Phytophthora boodjera sp. nov., a damping-off pathogen in production nurseries and from urban and natural landscapes, with an update on the status of P. alticola.

A new homothallic Phytophthora species, isolated in Western Australia (WA), is described as Phytophthora boodjera sp. nov. It produces persistent, papillate sporangia, oogonia with thick-walled oospores, and paragynous antheridia. Although morphologically similar to P. arenaria, phylogenetic analyses of the ITS, cox1, HSP90, β-tubulin and enolase gene regions revealed P. boodjera as a new species. In addition, P. boodjera has a higher optimal temperature for growth and a faster growth rate. Phytophthora boodjera has only recently been found in Western Australia and has mostly been isolated from dead and dying Eucalyptus seedlings in nurseries and from urban tree plantings, and occasionally from disturbed natural ecosystems. It is found in association with declining and dying Agonis flexuosa, Banksia media, B. grandis, Corymbia calophylla, Eucalyptus spp,. and Xanthorrhoea preissii. The status of P. alticola was also reviewed. The loss of all isolates associated with the original description except one; discrepancies in both sequence data and morphology of the remaining isolate with that presented the original description, and inconclusive holotype material places the status of this species in doubt.

INTRODUCTION

Numerous Phytophthora species have been associated with damping-off and seedling diseases in plant production nurseries worldwide (Hardy & Sivasithamparam 1988, Davison , Warfield , Moralejo , Goss , Lilja , Leonberger , Pérez-Sierra & Jung 2013, Prospero , Schoebel ). Phytophthora species are dispersed via the roots of infected plants, soil from potted plants, growth media and water, and in some cases by aerial transmission. Transfer of plants and plant products by human activity and through globalisation in trading is now generally accepted as the main method of introduction of exotic pathogens and pests. The most high-risk pathway for the movement of Phytophthora is “plants for plantings” (Brasier 2008, Liebhold , Scott ). Plants infected at production nurseries can potentially distribute Phytophthora species to parks and reserves, amenity plantings, plantations, rehabilitation and biodiversity plantings, wildflower farms, retail nurseries, and gardens. Many Phytophthora species, such as P. nicotianae, P. plurivora (often reported as P. citricola), P. cactorum and P. citrophthora, tend to be the most commonly recovered from nurseries worldwide, strongly supporting their dissemination through the nursery trade. Because of the level of attention that has been given to this important topic, it is now rare for a new species to be detected in nurseries (Moralejo ). Nevertheless the number of reports of Phytophthora species damaging to nursery trees, forests and natural ecosystems is increasing and this has significant implications for international plant biosecurity and plant health practice (Kroon ).

The most significant new detection of the past 20 years is Phytophthora ramorum (Grünwald , Parke & Grünwald 2012). Phytophthora ramorum was first detected infecting Viburnum and Rhododendron in plant nurseries in Germany and The Netherlands in 1993 (Werres ), and has subsequently been found in various nurseries all over Europe and North America. It has been recognized as an alien aggressive species in natural areas of the west coast of the USA where it causes sudden oak death, and in Cornwall in the UK (Rizzo , Brasier ). Spread through the international nursery trade, P. ramorum poses a serious risk to plant biosecurity worldwide (Brasier 2008, Parke & Lucas 2008, Parke & Grünwald 2012).

In recent years, many new Phytophthora species have been described from natural ecosystems in Western Australia (WA) (Burgess , Scott , Rea , Jung , b, Rea , Aghighi , Burgess , Crous , Hüberli ). In 2011, a new damping-off disease was reported in WA nurseries growing Eucalyptus and other species for restoration of agricultural land. ITS sequence data of the isolates did not match any known species, but were closely related to P. alticola and P. arenaria and were an exact match for a single WA isolate designated as “P. taxon arenaria-like” by Rea .

Phytophthora arenaria has been isolated primarily from Kwongan vegetation and mainly from Banksia species on the northern sandplains in south-west WA (Rea). Phytophthora alticola was first isolated and described by Maseko from cold-tolerant Eucalyptus species (E. dunnii, E. bajensis, and E. macarthurii) with collar and root rot in South African plantations at an altitude above 1150 m. The new taxon has been isolated in WA from dead and dying Eucalyptus seedlings in nurseries and from adult plants in the urban landscape, predominantly from eucalypts, and occasionally from Banksia species and Corymbia calophylla in natural ecosystems.

Further investigation of isolates thought to be P. arenaria in the Vegetation Health Service (VHS) collection of the WA Department of Parks and Wildlife (Burgess ) and other recent collections from urban surveys (Barber ) revealed two distinct groups of isolates. The first group were of P. arenaria, while the second appeared to be a new species related to P. alticola (Maseko ). In the current study, the P. alticola/P. arenaria species complex was re-evaluated using a combination of morphology and a multi-gene phylogeny resulting in the recognition of a new species, described here as P. boodjera sp. nov., and an investigation into the status of P. alticola.

MATERIALS AND METHODS

Isolates

The majority of isolates used were obtained from the Vegetation Health Service (VHS) Collection, Department of Parks and Wildlife, Perth, Western Australia. All isolates were baited from soil and root material using Eucalyptus sieberi cotyledons. The isolates were maintained in 90 mm Petri dishes on V8 agar (V8A, 0.1 L filtered V8 juice, 17 g agar, 0.1 g CaCO3, 0.9 L distilled water) and on 5 mm V8A discs stored in 20 mL sterile water in McCartney bottles at room temperature. The ex-type isolates of P. alticola were obtained from CBS (CBS-KNAW Fungal Biodiversity Centre, Utrecht). Sequence data from related species were obtained from GenBank (www.ncbi.nlm.nih.gov/genbank) the Phytophthora Database (PD; www.phytophthoradb.org), and q-bank (www.q-bank.eu). When all isolates in the CMW collection (Forestry and Agriculture Biotechnology Institute, University of Pretoria, SA) were evaluated and it was found that all isolates of P. alticola except CMW 19425 had perished, that isolate was re-numbered CMW 34279. All isolates used in this study are detailed in Table 1, and the status of all P. alticola isolates is given in Table 2.

Table 1.

Identity, date and location of isolation, host information and GenBank accession numbers (where available) for Phytophthora spp. considered in this study.

