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Phytophthora bisheria sp. nov., a new species identified in isolates from the Rosaceous raspberry, rose and strawberry in three continents

     United States Department of Agriculture, USDA-APHIS-PPQ-PHP-PSPI, National Identification Service (NIS), Molecular Diagnostics Laboratory (MDL), Bldg. 580, BARC-E, Powder Mill Road, Beltsville, Maryland 20705

Jorge A. Abad

     United States Department of Agriculture, USDA-APHIS-PPQ-PHP-PSPI, Plant Germplasm Quarantine Program (PGQP), Bldg. 580, BARC-E, Powder Mill Road, Beltsville, Maryland 20705

Michael D. Coffey

     World Phytophthora Collection, Department of Plant Pathology, University of California, Riverside, California 92521

Peter V. Oudemans

     P.E. Marucci Center for Blueberry Cranberry Research and Extension, Rutgers State University, Chatsworth, New Jersey 08019

Willem A. Man in ’t Veld
Hans de Gruyter

     Plant Protection Service, Department of Mycology, P.O. Box 9102, 6700 HC, Wageningen, the Netherlands

James Cunnington

     Department of Primary Industries-Knoxfield, Private Bag 15, Ferntree Gully Delivery Centre, Victoria, 3156, Australia

Frank J. Louws

     Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 

A homothallic semipapillate slow growing Phytophthora species associated with root rot of strawberries from greenhouse-grown plants in North Carolina, USA, root rot of roses in the Netherlands, and root rot of raspberry in Knoxfield, Australia, was identified. The main character of this organism is the production of paragynous antheridia with broad attachment to the oogonial wall. The morphology of the pathogen does not match that of any of the more than 85 described Phytophthora species. Phylogenetic analysis based on sequences of the internal transcribed spacer rDNA region (ITS1-5.8S-ITS2) of this taxon and those from other Phytophthora species from GenBank supports the conclusion that this organism is an unreported new species. In the phylogenetic tree with other reported Phytophthora species at the GenBank, the new species is more closely related to others in ITS clade 2 comprising semipapillate taxa including P. botryosa, P. citrophthora, P. colocasiae, P. meadii, P. citricola, P. inflata, P.tropicalis, P. capsici, Phytophthora sp. ‘glovera’ and P. multivesiculata. The most closely related species is P. multivesiculata isolated from Cymbidium orchid in the Netherlands. In this paper we describe the morphological characteristics and the phylogenetic relationships that support the description of this taxon as a new species Phytophthora bisheria sp. nov.

