both mating types of P. cinnamomi in native healthy vegetation and the presence of resistant or tolerant species in
plant families that contain highly susceptible species in other regions of the world, South-East Asia and, in particular, Ha-
inan, South China, Taiwan and New Guinea are considered as centre of origin of this pathogen (Crandall and Gravatt 1967;
Ko et al. 1978; Arentz and Simpson 1986; Chang 1993; Zeng et al. 2009; Brasier et al. 2010). A possible origin of P. parvis-
pora in South-East Asia is also supported by the fact that the earliest findings of this species came from Taiwan (Ann and
Ko 1985).
 Phytophthora parvispora sp. nov. 17

Although P. cinnamomi and P. parvispora may originate from the same region and share a similar phenotype and life-
style, their global spread shows significant differences, which might have evolutionary implications. Early records of disease
symptoms resembling ink disease in Castanea dentata and C. sativa, respectively, indicate that the causal organism P. cin-
namomi had already been introduced to both the southern United States and the Iberian Peninsula during the 18th Century
(Crandall and Gravatt 1967; Zentmyer 1980). In contrast, the escape of P. parvispora from its unknown native environment
must have happened only recently as indicated by the first record of P. parvispora in Europe dating back only 20 years and
all other findings outside of Asia having occurred after 2001 (Davison et al. 2006; Bezuidenhout et al. 2010; Dos Santos
2010; Horta et al. 2012; http://www.fishingforphytophthora.murdoch.edu.au; this study). During their introduction and
establishment in new environments, populations of invasive Phytophthora species usually go through a genetic bottleneck
resulting from the introduction of a low number of genotypes from a limited area within their centre of origin, and subse-
quent strong selection and genetic drift in the founder population resulting in a clonal population structure (Brasier 1992;
Goodwin 1997). This is particularly the case for P. cinnamomi where the global invasion was mainly achieved by clonal
spread of two genotypes from the A2 mating type (Old et al. 1984; Oudemans and Coffey 1991; Dobrowolski et al. 2002).
In the phylogenetic analysis of the present study, isolates from the two mating types of P. cinnamomi grouped in two sepa-
rate clusters confirming results from a recent mitochondrial haplotype analysis of a large global collection of A1 and A2
isolates (Martin and Coffey 2012) and from isozyme studies with large sets of P. cinnamomi isolates from Australian and
worldwide origin, respectively (Old et al. 1984; Oudemans and Coffey 1991). In all three studies, the two mating types
were clearly separated, and no phylogenetic evidence for gene flow between them could be found. Using microsatellite
markers, Dobrowolski et al. (2002) demonstrated high genetic variability within the geographically limited A1 mating type
of P. cinnamomi, while the global population of the widespread A2 mating type consisted of two microsatellite genotypes.
Furthermore, no sexual recombinants could be found even in locations where both mating types co-occurred within the
same rhizosphere (Dobrowolski et al. 2002) or location (Chang et al. 1996). These results suggest that long-term separation
resulted in reproductive barriers between the two mating types of P. cinnamomi, which most likely form distinct evolution-
ary lineages. In contrast, in P. parvispora, a higher degree of genetic variability compared with P. cinnamomi was shown in
the phylogenetic analysis of the present study, and the occurrence of both mating types in one greenhouse in Germany and
in several nurseries and plantings in Sardinia (Kr€ ober and Marwitz 1993; this study) suggests either multiple introductions
or the spread of a diverse founder population. The presence of both mating types in the same environment provides P. par-
vispora with an evolutionary advantage by creating new genetic variation via sexual recombination and thus facilitating
rapid adaptation to changes in both the host populations and the environment (Brasier 1992; Goodwin 1997; Cooke et al.
2012). In addition, the production of oospores enables long-term survival and the build-up of a genetic reservoir in the soil