Species	Location	Isolation date	Host association	Isolate number2	GenBank Accession No.
					ITS	BT	HSP	ENO	cox1
P. alticola1	Midillovo, KwaZulu-Natal	2000-20046	Eucalyptus badjensis	CMW 19417
ex-holotype	(KZN), South Africa			CBS 121937	q-bank5				q-bank5
P. alticola1	Midillovo, KZN, South Africa	2000-2004	E. macarthurii	CMW 19424	DQ988197	DQ988236
ex-paratype				CBS 121938
P. alticola1	Paulpetersburg, KZN,	2000-2004	E. dunnii	CMW 19425	DQ988196	DQ988235
ex-paratype	South Africa			CBS 121939	q-bank5				q-bank5
				CMW 342794	HQ013214	KJ372275	KJ396703	KJ396731	KJ396686
P. alticola1	unknown		Unknown	P 16052	GU259141				HQ261245
P. boodjera	Mt Claremont, Perth, WA	05/2011	Agonis flexuosa	PAB 11.564	KC748460	KJ372280	KJ396708	KJ396736	KJ396687
	Dalkeith, Perth, WA	05/2011	Eucalyptus marginata	PAB 11.674	KC748461	KJ372276	KJ396704	KJ396732	KJ396682
	Ravensthorpe, WA	08/2006	Banksia media	VHS 162823	EU301117	KJ372281	KJ396709	KJ396737	HQ013198
	Kensington, Perth, WA	02/2012	Eucalyptus sp.	VHS 266314	KJ372240	KJ372277	KJ396705	KJ396733	KJ396683
ex-holotype	Tincurrin, WA	03/2012	Soil dump	VHS 268064	KJ372244	KJ372283	KJ396710	KJ396738	KJ396688
				CBS 138637
	Tincurrin, WA	04/2012	Eucalyptus sp.	VHS 270164	KJ372245
	Tincurrin, WA	04/2012	Eucalyptus sp.	VHS 270174	KJ372246	KJ372284	KJ396711	KJ396739	KJ396689
	Tincurrin, WA	04/2012	Eucalyptus sp.	VHS 270184	KJ372247	KJ372285	KJ396712	KJ396740	KJ396690
	Tincurrin, WA	04/2012	Eucalyptus sp.	VHS 270204	KJ372248	KJ372286	KJ396713	KJ396741	KJ396691
	Tincurrin, WA	04/2012	Eucalyptus sp.	VHS 270214	KJ372249	KJ372287	KJ396714	KJ396742	KJ396692
	Tincurrin, WA	04/2012	Eucalyptus sp.	VHS 270224	KJ372250	KJ372288	KJ396715	KJ396743	KJ396693
	Tincurrin, WA	04/2012	E. polybractea	VHS 271714	KJ372241	KJ372278	KJ396706	KJ396734	KJ396684
	Stirling, Perth, WA	11/2012	Xanthorrhoea preissii	VHS 273824	KJ372242	KJ372279	KJ396707	KJ396735	KJ396685
	Gingin, WA	11/2012	B. grandis	VHS 28352
	Northam, WA	09/2013	Corymbia calophylla	TP 13.39
P. arenaria	Kalbarri, WA	06/1986	Kwongan heathland	DDS 12214	EU593266	KJ372297	KJ396724	KJ396752	HQ013201
	Eneabba, WA	02/2009	E. drummondii	CBS 1258004	HQ013205	KJ372296	KJ396723	KJ396751	HQ013215
ex-holotype	Eneabba, WA	02/2009	E. drummondii	CBS 1279504	HQ013219	KJ372289	KJ396716	KJ396744	HQ013203
	Lancelin, WA	11/2001	B. menziesii	VHS 98614	EU301118	KJ372290	KJ396717	KJ396745	HQ013202
				IMI 389662
	Bunbury, WA	02/2002	B. littoralis	VHS 10154	EU301114	KJ372298	KJ396725	KJ396753	KJ396697
				IMI 389663
	Badgingarra, WA	04/2006	B. attenuata	VHS 154534	EU301115	KJ372291	KJ396718	KJ396746	HQ013199
	Badgingarra, WA	04/2006	B. attenuata	VHS 154894	HQ013216	KJ372292	KJ396719	KJ396747	HQ013200
	Eneabba, WA	06/2008	B. attenuata	VHS 199314	HQ013217	KJ372293	KJ396720	KJ396748	KJ396694
	Eneabba, WA	11/2008	B. attenuata	VHS 205374	KJ372253	KJ372299	KJ396727	KJ396754	KJ396698
	Ellenbrook, Perth, WA	09/2011	Banksia sp.	VHS 253704	KJ372254	KJ372300	KJ396726	KJ396755	KJ396699
	Dongara, WA	11/2012	Banksia sp.	VHS 28145	KJ372251	KJ372294	KJ396721	KJ396749	KJ396695
	Muchea, WA	12/2012	X. preissii	VHS 28269	KJ372252	KJ372295	KJ396722	KJ396750	KJ396696
P. frigida	South Africa		Eucalyptus sp.	P 16059	GU259147				HQ261313
P. palmivora	United States			P 0113	GU259121	EU080465	EU080468	EU080467	HQ261383
P. heveae	United States			P 10167	GU259516	EU080796	EU080799	EU080798
P. quercetorum	United States			MD 9.2		EU080901	EU080904	EU080903
P. castaneae	Japan			P 10187	FJ801304	EUO80803	EU080806	EU080805	HQ261348
P. megakarya	Sao Tome and Principe			P 8516	PD5	EU079970	EU079973	EU079972	HQ261356
P. nicotianeae	Australia		Nicotiana tabacum	332					AY129169
P. cactorum	United States		Malus sylvestris	NY 568					AY129174
P. plurivora	Germany		Quercus robur	CBS 124087					FJ237510

1See Table 2 for explanation on the status of these isolates.

2Abbreviations of isolates in culture collections (where known): CBS = Centraalbureau voor Schimmelcultures, The Netherlands; IMI = CABI Bioscience (formerly International Mycological Institute), UK; VHS = Vegetation Health Service Collection, Department of Parks and Wildlife, Perth, Australia; DDS = earlier prefix of VHS Collection; PAB = Paul Barber, in Murdoch University (MU) Culture Collection; TP = Trudy Paap, in Murdoch University (MU) Culture Collection; CMW = culture collection of Forestry and Agriculture Biotechnology Institute, University of Pretoria, South Africa; P = isolate codes from World Phytophthora Collection, University of California, Riverside.