Key words: ITS, morphology, Oomycetes, pathogenicity, phylogenetics, Phytophthora, Straminipiles, taxonomy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Phytophthora is a fungus-like genus in the Oomycetes recently placed in the new kingdom Straminipila (Straminipiles) that includes among other genera, the diatoms, brown algae and golden brown algae (Dick 2001). The genus consists of more than 80 species and harbors a group of important plant pathogens including some historically devastating species (P. cinnamomi Rands, P. infestans [Mont.] de Bary and P. ramorum Werres, de Cock & Man in ’t Veld]. Interest in Phytophthora species identification has been stimulated in the past 5 y with the introduction of powerful phylogenetic analyses (Cooke et al 2000, Martin and Tooley 2003, Kroon et al 2004). Sequence analysis of internal transcribed spacer region (ITS) of the nuclear ribosomal DNA (rDNA) gene repeat (Cooke et al 2000), translation elongation factor (TEF) 1 alpha, β-tubulin and the mitochondrial encoded cytochrome oxidase I (Cox I) (Kroon et al 2004) and Cox II (Martin and Tooley 2003) genes have been used for phylogenetic studies and for the validation of the new Phytophthora taxa reported in recent years. The relationships among Phytophthora species on the basis of ITS analysis broadly matched those based on the nuclear encoded elongation factor EF-1, β-tubulin, Cox I and Cox II. However groupings based on sequence analysis did not concur with those based on morphology (Waterhouse 1963). Integration of morphological and molecular identification for the description of new taxa has greatly enhanced the knowledge of the taxonomy of the Oomycetes. Using molecular methods hybrids also have been discovered in recent years (Man in ’t Veld et al 1998; Brasier et al 2003, 2003b, 2004). In the past 10 y 19 species have been described on the basis of morphological and molecular supporting data. These species are in addition to the 64 morphological species presented in the Phytophthora treatise of Erwin and Ribeiro (1996) and include P. alni with subspp. alni, multiformis, and uniformis Brasier & Kirk, P. brassicae de Cock & Man in ’t Veld, P. captiosa Dick & Dobbie, P. europaea Hansen & Jung, P. fallax Dobbie & Dick, P. foliorum Donahoo & Lamour, P. hedraiandra de Cock & Man in ’t Veld, P. inundata Brasier, Sánch. Hern. & Kirk, P. ipomoeae Flier & Grünwald, P. kernoviae Brasier, Beales & Kirk, P. multivesiculata Ilieva, Man in ’t Veld, Veenb.-Rijks & Pieters, P. nemorosa Hansen & Reeser, P. oryzobladis Wang & Lu (ex H.H. Ho), P. pistaciae Mirab., P. polonica Belbahri, Moralejo, & Leford, P. psychrophila Jung & Hansen, P. quercina Jung, P. ramorum Werres, de Cock & Man in ’t Veld, P. tropicalis Aragaki & Uchida, and P. uliginosa Jung & Hansen (Aragaki and Uchida 2001; Belbahri et al 2006; Brasier et al 2003a–2005; Dick et al 2006; de Cock and Lévesque 2004; Donahoo et al 2006; Flier et al 2002; Hansen et al 2003; Ho 2001; Ilieva et al 1998; Jung et al 1999, 2002; Man in ’t Veld et al 2002; Mirabolfathy et al 2001; Werres et al 2001). Sequences from the ITS, TEF, β-tubulin, Cox I and Cox II for more than 65 of the 80 reported Phytophthora species are deposited in GenBank and compiled at the National Center for Biotechnology Information (NCBI). Multilocus analysis based on isozymes also provides a powerful method to delineate species (Oudemans and Coffey 1991).

Strawberry (Fragaria x ananassa) is an economically important fruit crop in the United States and around the world. At least seven Phytophthora species have been isolated from roots, crowns, stolons and fruits of affected strawberry plants in different regions of the world (Erwin and Ribeiro 1996, Mass 1998). Species of Phytophthora associated with strawberries include P. cactorum (Lebert & Cohn) Schröt, P. citricola Sawada, P. citrophthora (R.E. Sm. & E.H. Sm.) Leonian, P. cryptogea Pethybr. & Laff., P. fragariae Hickman var. fragariae, P. megasperma Drechsler and P. nicotianae Breda de Hann (=P. parasitica Dastur). From this group P. cactorum, the cause of "leather rot of fruit", stem, crown and root rot, and P. fragariae var. fragariae, the cause of red stele or red core, are the most important pathogens and occur in most countries where strawberries are cultivated (Diekmann et al 1994, Mass 1998). Phytophthora nicotianae has been reported in USA (California, Hawaii, Tennessee), Bulgaria, Japan and Taiwan (Matsuzaki et al 1980, Chang 1988, Ilieva 1995, Winterbottom et al 1996, Mass 1998). Phytophthora citricola and P. megasperma have been reported in USA (California), Bulgaria and Taiwan (Chang 1988, Ilieva 1995). Phytophthora citrophthora has been reported in Bulgaria (Ilieva 1995) and Taiwan (Chang 1988) where it causes a type of leather rot on strawberry fruits. Phytophthora cryptogea has been reported from Bulgaria (Ilieva 1995). During spring 1999 strawberry plants growing in two greenhouses in Raleigh, North Carolina, showed symptoms of slight yellowing and root rot. Ten isolates of a semipapillate homothallic species, morphologically distinct from other Phytophthora species, were isolated from the symptomatic roots (Abad et al 2001). The US authors deposited their ITS sequences in GenBank-NCBI before publication. This enabled the Dutch and Australian authors to match their sequences with those from the US, which revealed that all three groups were working independently on the same undescribed species.