(Brasier 1992). Phytophthora cinnamomi has an extremely wide host range that includes almost 4000 woody and herba-
ceous plant species (Zentmyer 1980; Shearer et al. 2004). This pathogen is a notorious threat to the production of nursery
stock on a global scale (MacDonald et al. 1994; Erwin and Ribeiro 1996; Ferguson and Jeffers 1999; Moralejo et al. 2008;
Perez-Sierra and Jung 2013). After its escape from its native area, P. cinnamomi caused some of the most devastating die-
backs of natural ecosystems, for example, ink disease of native chestnut and oak decline in Southern Europe, the south-
eastern United States and Mexico (Brasier et al. 1993; Tainter et al. 2000; Vettraino et al. 2005; Balci et al. 2007; Jung et al.
2013b); little leaf disease of pine species in the south-eastern United States and the Caribbean (Oak and Tainter 1988; Jung
and Dobler 2002); dieback of Fynbos heathlands in the Cape province of South Africa (Von Broembsen and Kruger 1985);
and dieback of eucalypt forests, Banksia woodlands and heathlands in Australia (Weste and Marks 1987; Shearer and Tipp-
ett 1989; Cahill et al. 2008). In contrast, P. parvispora has apparently thrived in the nursery trade for only 20 years and
has never been recorded from natural environments. However, this might likely be related to the fact that the time avail-
able for the escape of P. parvispora from nurseries or plantings to natural ecosystems and subsequent establishment and
expression of disease symptoms has simply been too short. Whether P. parvispora has a similarly wide host range and
potential to cause devastation of natural ecosystems as P. cinnamomi therefore remains unclear. However, with seven
known host species in six monocotyledonous, dicotyledonous and coniferous plant families, that is, Apocynaceae (Mandevil-
la sp.), Asparagaceae (Beaucarnea sp.), Ericaceae (A. unedo), Fabaceae (V. unguiculata), Pinaceae (P. pinea) and Rutaceae
(A. betulina and Citrus sp.), P. parvispora apparently has the potential to overcome resistance mechanisms of a quite
diverse array of host plants. Consequently, extensive testing of the potential host range of P. parvispora amongst ecologi-
cally and economically important tree species, ornamentals and horticultural crops is urgently required to assess the risk
posed by this pathogen.
In the present study, P. parvispora is reported for the first time causing root and collar rot on A. unedo, and in the soil
infestation test, all isolates showed similar aggressiveness to the root system of A. unedo as P. cinnamomi. Only one
P. parvispora isolate from A. betulina in South Africa showed no pathogenicity to A. unedo, but this isolate was also non-
pathogenic to A. betulina (Bezuidenhout et al. 2010) and might have a generally reduced fitness. Because P. cinnamomi is a
major pathogen of many species from the Ericaceae such as Erica, Gaulteria, Pieris, Rhododendron and Azalea (Erwin and
Ribeiro 1996), knowledge about pathogenicity of P. parvispora to members of this important plant family will be of high
interest.

Acknowledgements
The authors would like to thank G. Magnano di San Lio, A. Pane and S.O. Cacciola (University of Catania, Italy) and C.M. Bezuidenhout
(Agricultural Research Council in Stellenbosch, South Africa) for contributing further P. parvispora isolates to this study. They are also
grateful to C.M. Brasier and J.F. Webber (Forest Research, Farnham, UK) for their constructive discussions and valuable suggestions; A.M.
18 B. Scanu, G. C. Hunter, B. T. Linaldeddu et al.

Perez-Sierra for providing the ITS sequence of the Portuguese P. parvispora isolate; S.A. Kirk and S. Franceschini (Forest Research, Farn-
ham, UK) and S. Seddaiu and A. Deidda (University of Sassari, Italy) for help with laboratory routines. B.S. was supported by a grant from
the Regional Government – Legge Regionale 7 agosto 2007, N. 7 – CRP1_629. T.J. acknowledges support by the Regione Autonoma della
Sardegna, Visiting Professor Program at the University of Sassari, Italy. This work was supported in part by grants from Fondazione Banco
di Sardegna and Parco Nazionale dell’Arcipelago di La Maddalena.

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