3Designated as Phytophthora taxon arenaria-like by Rea .

4Isolates used in the morphological study.

5Sequence available on Phytophthora database (http://www.phytophthoradb.org/) or q-bank (http://www.q-bank.eu/).

6No specific dates provided by Maseko et al. (2004), just date range under ‘sampling and isolation’.

Table 2.

Status of Phytophthora alticola isolates submitted to different culture collections.

Isolate	Sequence1	Notes on status of isolate
CMW 19416	no sequence (OD)	Lost in CMW collection. Only papillate, caducous sporangia and chlamydospores observed from preserved slide associated with PREM 59214
PREM 59214-paratype
CMW 19417	no sequence (OD)	Lost in CMW collection. Supposed corresponding isolate in CBS is actually P. palmivora and all sequence data on q-bank associated with this isolate is P. palmivora. Only papillate, caducous sporangia and chlamydospores observed from preserved slide associated with PREM 59215
PREM 59215-holotype
CBS 121937	ITS, CO, YPT1, TEF (q-bank)
CMW 19419	ITS and BT (OD)	Lost in CMW collection
PD 01642
CMW 19421	ITS and BT (OD)	Lost in CMW collection
PD 01641
CMW 19422	ITS and BT (OD)	Lost in CMW collection
PD 01640
CMW 19423	ITS and BT (OD)	Lost in CMW collection
PD 01639
CMW 19424	ITS and BT (OD)	Lost in CMW collection. Sequence on q-bank of ITS and BT is from the original description. The ITS of isolate re-sequenced in this study corresponds to P. frigida. Only aplerotic oospores and amphigynous antheridia observed from preserved slide associated with PREM59216
PREM 59216-paratype
CBS 121938
PD 01638
CMW 19425	ITS and BT (OD)	Living in CMW collection and renamed CMW 35429. ITS and BT of re-sequenced isolate differ from original description by 3 and 2 bp respectively. ITS and CO sequence on q-bank is identical to sequence of isolate CMW 35429 obtained in the current study. Only aplerotic oospores and amphigynous antheridia observed from preserved slide associated with PREM 59217
PREM 59217-paratype
CBS 121939	ITS, CO, YPT1, TEF (q-bank)
PD 01637
CMW 35429	ITS, cox1, ENO, HSP, BT	Was sent to WPC as CMW 35429 as a replacement for P. alticola and named WPC 16948. ITS sequence supplied by Gloria Abad is identical to that obtained in the current study for isolate CMW 35429
P16948	ITS (GA)
PD 01914	cox2 and cox1 (PD)	Was sent to WPC as P. alticola isolate CMW 19424 but when sequenced it was identified as being an isolate of P. frigida
P16053
PD 02043	cox2 and cox1 (PD)	Was sent to WPC as P. frigida isolate CMW 19433 and when sequenced it was identified as being an isolate of P. frigida
P16051
PD 02044	cox2 and cox1 (PD)	Was sent to WPC as P. alticola isolate CMW 19425 but when sequenced it was identified as being an isolate of P. frigida
P16054
PD 02775	cox1 (PD)	Was sent to WPC as P. frigida isolate CMW 20311 but when sequenced it was identified as being an isolate of P. alticola and thus cannot be linked to any isolate from CMW collection
P16052
VHS 26631	ITS, cox1, ENO, HSP, BT	List in WPC as a neotype for P. alticola, but this is not recommended as the isolate is from a different host and a different country from the original description. In current study this is considered an isolate of P. boodjera.
P19861

1OD = original description (Maseko ), WPC = World Phytophthora Collection (http://phytophthora.ucr.edu/), GA = supplied by Gloria Abad, PD = Phytophthora database http://www.phytophthoradb.org/, q-bank = http://www.q-bank.eu/.

DNA isolation, amplification and sequencing

The Phytophthora isolates were cultured on half-strength potato dextrose agar (PDA) (Becton Dickinson, Sparks, MD), 19.5 g PDA, 7.5 g agar and 1 L of distilled water) at 20 °C for 2 wk. Mycelium was collected by scraping from the agar surface with a sterile blade and placing in a 1.5 mL sterile Eppendorf® tube. It was frozen in liquid nitrogen and crushed to a fine powder, and genomic DNA was extracted following the method of Andjic . In all cases, the PCR reaction mixtures were as described previously (Andjic ) but using the PCR conditions described in the original papers (cited below). The region spanning the internal transcribed spacer (ITS1-5.8S-ITS2) region of the ribosomal DNA was amplified using the primers DC6 (Cooke ) and ITS-4 (White ). The mitochondrial gene cox1 was amplified with primers FM77 and FM 84 (Martin & Tooley 2003). Heat shock protein 90 (HSP) was amplified with HSP90-F int and HSP90-R1 primers (Blair ). β-tubulin (BT) was amplified with primers BTF1A and BTR1, and enolase (ENO) was amplified with primers Enl Fy and Enl R1 according to Kroon .

All gene regions were sequenced in both directions with the primers used in amplification. The clean-up products and sequencing were accomplished as described previously (Sakalidis). All sequences derived in this study were added to GenBank, and the accession numbers are provided in Table 1.

Phylogenetic analysis

The data set consisted of sequences of Phyophthora boodjera sp. nov., P. alticola and P. arenaria isolates used in this study, and other closely related species in ITS clade 4 (Table 1) which were compiled and manually edited in Geneious v. R7 (http://www.geneious.com/) and Bayesian analysis conducted using a MrBayes (Ronquist ) plugin within Geneious after determining the most appropriate substitution model with jModelTest-2.1.4 (Darriba ). Alignment files and trees can be viewed on TreeBASE (http://www.treebase.org/).