In the Netherlands the slow growing Phytophthora sp. was isolated from the roots of young rose cuttings suffering from root rot received at the Dutch Plant Protection Service in 1990. These infected cuttings were stunted, had fewer side shoots, smaller dull green leaves that yellowed and shed prematurely. Newly formed roots were rotted and microscopic examination of such squashed infected rootlets revealed numerous scattered oogonia and thick walled oospores with occasionally semipapillate sporangia. Many attempts were made to isolate the Phytophthora, but only twice, in 1990 and 1998, was isolation successful. Roses are economically important both for the domestic market as well as for export in the Netherlands and many other countries worldwide. Numerous varieties of roses are grown on a large scale by specialized growers, usually on artificial substrates in greenhouses year around. Several Phytophthora species have been reported to be pathogenic on roses, such as P. cactorum, P. citrophthora and P. megasperma (Erwin and Ribeiro 1996). They all may cause root rot resulting in wilting of the leaves and eventually plant death.

In Australia Phytophthora root rot is one of the most important diseases of raspberry. Phytophthora cryptogea and P. fragariae Hickman var. rubi Wilcox & Duncan are the most commonly encountered species (Washington 1999). Phytophthora megasperma, P. cactorum, P. citricola, P. drechsleri Tucker, P. cambivora (Petri) Buisman and P. idaei Kennedy have been recorded from raspberry in other parts of the world (Ellis et al 1991, Kennedy and Duncan 1995). In 1996 routine pathogen screening of the raspberry germ-plasm collection at the Knoxfield research centre of the Victorian Department of Primary Industries isolated several species of Phytophthora from rotting roots. While most of these isolates were P. fragariae var. rubi, one slow growing isolate was identified as P. idaei. The P. idaei affected plant was wilting and the leaves becoming chlorotic with necrotic margins. DNA sequence data obtained in 2005 revealed that this isolate was not P. idaei but instead an undescribed species.

The morphological features of the isolates obtained from raspberry, rose and strawberry were identical and closely resembled those of P. citricola and P. inflata. In this study we integrated morphological and molecular data to describe this taxon as a new Phytophthora species.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Isolation and morphological characterization.— – Roots of symptomatic strawberry plants were washed thoroughly with tap water to remove soil and plant debris. Small sections from the edge of rotted and healthy tissue were selected and blotted on sterile paper towels. Root pieces were placed on CMA P₁₀ARP selective medium containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene with or without hymexazol (Kanwischer and Mitchell 1978), and CMA-P₁₀VP selective medium containing pimaricin, vancomycin, and pentachloronitrobenzene with or without hymexazol (Tsao and Guy 1977). The plates were kept at room temperature (ca. 25 C) in the dark for 14 d. Coralloid mycelial growth on the media (typical for Phytophthora species) was observed under a microscope and individual hyphal tips were marked and transferred into new CMA-P₁₀ARP or CMA-P₁₀VP. The emerging colonies were subcultured to CMA and incubated for 15 d at room temperature (ca. 25 C). Ten isolates from strawberries of the unknown slow growing Phytophthora were selected for further analysis.

Production of sporangia was induced by placing small mycelial plugs from the edge of active growing colonies in CMA (12–15 d) into 10% soil solution and incubating under continuous fluorescent light at room temperature (22–25 C) for 4–7 d. Isolates were also grown on V8 juice agar (Gams et al 1998), baby carrot agar (B-CA) and baby lima bean agar B-LBA. Both media were prepared with 50 g of the substrate/500 mL water, autoclaved 5 min, filtered and made up to 1 L, plus 17 g of agar. Fifty sporangia and oogonia were measured in each isolate after 15 d of growth in the dark for the morphological characterization of the species.

Isolates were evaluated morphologically to species level with published keys for identification of Phytophthora species (Waterhouse 1963, Stamps et al 1990, Erwin and Ribeiro 1996) and a Morphological/Molecular (Pictorial/Phylogenetic) key (Abad ZG unpublished). Colony morphology and analysis of the minimum, optimum and maximum cardinal growth temperatures were performed in CMA, V8, B-CA and potato-dextrose agar 30 (PDA, Difco 30 gr/lt). Cultures of the organism were maintained on slants of CMA and in sterile water cultures (glass tubes with 15 mL distilled sterilized water) during this study.