Culture characteristics

Circular inoculum plugs (5 mm diam) were taken from the margin of 7 d-old cultures on V8A and placed in the centre of 90 mm Petri dishes of the test media. Morphology of hyphal and colony growth patterns were defined from 7 d-old cultures grown at 20 °C in the dark on V8A, malt extract agar (MEA), carrot agar (CA; 0.1 L filtered carrot juice, 17 g agar and 0.9 L distilled water) and half-strength PDA (all from BBL, Becton Dickinson, Sparks, MD). Colony morphology was described according to Erwin & Ribeiro (1996). For temperature growth studies, all isolates were subcultured onto V8A plates and incubated for 24 h at 20 °C for growth stimulation. The plates were then moved to incubators fixed at 4, 10, 15, 20, 25, 30, 32.5, 35 and 37.5 °C. Plates were observed daily to ensure that the colonies did not reach the edge of the Petri dish; the radial growth rate was measured after 4–7 d, along two lines crossing the middle of the inoculum plug at right angles, and the mean growth rates (mm per day) were assessed. After 7 d, plates with no colony growth at 35 °C and 37.5 °C were returned to 20 °C for 7 d to check the isolate viability.

Morphology

Sporangia were produced by flooding 15 × 15 mm square agar discs, removed from the growing edge of 3–5-d-old colonies on V8A in 90 mm Petri dishes, with sterile water at 18–25 °C with their surfaces submerged, in natural daylight. This water was decanted and replaced twice (after 4 and 6 h). In the final change, 1 mL of non-sterile soil extract was also added and the Petri dishes were incubated overnight. The soil extract was made by suspending 100 g of pine (Pinus radiata) bark potting mixture in 1 L distilled water and incubating this on an orbital shaker for 24 h at 20 °C before filtering through Whatman no. 1 paper to remove soil particles. After 18–36 h, dimensions and characteristic features of 50 mature sporangia of each isolate, selected at random, were ascertained at 400 × in a B ×51 Olympus microscope.

Gametangia were produced by all isolates on V8A in the dark at 20 °C after 7 d. After 14 d, dimensions and characteristic features of 50 randomly-selected mature oogonia, oospores and antheridia were measured at 400 ×. The oospore wall index was calculated as the ratio between the volume of the oospore wall and the volume of the whole oospore (Dick 1990).

The preserved type materials of P. alticola available from the National Mycological Herbarium in Pretoria (PREM 59214, PREM 59215, PREM 59216, PREM 59217) were re-examined. The slides were rehydrated with 85 % lactic acid and observed with a Zeiss Axioskop 2 Plus compound microscope fitted with an Axiocam MRc camera. Dimensions were measured using Axiovision v. 4.8 software.

RESULTS

CMW 19417 was designated as the type isolate of Phytophora alticola by Maseko , but no sequence data were provided for this isolate. A subsequent sequence of this same isolate, CBS 121937 available on q-bank, actually corresponds to P. palmivora (Fig. 1). CMW 19424 and CMW 19425 were originally designated as paratypes and ITS sequence data were provided for these isolates. All of these isolates were subsequently lost except CMW 19425 (= CBS 121939 = CMW 34279 = P19861). ITS sequence data for isolates presented with the original description, including CMW 19425 (DQ988196), differ by 3 bp from all recent sequences of CMW 34279, CBS 121939 and P19861 (Fig. 1). However, when resequenced CMW 19424 (= CBS 121938) was found to actually be an isolate of P. frigida (Fig 1). Based on ITS sequence data, the WA isolates investigated in this study cluster with either isolate CMW 34279 or with P. arenaria (Fig. 1).

Fig. 1.

Bayesian inference tree based on ITS sequence data generated in MrBayes using the GTR +G substitution model showing relationship between P. alticola nom. dub. (green), P. boodjera sp. nov. (blue) and P. arenaria (red). Isolates designated as P. alticola in CBS correspond to P. palmivora (purple) and P. frigida (orange). The posterior probability is shown at the nodes. Phytophthora castaneae and P. heaveae were used as outgroup taxa. Asterisks indicate the re-sequenced isolates CBS 121937 and CBS 121938. CBS 121939 was resequenced, but not included as it was identical to the sequence on q-bank for this isolate.

BT sequences data was also provided in the original description (Maseko ): all isolates assigned to P. alticola were identical, but differ by 2 bp from the new sequence of isolate CMW 34279 and by 4 bp from P. boodjera sp. nov. (figure available on request from the authors). The coxI sequence of isolate CMW 34279 from three separate databases is identical and clusters separately from isolates assigned to P. boodjera sp. nov. (figure available on request from the authors). Isolates of P. arenaria cluster together, although intraspecific sequence variation is observed. In the concatenated dataset (Fig. 2), isolate CMW 34279 clusters with isolates of P. boodjera sp. nov., although it differs by 8 bp across the five gene regions examined. If the isolate is duplicated it forms a strongly supported cluster on its own (data not shown). Isolates of P. arenaria also reside in a strongly supported clade, although intraspecific variation is observed (Fig. 2).

Fig. 2.

Bayesian inference tree based on concatenated sequence data from ITS, β-tubulin, HSP90, enolase and coxI gene regions generated in MrBayes using the GTR +G substitution model showing relationship between P. alticola nom. dub. (green), P. boodjera sp. nov. (blue) and P. arenaria (red). The posterior probability is shown at the nodes. Phytophthora castaneae and P. heaveae were used as outgroup taxa.

Status of Phytophthora alticola

In 2008, the World Phytophthora Collection (WPC; http://phytophthora.ucr.edu/default.html) was sent four isolates from the CMW collection, two isolates each of P. alticola and P. frigida. When the WPC sequenced them, they realised the identities were incorrect and informed the CMW collection (Table 2). Isolates of P. alticola and P. frigida were then checked in the CMW collection and it was discovered that all isolates of P. alticola had perished or were incorrectly identified, except for CMW19425 which was cleaned and renumbered CMW34279. This isolate was then sent to WPC where it was given the code P19861. Also in 2007, three isolates were sent to CBS; of these, the ex-holotype isolate CBS 121937 (= CMW 19417) is actually P. palmivora (the sequence associated with this isolate is available from q-bank), the ex-paratype isolate CBS 121938 (= CMW 19424) was not re-sequenced but is now determined as of P. frigida, leaving the same single isolate CBS 121939 (= CMW 34279) (Table 2).