Isolates from rose were processed with a similar methodology in the Netherlands. Isolates were obtained from diseased cuttings of roses sent to the Plant Protection Service for diagnosis. Small pieces of infected fine roots were washed in tap water and then in 50% v/v alcohol for 30 s before rinsing in distilled water and drying on filter paper. Small pieces (3–5 mm) were plated on cherry decoction agar (CHA) and water agar (WA) (Gams et al 1998) as well as on the selective medium P10VP (Tsao and Guy 1977). For colony characterization, emerging colonies on V8 juice agar (V8) were subcultured on V8, CMA, cherry decoction agar (CHA), oatmeal agar (OA), synthetic nutrient-poor agar (SNA) and water agar (WA) (Gams et al 1998). The agar plates were incubated in the dark at 22 C and the colonies was measured after 7 d and 14 d respectively. Sizes of zoosporangia, oogonia and oospores were evaluated on isolates on V8. The methods used to isolate the culture from raspberry in Australia during 1996 were not recorded. However, based on current protocols in the same laboratory, it is likely that roots were surface sterilized and plated onto both V8 and water agar. Morphological and cultural characteristics of this isolate were determined as for those from strawberry.

Molecular characterization.— – Ten isolates of the putative new Phytophthora species were grown 14 d in potato-dextrose broth (Difco, USA) and DNA was extracted with the PUREGENE DNA isolation kit (Gentra Systems Inc. Minneapolis, Minnesota) according to manufacturer’s protocols. Concentration and quality of total DNA were estimated by electrophoresis. Two oligonucleotide universal primers, ITS5 and ITS4 (White et al 1990), were used to amplify the ITS rDNA region (ITS 1, 5.8S, ITS2). PCR amplification was performed following the protocol described by Cooke and Duncan (1997). Amplicons were purified with the QIAGEN kit according to manufacturer’s protocol (QIAGEN Inc., Chatsworth, California). Sequences of the amplified ITS region of two isolates obtained from strawberry (Cg 2-3-3 and P 1.33) were determined at the Genomic Research Laboratory (GRL), North Carolina State University, USA. Sequences of raspberry (1680) and rose (PD 90/444) isolates were obtained respectively in Australia and in the Netherlands. To ascertain the phylogeny of this organism sequences were preliminary aligned and compared with a selected Phytophthora database of Z.G. Abad that includes sequences of selected groups of Phytophthora species from GenBank (NCBI) and selected putative new species, in the collection of Z.G. Abad, under progress for official description (data not shown). Sequences of four isolates of the putative new Phytophthora species, eight Phytophthora in Clade 2 (Cooke et al 2000) and another five species in Clades 1, 6 and 7 reported isolated from strawberry and raspberry were selected for final phylogenetic analysis (TABLE I). A sequence of P. lateralis in Clade 8 was used as outgroup. Phylogenetic analysis with the group of selected sequences was carried out in TOPALi (Milne et al 2004) using the Felsenstein-84 nucleotide substitution plus gamma rates heterogeneity model to estimate pairwise distances. The tree was estimated with neighbor joining. Bootstrap support values were derived from 500 replicates for the NJ and the generated tree was exported via TREEVIEW (Page 1996).

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Phytophthora bisheria Z.G. Abad, J.A. Abad & F.J. Louws, sp. nov. FIGS. 1–40

View larger version (168K): [in this window] [in a new window]   FIGS. 1–9. Phytophthora bisheria. 1. Colony morphology on CMA culture medium. 2–9. Asexual stage. 2–3. Direct germination of sporangium producing a pair or a single new sporangia. 4–7. Arrangement of sporangiophore, primary and secondary sporangia. 8–9. Coralloid long and nodose hyphae occasionally present in CMA water cultures. Bar = 10 µm.

View larger version (153K): [in this window] [in a new window]   FIGS. 10–22. Phytophthora bisheria sporangia (asexual stage). Typical shapes of sporangia in strawberry isolate Cg 2.3.3. Bar 10 = µm.

View larger version (147K): [in this window] [in a new window]   FIGS. 23–34. Phytophthora bisheria gametangia and oospores (sexual stage). 23–29. Oogonia with paragynous antheridia with broad-apical attachment. 28–29. Oogonia containing aplerotic oospores with tapering base. 30–31. Oogonia with multiple paragynous antheridia up to three per oogonium. 32–34. Oogonia with distorted forms of wall projections. Bar = 10 µm.

View larger version (116K): [in this window] [in a new window]   FIGS. 35–40. Phytophthora bisheria clusters of young, mature, and abortive oospores.

Coloniae in agaro lentissime crescentes. Coloniae in CMA 1.6–1.9 mm d^–1, submersae lobis radiantibus. Mycelium ramosum, hyphae primariae 3–7 µm latae. Chlamydosporae ignotae. Sporangiophora simplicia. Sporangia nonpedicellata, persistentia, papillae semi conspicuae. Sporangia ovoidea-obpyriforme 26–44 (medio 34) µm longa, 21–30 (medio 27) µm latae, obpyriforme 42–72 (medio 49) µm longa x 24–36 (medio 30) µm latae, globosa (22.8 µm diam). Zoosporae encystatae 8.6 µm diam. Oogonia sphaerica, plerumque terminalia raro intercalaria, 24–46 (medio 35) µm diam. Antheridia sic diclina, paragyna 7–11 (medio 9.5) µm. Hyphae antheridiferae longae. Oosporae valde apleroticae, 25–31 (medio 28) µm diam, parietibus 4–6 (medio 5) crassis.

Phytophthora bisheria Z.G. Abad, J.A. Abad & F.J. Louws, sp. nov.

Colonies on solid media slow growing, on CMA growth-rate 1.6–1.9 mm d^–1. Colonies on CMA, light rosette pattern concentrated in the center of the colony. Mycelium branched, main hyphae 3–7 µm wide. Chlamydospores and hyphal swellings not observed. Unbranched sporangiophores. Sporangia semipapillate, some bipapillate; ovoid, obpyriform, ovoid-obpyriform, obturbinate, globose or irregular; persistent with lateral attachment to the sporangiophore; ovoid-obpyriform sporangia 26–44 (av. 34) µm long, 21–30 (av. 27) µm wide, obpyriform sporangia 42–72 (av. 49) µm long x 24–36 (av. 30) µm wide, globose on average 22.8 µm diam.

Encysted zoospores 8.6 µm diam. Oogonia spherical 24–46 (av. 35) µm diam, terminal, rarely intercalary. Antheridia paragynous with broad attachment to the oogonial wall 7–11 (av. 9.5) µm, frequently with long stalk and branched. Occasionally two or three antheridia per oogonium are observed. Oospores aplerotic, 25–31 (av. 28) µm diam, wall 4–6 (av. 5) µm thick.

Holotype. – UNITED STATES OF AMERICA, North Carolina, Raleigh, from roots of strawberry (Fragraria x ananassa) in greenhouse, 16 Nov 1999, collector Z.G. Abad. Isolate Cg 2.3.3. BPI 878369 (dried culture on baby carrot agar). Ex-type: CBS 122081, GenBank accession No. AY241924.