At the start of this project, it was known that the ex-holotype isolate of P. alticola had perished, as indeed had all other isolates except an ex-paratype isolate CMW 19425 (= CMW 35429, = CBS 121939, = P19861). The ITS sequence of this isolate from all collections is identical, although there are a few bp different from the ITS sequence of the same isolate in the original description (Fig. 1). The sequence in the original description is short and the differences are at the end of the sequence and could have been erroneously labelled. Controversially, sequence data of other isolates in various collections designated as P. alticola match different species (Table 2).

It was originally considered that epitypification would be possible with the intention to designate CMW 34279 as the epitype. However, morphological examination of this isolate revealed that it differed from the original description: the sporangia are not caducous and chlamydospores are not produced (Table 3). Subsequent examination of the holotype and paratypes from PREM were inconclusive (Table 3). Each of the PREM types consisted of a semi-dried agar disc kept at 4 °C and a microscopic slide. The agar disks were all contaminated with bacteria and a dark hyphomycete, most of the mycelia had lysed, but a few aborted oospores were observed in PREM 59216 (= CMW 19424) and PREM 59217 (= CMW 19425). Some reproductive structures were present on the slides. Sporangia and chlamydospores were present for PREM 59214 (= CMW 19416) and PREM 59215 (= CMW 19417). The sporangia were predominantly ovoid, caducous and papillate, and produced in close sympodia (Table 3, Fig. 3). The dimensions of these sporangia match the original description of P. alticola (Maseko ). However, in the original description the sporangia were described as borne on terminal or branched sporangiophores, while the slide associated with the holotype had sporangia borne in close sympodia. These sporangia and their branching patterns resemble more those produced by P. palmivora rather than those of living isolate CMW 34279 (Table 3, Fig. 3). Oospores only were present in paratypes PREM 59216 (= CMW 19424) and PREM 59217 (= CMW 19425). The dimensions of these aplerotic oospores match the original description and those of living isolate CMW 34279, however antheridia of the types are amphigynous, while those of CMW 34279 are paragynous (Table 3, Figs 3–4). Both P. frigida and P. alticola were described as having aplerotic oospores with amphigynous antheridia (Table 3), therefore the slides associated with the paratypes are inconclusive.

Table 3.

Comparison of morphological characters and dimensions, and temperature-growth relations of Phytophthora palmivora, P. frigida, P. alticola (from original description, holotype and paratype material and living isolate CMW 19425 = CMW 34279), P. boodjera, and P. arenaria.

Species and sources of data	P. palmivora (Erwin & Ribero, 1995)	P. frigida (Maseko 2007)	P. alticola (holotype1)	P. alticola (paratype2)	P. alticola (Maseko 2007)	CMW 342793 (this study)	P. boodjera (this study)	P. arenaria (Rea 2011)	P. arenaria (this study)
			PREM 59215	PREM 59217
No of isolates		10			10	1	12	10	9
Sporangia (mm)
L × B mean	45.3 × 29.8	33 × 37	31.1 ± 5.0 × 30.9± 4.5		36 × 28	38.9 ± 5.4 × 28.6 ± 4.3	39.2 ± 4.4 × 29.7 ± 3.4	31.8 ± 4.6 × 23.7 ± 3.5	23.9 ± 3.1 × 19.8 ± 3.4
Range	40–60 × 25–35	24–40 × 20–33	27.7–45.7 × 23.0–29.4		30–45 × 20–35	20.4–60.7 × 19.0–38.9	15.2–64.5 × 13.9–42.5	20.2–53.0 × 12.5–35.0	12.7–38.5 × 9.9–30.7
Range of isolates means					na	na	32.6–44.6 × 24.7–33.3	28.9–34.8 × 21.4–28.3	19.5–24.9 × 16.0–23.1
L/B ratio	1.2–1.8	1.22	1.21 ± 0.12		1.4 (<1.6)	1.35 ± 0.03	1.27 ± 0.16	1.40 ± 0.17	1.22 ± 0.20
Range of isolates means					na	na	1.19–1.35	1.2–1.5	1.08–1.65
Sporangial characteristics	Papillate	Papillate, rarely bipapillate	Papillate		Papillate, rarely bipapillate	Papillate, rarely bipapillate or bilobed	Papillate, rarely bipapillate or bilobed	Papillate, rarely bi/tripapillate or bilobed	Papillate, rarely bipapillate or bilobed
Persistence	caducous	caducous	semi-caducous		caducous	persistent	persistent	persistent	persistent
Sporangiophores	Lax or close sympodia	simple	Lax or close sympodia		simple or branched sympodia	simple or branched sympodia often with bulbous base, very often laterally attached	simple or branched sympodia often with bulbous base, very often laterally attached	simple or branched sympodia often with bulbous base	simple or branched sympodia often with bulbous base, very often laterally attached
Sporangia shape	ellipsoid, ovoid spherical	ovoid, sometimes obpyriform	Usually ovoid to broad ovoid		usually ovoid or ellipsoid, sometimes obpyriform or peanut-shaped	ovoid 66 %, limoniform 14 %, peanut-shaped 8 %, obpyriform 6 %, distorted 6 %	ovoid 64 %, limoniform 20 %, peanut-shaped 10 %, distorted 6 %	usually ovoid, also obpyriform or distorted	ovoid 40 %, subglobose 20 %, globose 14 %, obpyriform 12 %, distorted 4 %
Proliferation	absent	absent	absent		absent	absent	absent	absent	absent
Exit pores (mm)
Width		5–6			6	6.21 ± 0.53	6.09 ± 1.02	6.00 ± 1.00	5.50 ± 0.95
Width range		5–10			4–8	5.00–7.10	4.85–8.89	3.40–8.90	3.88–7.10
Chlamydospores (mm)	32–42	24–26	42.6 ± 5.8		Some isolates	absent	absent	absent	absent
					28 (20–35)
Hyphal swellings		Spherical			Irregular	Catenulate, some with radiating hyphae	Catenulate, some with radiating hyphae	Catenulate, globose to sub-globose, some with radiating hyphae	Catenulate, globose to sub-globose, some with radiating hyphae
Mean diameter (mm)					na	14.7	15.2	na	12.8
Breeding system	Heterothallic	Heterothallic		Homothallic	Homothallic	Homothallic	Homothallic	Homothallic	Homothallic
Oogonia (mm)
Mean diameter		38		26.2 ± 2.3	284	27.3 ± 1.9	29.4 ± 2.3	25.3 ± 2.2	26.6 ± 1.6
Diameter range	22–34.8	24–48		24–37	20–35	22.03–31.07	24.3–33.9	19.6–34.3	20.5–29.6
Range of isolates means					na	na	24.6–33.4	24.3–28.1	23.6–28.8
Oospores (mm)
Mean diameter	22.8 ± 0.1	33		26.2 ± 2.1	30 (28.3 × 30.5)	24.9 ± 2.1	25.5 ± 1.9	22.3 ± 1.8	23.8 ± 1.6
Diameter range	22.8	25–42		21–31	24–36	20.3–29.5	20.92–29.3	16.0–28.3	17.8–28.6
Range of isolates means					na	na	21.3–29.5	21.4–23.9	21.5–25.9
Wall thickness					na	2.51 ± 0.4	2.47 ± 0.33	2.30 ± 0.34	2.57 ± 0.22
Oospore wall index		na		0.57± 0.01	na	0.54 ± 0.05	0.47 ± 0.05	0.50 ± 0.05	0.53 ± 0.06
Oogonial characteristics	Aplerotic	Aplerotic		Aplerotic	Markedly aplerotic, oospores with thick inner walls	Aplerotic oospores Mature oogonia with a slightly wavy surface and golden-brown discoloration often with tapering base	Aplerotic oospores Mature oogonia with a slightly wavy surface and golden-brown discoloration	Aplerotic oospores Mature oogonia with a slightly wavy surface and golden-brown discoloration	Aplerotic oospores Mature oogonia with a slightly wavy surface and golden-brown discoloration
Antheridia	Amphigynous	Amphigynous		Amphigynous	Mainly amphigynous	Paragynous, often with finger-like projections	Paragynous	Paragynous, often with finger like projections	Paragynous
L × B mean (mm)					na	10.6 ± 2.3 ×	10.4 ± 1.9 ×	11.2 ± 1.7 ×	10.0 ± 2.1 ×
						8.3 ± 1.4	8.3± 1.5	8.4 ± 1.3	7.5 ± 1.3
L × B range (mm)					na	Na	8.2–10.9 ×	7.9–16.4 ×	6.4–13.8 ×
							7.3–10.6	6.0–10.5	5.6–12.8
Growth Characteristics
Max temp (°C)	34	30 to <35			30 to <35	35	35	32.5	35
Opt temp (°C)	27.5–30	25			25	20–25	25–30	30	25
Min temp (°C)	11	>5<10			>10<15	>10<15	>10<15	>10<15	15
Lethal temp (°C)					na	>37.5	>37.5	na	<37.5
Growth rate at optimum (mm/day)		ca. 7.5 (CA),			ca. 4.5 (CA),	8.20 (V8A)	9.18 (V8A)	5.9–7.4 (CA)	8.65 (V8A)
		ca. 8 (V8A)			ca. 7 (V8A)
Growth rate at 20°C (mm/day)		5 (V8A),			4.5 (V8A),	7.75 (V8A)	6.12 (V8A)	3.8–5.2 (CA)	5.96 (V8A)
		3.0 (CA)			3.0 (CA)
Colony morphology	On CA, stellate, defined edge, aerial mycelium in centre	Stellate-petaloid on V8, CA, PDA and MEA, moderately fluffy			Uniform and fluffy on MEA and V8A, stellate with limited aerial mycelium on CA and PDA	Appressed and cottony with no distinctive growth pattern and regular smooth margins on CA, V8A and PDA; sparse, slow growth on MEA	Appressed with no distinctive growth pattern and regular smooth margins on CA, V8A, MEA and PDA	Radiate to faintly radiate with very limited aerial mycelium and regular smooth margins on CA, V8A, MEA and PDA	Appressed with no distinctive growth pattern and regular smooth margins on CA, V8A and PDA, sometimes slightly petaloid on V8A; sparse on MEA

1Morphological features of paratype PREM 59214 = CMW 19416 same as holotype PREM 59215 = CMW 19417, caducous, papillate sporangia in close sympodia and chlamydospores present. No oospores observed.

2Morphological features of paratype PREM 59216 = CMW 19424 same as paratype PREM 59217 = CMW 19425 = CMW 35479; amphigynous, aplerotic oospores turning brown on maturity. No sporangia or chlamydospores observed.

3Isolate CMW 19425 = CMW 35429 = CBS 121939 = WPC 16948 is the only isolate still surviving from the original description of P. alticola (Maseko ) and it is linked to PREM 59217. Note: when all isolates were lost in the CMW collection, the remaining isolate CMW 19425 was renamed CMW 35429 and it is this isolate that was sent to the World Phytophthora Collection and given the code P 16948.

4Measurements from Maseko where oospores were misrepresented to be larger than oogonia.

Fig. 3.

Rehydrated slides of P. alticola nom. dub. (type specimens). Sporangia of paratype PREM 59214 = CMW 19416: (a) close sympodia with papillate, ovoid sporangia, (b) papillate, ovoid caducous sporangia with short pedicels, (c) papillate ovoid sporangia. Sporangia and chlamydospores of holotype PREM 59215 = CMW 19417: (d) Papillate, ovoid sporangia, (e–f) chlamydospores. Oospores of paratype PREM 59216 = CMW 19424: (g–h) aplerotic oospores with amphigynous antheridia. Oospores of paratype PREM 59217 = CMW 19425: (i–l) aplerotic oospores with amphigynous antheridia. Bar = 50 μm.

Fig. 4.

(a–k) Papillate sporangia of Phytophora boodjera formed on V8A flooded with soil extract. Ovoid to broadly ovoid (a, b, e, f, g), limoniform (d right, h) bipapillate (i) distorted and bipapillate (j) often with laterally attached sporangiophore (c, k). Branching sporangiophores were rarely observed (d), occasional constriction of sporangiophore near base of sporangia (e), or bulbous sporangiophore (f). Hyphal coils rarely observed (l). Oogonia of P. alticola nom. dub. isolate CMW34279 with tapering bases, wavy margins and turning golden brown at maturity, with aplerotic oospores and paragynous antheridia (m–q). Aplerotic oospores of P. boodjera with paragynous antheridia (r–v). Scale bar = 20 μm.