Additional representative cultures. – United States of America: Isolate P 1.33 from greenhouses at Raleigh, North Carolina; from strawberry roots (Fragaria x ananassa); collected 16 Nov 1999; collector Gloria Abad. GenBank accession No. AF408625. Australia: Isolate VPRI 21375 from greenhouses at Department of Primary Industries, Knoxfield, Victoria; from Rubus idaeus L. (Rosaceae), isolated from germplasm collection; collected 16 Nov 1999; collector N. Collins. GenBank accession No. DQ298447. The Netherlands: isolate CBS 253.93 (PD 90/444 ex roots of Rosa cv. ‘Spectaculair’) from a greenhouse at Aalsmeer; collected 6 Mar 1990; collector J. de Gruyter, Dutch Plant Protection Service. GenBank accession No. DQ302411. Phytophthora bisheria has never been found in the field in Australia, the Netherlands or USA.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Morphological characterization.— – Colonies of isolates of this organism grew slowly in all culture media. On CMA growth was 1.6–1.9 mm d^–1, and on V8 agar 3.7–4.9 mm d^–1. On CMA, minimum temperature for growth was 10 C, optimum 26 C and maximum 32 C. Colonies on CMA were flat and grew with a uniform pattern at the border of the colony (FIG. 1). Colonies on V8 agar were slightly stellate, on B-CA, B-LBA and PDA30 cottony without pattern. Main hyphae in CMA were 3.0–7.2 µm wide. Chlamydospores and hyphal swellings were not produced in water or in culture media. Moderate amounts of sporangia and oospores were produced in CMA and B-CA and more abundantly in B-LBA. Sporangia were produced abundantly when plugs from the edge of colony in CMA were incubated in water or 10% soil water extract for 24 h. Sporangia were persistent and originated by direct germination of primary sporangia (FIGS. 2–3) or individually in long sporangiophores that occasionally branched under the primary sporangia (FIGS. 4–7). Sporangia frequently were attached laterally to the sporangiophores. Irregular branched coralloid, nodose hyphae were observed frequently (FIGS. 8–9). Typical sporangia under water and 10% soil-water extract were semipapillate with an exit pore of about 7–9 µm (av. 7.2 µm) wide and 2.4 µm deep. Sporangia ranged from ovoid, ovoid-obpyriform, obpyriform, obturbinate to globose or irregular with an elongated neck (FIGS. 10–22). Ovoid-obpyriform sporangia were 26–44 (av. 34) µm long x 21–30 (av. 27) µm wide. Obpyriform sporangia were 42–72 (av. 49) µm long x 24–36 (av. 30) µm wide. Globose sporangia occasionally were produced (av. 22.8 µm). Larger distorted sporangia occasionally were produced and were 72–90 (av. 81) µm long x 26–32 (av. 29) µm wide. Bipapillate sporangia occasionally were produced (FIG. 18). A large vacuole was observed in many sporangia (FIGS. 11–12, 14–15, 19–20).