In the original description (Maseko ), no sequence data were provided for PREM 59214 (= CMW 19416) and PREM 59215 (= CMW 19417). When the ex-holotype isolate was submitted to CBS and sequenced for q-bank (CBS 121937) it was found to be an isolate of P. palmivora (Fig. 1, Table 2). Caducous, papillate sporangia and chlamydospores matching P. palmivora were observed in PREM 59214 (= CMW 19416) and PREM 59215 (= CMW 19417) (Fig. 3). When the ex-paratype isolate CMW 19424 was submitted to CBS it was found to be P. frigida, as were several isolates labelled as P. alticola that were sent to WPC (Fig. 1, Table 2). Phytophthora frigida also has aplerotic oogonia with amphigynous antheridia, as observed for PREM 59216 (= CMW 19424) and PREM 59217 (= CMW 19425). Thus, we believe that while in the original description of P. alticola the sequence data provided was identical for all isolates, the actual morphological description is based on a set of isolates from more than one species; these are most probably P. palmivora, P. frigida, and a species represented by isolate CMW 34279. As there are no other living isolates linked to the original description available for examination and as no more isolates have been recovered in South Africa, despite extensive sampling, it is not possible to amend the description of P. alticola or to designate PREM 59217 (= CMW 19425, = CMW 35429) as an epitype. At this point in time the application of the name P. alticola is in doubt and will remain so until more isolates from similar hosts or locations can be made and this taxon will be referred to hereafter as P. alticola nom. dub.

Compared with the description of P. alticola nom. dub., CMW 34279 has a higher optimum temperature for growth, faster growth rate, persistent sporangia, no chlamydospores and paragynous antheridia, and is very similar in morphology to isolates from Australia described here as P. boodjera.

TAXONOMY

Phytophthora boodjera A.V. Simamora & T.I. Burgess, sp. nov.

MycoBank MB809223

(Figs 4–5)

Fig. 5.

Colony morphology of (top to bottom) isolate CMW 34279, Phytophthora boodjera (VHS 27171, CBS 138637), and P. arenaria (CBS 127950, VHS 25370) after 7 d growth at 20 °C on different media: CA, V8A, MEA and half strength PDA (left to right).

Etymology: the species name is derived from the Noongar (local Aboriginal) name for earth, ground, or sand plain.

Type: Australia: Western Australia: Tincurrin, from nursery soil dump, Mar. 2012, collected by the Vegetation Health Service of the Department of Parks and Wildlife (MURU 470–holotype; cultures ex-type CBS 138637 = VHS 26806). ITS, β-tubulin, HSP90, enolase and coxI sequence GenBank KJ372244, KJ372283, KJ396710, KJ396738 and KJ396688 respectively).

Diagnosis: P. boodjera is phylogenetically closely related to P. alticola nom. dub. but differs in having persistent sporangia, paragynous antheridia and no chlamydospores. P. boodjera is morphologically similar to P. arenaria but differs in having a higher lethal temperature and larger sporangia and oogonia.

Description (type): Papillate, persistent predominantly ovoid sporangia (52 %) but also limoniform (45 %) and distorted shapes (3 %). Sporangia averaged 34.7 ± 1.16 × 27 ± 0.78 μm and ranged 15.2–62.3 × 14.6–42.5 μm. Homothallic; aplerotic oogonia averaged 28.9 ± 2.13 μm, ranging from 24.3–34 μm. Oospores averaging 26.3 ± 1.42 μm diam, range 20.9–29.4 μm. Growth rate at optimum of 25 °C was 11.2 mm/d. Colonies were appressed with no pattern and had regular smooth margins on CA, V8A, MEA and PDA.

Description (species): Sporangia papillate, persistent, abundantly produced in soil extract water on simple sporangiophores frequently with globose swellings close to the sporangial base (Fig. 4f). Although predominantly ovoid (64 %, Fig. 4a–g), various sporangial shapes were observed including limoniform (20 %, Fig. 4d right, 4h), peanut-shaped (10 %) and distorted shapes (6 %, Fig. 4i, j). Bipapillate (Fig. 4i) sporangia were also occasionally observed. Sporangiophores often laterally attached to sporangia (Fig. 4c, k), and sometimes constricted (Fig. 4e); branched sporangiophores rare (Fig. 4d). Sporangia from 12 isolates averaged 39.2 ± 4.4 × 29.7 ± 3.4 μm (range 32.5–44.5 × 24.5–33.5 μm), exit pores narrow, 6 ± 1 μm, length:breadth ratio 1.27 ± 0.16 (Table 3). Chlamydospores absent.

Homothallic, readily producing oogonia (and sporangia) in single culture on CA and V8A. Oospores matured within 14 to 21 d. Oogonia averaged 29.4 ± 2.3 μm diam with isolate means ranging from 24.6 to 33.4 μm (Table 3). Oospores aplerotic in all isolates, containing ooplasts when semi-mature to mature (Fig. 4s–v). Oospores averaged 25.5 ± 1.9 μm diam with isolate means ranging from 21.3 to 29.5 μm (Table 3). Oospore walls thick (2.5 ± 0.33 μm) (Fig. 4s–v), oospore wall index 0.47 ± 0.05 μm (Table 3). Antheridia paragynous (Fig. 4r–v), averaging 10.4 ± 1.9 × 8.3 ± 1.5 μm. Hyphal swellings catenulate, some with radiating hyphae, formed rarely in non-sterile soil extract water.

Cultures: All isolates produced colonies that were appressed with no distinctive growth pattern and regular smooth margins on CA, V8A, MEA, and PDA (Fig. 5). Growth on MEA was sparser than on the other media. Optimum temperature for the growth on V8A 25–30 °C, where the average growth rate was 9.18 ± 0.56 mm/d (Fig. 6). The maximum temperature for growth was 35 °C (Table 3). Although no growth occurred at 37.5 °C, this temperature was not lethal since isolates resumed growth when subsequently incubated at 20 °C.

Fig. 6.

Average growth rate (mm/d ± SE) of Phytophthora boodjera (blue), CMW 34279 (green) and P. arenaria (red) on V8A across the temperature range from 4–37.5 °C.