All isolates were homothallic and abundant oo-spore production was observed on V8 and B-LBA with fewer oospore on CMA and B-CA after 10 d culture in the dark. Isolate Cg 2.3.3 produced more abundant oospores in V8 agar and B-LBA culture media than isolate P 1.33. Oogonia were formed rarely in water cultures in both isolates. Antheridia were predominantly paragynous with broad-apical attachment (FIGS. 23–29). Oospores were aplerotic and 25–31 (av. 28) µm diam with a wall thickness of 4–6 (av. 5) µm. Oogonia, containing aplerotic oospore, with a tapering base, occasionally were observed (ca. 5%) (FIGS. 28–29). Oogonia with paragynous antheridia up to three per oogonium were observed rarely (FIGS. 30–31). Oogonia were smooth-walled, 24–46 (av. 35) µm diam. Oogonia with unusual forms of wall projections were observed rarely (FIGS. 32–34). Some crescent forms of aplerotic oospores were observed (FIGS. 25, 28). The presence of clusters of oospores containing young, mature and abortive oospores is another character of the species (FIGS. 35–40). Oospores in clusters were slightly smaller than individual oospores, 21.6–27.6 (av. 25) µm. ITS sequence analysis demonstrated that the Phytophthora species, isolated from diseased roots of raspberry, rose and strawberry, possessed novel molecular characters (FIG. 41). The full sequences of the ITS rDNA region (ITS1 5.8S ITS2) of four isolates of P. bisheria were determined (TABLE I). In each isolate the ITS rDNA region was identical at 804 bp. Alignment of these ITS sequences with published GenBank accessions and a Phytophthora database of Z.G. Abad confirms that P. bisheria is a solid new species (FIG. 41). Phylogenetic analisis of the ITS rDNA region shows that P. bisheria is in Clade 2 and closely related to, but distinct from, P. multivesiculata Ilieva, Man in ’t Veld, Veenb.-Rijks & Pieters (1998). Both species differ in 61 bp. P. bisheria and P. multivesiculata are also more closely related to species in Clade 2 including P. botryosa, P. citrophthora, P. colocasiae, P. meadii, P. citricola, P. inflata, P.tropicalis, P. capsici, Phytophthora sp. glovera (FIG. 41) than to other members in main cluster of Phytophthora (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 41. Neighbor joining phylogenetic tree based on the ITS1-5.8S-ITS2 region of Phytophthora major Clade 2 (Cooke et al 2000) showing the position of representative isolates of P. bisheria in relation to other known taxa in the clade. Species in Clades 1, 6 and 7 are representatives isolated from strawberry, and raspberry. P. lateralis (Clade 8) is outgroup. Scale bar unit: number of nucleotide substitutions per site.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
A new species was isolated respectively from strawberry, rose and raspberry roots in USA, the Netherlands and Australia and described as Phytophthora bisheria. Phytophthora bisheria is a self-fertile (homothallic) slow growing species that produces semipapillate, persistent sporangia and unique paragynous antheridia that broadly attach to the oogonial walls. Morphological comparison of the isolates of this species with those published for the more than 80 reported taxa showed P. bisheria is fairly distinct. Alignment of ITS sequences of P. bisheria and those from 70 other Phytophthora species from GenBank and putative new species under progress for official description validates the results of morphological comparison and strongly supports the description of P. bisheria as a new species. In the phylogenetic ITS tree, P. bisheria groups in Clade 2 (Cooke et al 2000) that contains semipapillate and papillate species and forms a single clade with its closest relative being P. multivesiculata, isolated from Cymbidium orchid in the Netherlands (Ilieva et al 1998), from which it differs at 61 positions. The main morphological differences between the two species are the amphigynous antheridia, catenulate hyphal swellings and sporangia with internal proliferation produced only by P. multivesiculata. Morphologically P. bisheria shows some similarities to other Clade 2 taxa, the semipapillate P. citricola and P. inflata. However P. bisheria clearly can be discriminated from these taxa on the basis of the structure of the antheridia, oogonia and oospores. The oospores of P. bisheria average 28 µm diam, compared with those of P. citricola, which average 22 µm and P. inflata which average 31.3 µm (Erwin and Ribeiro 1996). Also isolation of P. bisheria from diseased roots of strawberry and roses proved to be difficult whereas P. citricola is not difficult to isolate from different hosts. The main difference from P. inflata is the presence of contorted, inflated, variously lobed or branched paragynous antheridia in this species (Erwin and Ribeiro 1996) compared to the wide antheridia with broad attachment of P. bisheria. Isozyme analysis also confirmed the differences between this new Phytophthora sp. and P. citricola because both species contained several private alleles (Oudemans and Coffey 1991).

The independent findings of P. bisheria as a root pathogen on three different Rosaceous plants suggests that this organism has a preference for Rosaceous hosts because all the cultures found in this study were isolated from plants in the Rosaceae. But without further host range experiments it is not known whether P. bisheria is specific to this family. Regardless of where it originated P. bisheria probably has been spread around the world on nursery material because that is where all the isolates in this study were found. It could be quite widespread but has gone unnoticed because it is a slow growing species and difficult to isolate. The spread of this pathogen by infected plant material is possible. Stock material is grown and delivered by highly specialized growers, and theoretically the pathogen can be spread from such a site with infested plant material. Because it has been isolated only from glasshouse grown plants it might not be aggressive in the field. Now that this species has been described it is likely that further records will be made around the world and the distribution of the pathogen will be elucidated.

In conclusion, by integrating morphological and molecular taxonomy, we confirm that Phytophthora bisheria Z.G. Abad, J.A. Abad & F.J. Louws, isolated from strawberry, rose and raspberry roots in USA, the Netherlands and Australia, is a distinct new Phytophthora species.

	ACKNOWLEDGMENTS

 
The work was supported in part by a grant from the North Carolina Strawberry Growers Association. We are grateful for revision of the manuscript by North Carolina State University scientists David Shew and Mike Benson and for the laboratory assistance of Jennifer Phillips. We thank Dr Rod Jones at the Department of Primary Industries-Knoxfield, Australia, for sequencing isolate VPRI 21375.

	FOOTNOTES

 
Accepted for publication September 21, 2007.

² Former address: Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695.

¹ Corresponding author. E-mail: Gloria.Abad{at}aphis.usda.gov Former address: Plant Pathogen Identification Laboratory, Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
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