Additional specimens examined: Australia: Western Australia: Mt Claremont, Perth, from roots of dying Agonis flexuosa, May 2011, Paul Barber (PAB 11.56, private collection); Dalkeith, from roots of dying Eucalyptus marginata, May 2011 Paul Barber (PAB 11.67, private collection); Northam, from Corymbia calophylla, Sept. 2013,Trudy Paap (TP13.39, private collection). Ravensthorpe, from Banksia media, Aug. 2006, (VHS 16282); Kensington, Perth, WA, from Eucalyptus sp., Feb. 2012, (VHS 26631); Tincurrin, from Eucalyptus spp., Apr. 2012, (VHS 27016, VHS 27017, VHS 27018, VHS 27020, VHS 27021, VHS 27022); Tincurrin, from roots of E. polybractea, Apr. 2012, (VHS 27171); Stirling, Perth, from Xanthorrhoea preissii, Nov. 2012, (VHS 27382); Gingin, from Banksia grandis, Nov. 2012, (VHS 28352); All VHS isolates were collected and are maintained by the Vegetation Health Service of the Department of Parks and Wildlife, Western Australia.

Notes: Phytophthora boodjera is morphologically very similar to isolate CMW 34279 linked to P. alticola nom. dub.; all measurements overlap, although CMW 34279 produces on average smaller sporangia, oogonia and oospores (Table 3). Colony morphologies on malt extract agar also differ (Fig. 5), and P. boodjera has a higher optimal temperature for growth and grows faster at higher temperatures (Fig. 6). Isolates of P. boodjera differ from CMW 34279 by one fixed single nucleotide polymorphism (SNP) in the ENO gene region, two in HSP and two in BT; three fixed SNPs separate the species in the cox1 gene region.

Phytophthora boodjera is closely related to P. arenaria. Morphologically, these species are very similar producing abundant thick walled oospores and sporangia of similar shapes and sizes (Table 3). The most marked differences between these species are: (1) 37.5 °C is lethal to P. arenaria but not to P. boodjera; (2) sporangia as well as oogonia and oospores are smaller in P. arenaria; and (3) 34 % of sporangia of P. arenaria are globose to subglobose while this shape is rare in P. boodjera (Table 3).

DISCUSSION

Phytophthora isolates from plant production nurseries in Western Australia (WA) were identified as closely related to P. alticola nom. dub. based on ITS sequence data. These isolates were compared to the single remaining isolate of P. alticola nom. dub. from the original description (Maseko ). Based on morphology and molecular data from four nuclear and one mitochondrial gene region, the isolates from WA were recognized as a new species and described as P. boodjera. Phytophthora boodjera has emerged as a pathogen in some WA plant production nurseries and is now regularly recovered also from urban environments. However, it has been recovered infrequently (VHS 16282 from Ravensthorpe, VHS 28352 from Gingin, and TP 13.39 from Northam) from natural ecosystems in WA, despite widespread sampling in the region (Burgess , Rea ).

Phytophthora alticola nom. dub. was originally described from Eucalyptus plantations in South Africa and has never been recovered from sampling within natural ecosystems in that region (Nagel , Oh ). This suggests that P. alticola has been introduced into South Africa. Morphological studies of the remaining isolate CMW 34279 revealed three major discrepancies with the original description: firstly, P. alticola nom. dub. was described as having caducous sporangia, and secondly, as producing chlamydospores; however, the remaining isolate CMW 34279 has persistent sporangia and produced no chlamydospores. Thirdly, P. alticola nom. dub. was described as producing mainly amphigynous and some paragynous antheridia; however, in the remaining isolate CMW 34279, only paragynous antheridia were observed. Although the ex-holotype isolate CMW 19417 has been lost, re-examination of the holotype PREM 59215 revealed sporangia and chlamydospores matching the original description of P. alticola nom. dub. except that they were produced in close sympodia rather than simple or branched sympodia (Maseko ). CMW 19417 was submitted to CBS and the sequence of this isolate reveals that it is P. palmivora. The dimensions and characteristics of sporangia and chlamydospores observed in the holotype match those of P. palmivora.

Discrepancies in sequence data were found between the original description of P. alticola nom. dub. and the remaining ex-paratype isolate CMW 19425 (= CMW 34279). Unfortunately only oospores can be observed on the paratype PREM 59217 (= CMW 19425), but even these differ from the original description in that all antheridia are amphigynous in the holotype material, but they are all paragynous for CMW 34279. Thus, after examining the holotype and paratype material and resequencing isolates submitted to CBS, we have concluded that the original description was based on a mix of species and, as no further isolates similar to CMW 34279 have been recovered in South Africa despite extensive sampling (Oh ), the status of P. alticola is in doubt.

Phytophthora arenaria (Rea ), the species most closely related to P. boodjera in Western Australia, has been recovered exclusively from natural Kwongan vegetation on the coastal sand plains of south-west WA, where it was mainly isolated from dead and dying Banksia species and from the rhizosphere soil associated with such plants. This species appears to be restricted to the Kwongan vegetation and to be adapted to this ecosystem, suggesting that P. arenaria is native to WA. Phytophthora boodjera has only recently been found in WA and has mostly been isolated from dead and dying eucalypt seedlings in plant production nurseries and from declining trees (predominantly Myrtaceae) in disturbed urban landscapes, and once from Xanthorrhoea preissii. It has been isolated from natural ecosystems on only three occasions (from Banksia media, B. grandis, and Corymbia calophylla) and currently we consider this to be an introduced species.

Recent outbreaks of the damping-off disease of young eucalypt seedlings, caused by P. boodjera, have raised new concerns about the risk of Phytophthora species in plant production nurseries in WA. The dispersal of Phytophthora from nurseries to field plantings in previously non-infested areas may result in serious threats to biodiversity in natural ecosystems in these areas.

ACKNOWLEDGEMENTS

Trudy Paap, Keith Parnell, and Paul Barber were all involved in the collection of isolates for this study. Mike Coffey, Gloria Abad, Seonju Marincowitz and Arthur de Cock have all provided valuable help and discussion in resolving the current status of Phytophthora alticola. Diane White is thanked for technical assistance. A.S. thanks DIKTI program from the Indonesian Government for a PhD Scholarship.