Identification of two powdery mildew fungi, Oidium neolycopersici sp. nov. and O. lycopersici,...

684

Levente KISS1*, Roger T. A. COOK2, Gregory S. SAENZ3, James H. CUNNINGTON4, Susumu TAKAMATSU5,

Ian PASCOE6, Marc BARDIN7, Philippe C. NICOT7, Y. SATO8 and Amy Y. ROSSMAN9

" Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, H-1525 Budapest, Hungary.#Central Science Laboratory, Ministry of Agriculture Fisheries and Food, York YO41 1LZ, UK.$Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA.%Department of Applied Biology}Biotechnology, RMIT University, Melbourne 3001, Australia.& Faculty of Bioresources, Mie University, Tsu 514-8507, Japan.' Institute for Horticultural Development, Victoria 3176, Australia.( INRA, UniteU de Pathologie VeUgeU tale, B.P. 94, 84143 Montfavet cedex, France.)College of Technology, Toyama Prefectural University, Toyama 939-0398, Japan.*USDA-ARS, Systematic Botany & Mycology Laboratory, Beltsville, MD 20705, USA.

E-mail : lkiss!nki.hu

Received 15 August 2000 ; accepted 9 January 2001.

A world-wide study of the Oidium species causing economic damage on tomato has identified two taxa using classical

morphological, scanning electron microscope (SEM) and molecular phylogenetic analyses. The material consisted of a total of 25

tomato powdery mildew isolates and 29 herbarium specimens coming from all continents where tomatoes are grown. A taxon with

non-catenate conidia widespread in Europe, Africa, North and South America and Asia was identified as an O. subgen. Pseudoidium

species (teleomorph : Erysiphe sect. Erysiphe). Formerly mistaken for O. lycopersicum (or O. lycopersici), it is now recognised as a distinct

species, O. neolycopersici sp. nov. A phylogenetic analysis of the internal transcribed spacer (ITS) sequences of the ribosomal DNA

(rDNA) indicated that O. neolycopersici is closely related to Erysiphe macleayae, E. aquilegiae and other Pseudoidium species. Only a

taxon with catenate conidia was found on Australian specimens. This was identified as a species of O. subgen. Reticuloidium

(teleomorph : Golovinomyces sp.). Phylogenetic analysis of the rDNA ITS sequences showed that this species is closely related to

O. longipes infecting eggplant. Because it is most likely to be the same species as the original O. lycopersicum, which was actually first

described in Australia, this is here neotypified as O. lycopersici.

INTRODUCTION

An apparently new powdery mildew disease caused by an

Oidium anamorph has appeared recently on tomato (Lyco-

persicon esculentum), on both greenhouse and field crops, and

has spread rapidly around the world (Whipps, Budge & Fenlon

1998, Mieslerova & Lebeda 1999). It is distinct from Leveillula

taurica (Le! v.) Arnaud 1921, the powdery mildew fungus

having an Oidiopsis anamorph and long known to affect

tomato in warmer regions. However, the published data on

the nature of the conidiogenesis of the Oidium species

associated with these new emerging epidemics are often

contradictory making the identification questionable in the

absence of any known teleomorph. Sometimes the conidia

have been described as maturing singly and at other times as

maturing in a chain. The nature of the conidiogenesis is a

stable character within species, yet within Oidium there are

two groups with fundamentally different types of conidio-

* Corresponding author.

genesis. One is a homogeneous group referred to in the

literature as ‘pseudoidium-type ’ where conidia mature one at

a time and the other is a heterogeneous group, ‘euoidium-

type ’, belonging to several genera having in common conidia

which mature in a chain (Blumer 1967, Hammett 1977,

Boesewinkel 1980, Braun 1987, 1995). Thus, in the recent

tomato powdery mildew outbreaks, at least two species of

Oidium were reported sometimes even in the same geo-

graphical region.

A recent comprehensive scanning electron microscope

(SEM) study (Cook, Inman & Billings 1997) has provided a

new tool for the identification of powdery mildew anamorphs

based on the surface patterns of their conidia and septa. It was

subsequently supported by phylogenetic analyses of nuclear

ribosomal DNA (rDNA) internal transcribed spacer (ITS)

sequences in the Erysiphaceae which have provided a large data

set for identification of powdery mildew species (Takamatsu,

Hirata & Sato 1998, Saenz & Taylor 1999, Takamatsu et al.

1999, Mori, Sato & Takamatsu 2000). Therefore, the classical

morphological criteria and host range data used in identi-

Mycol. Res. 105 (6) : 684–697 (June 2001). Printed in the United Kingdom.

Identification of two powdery mildew fungi, Oidiumneolycopersici sp. nov. and O. lycopersici, infecting tomatoin different parts of the world

L. Kiss and others 685

fication (Braun 1987, 1995) can now be supplemented with

both SEM and molecular methods.

This confluence of methods giving mutually supporting

results has triggered a complex revolution in powdery mildew

taxonomy. All ‘pseudoidium ’ anamorphs were shown to

belong to a homogeneous group and are now referred to a

single subgenus of Oidium viz O. subgen. Pseudoidium.

However, ‘euoidium ’ anamorphs are heterogeneous and were

shown to belong to six separate subgenera of Oidium viz O.

subgen. : Oidium, Reticuloidium, Striatoidium, Graciloidium,

Fibroidium, and Setoidium (Cook et al. 1997, Braun et al. 2001).

(Since ‘euoidium ’ is not a homogeneous group, we suggest

replacing this term with ‘catenate Oidium species ’.) These

findings have necessitated a re-structuring of many of the

teleomorphic genera. For instance, Erysiphe is now reserved

for all the teleomorphs of O. subgen. Pseudoidium thus

absorbing the old genera of Microsphaera and Uncinula which

are relegated to sections of Erysiphe. The remaining (chain

forming) members of the old genus Erysiphe have now been

subdivided into Golovinomyces having O. subgen. Reticuloidium

anamorphs, and Neoerysiphe having O. subgen. Striatoidium

anamorphs (Braun 1999, Braun & Takamatsu 2000).

The uncertainty regarding the identity of the causal agents

of tomato powdery mildew poses a major problem for plant

breeders. There is an urgent need to breed resistant tomato

cultivars against the new disease because all cultivars grown

world-wide are susceptible (Burgerjon et al. 1990, Neshev

1993, Lindhout, Pet & van der Beek 1994, Ciccarese et al.

1998, Huang et al. 2000). However, if different powdery

mildew species are involved, different mechanisms of resistance

could complicate the breeding strategy as in the case of certain

melon lines which could be infected by two distinct powdery

mildew species (Epinat, Pitrat & Bertrand 1993). Consequently,

there is a practical need for an exact identification of novel

pathogens for breeding purposes.

Reports suggesting that the pathogen was a catenate

Oidium species came from four continents : Australia (Price

1981, Wicks & Clare 1981) ; Asia : Japan (Abiko 1978) and

India (Kumar et al. 1995) ; Europe : Switzerland (Corbaz 1990,

1993), Bulgaria (Neshev 1993), and Hungary (Kiss 1996) ; and

North America : Canada (Be! langer & Jarvis 1994), and the

USA (Arredondo et al. 1996, Karasevicz & Zitter 1996, White

et al. 1997, Pernezny & Sonoda 1998). The names Erysiphe

cichoracearum DC. 1805 (Abiko 1978, Wicks & Clare 1981,

Corbaz 1990, 1993), Oidium sp. (Price 1981, Neshev 1993)

and Erysiphe sp. (Be! langer & Jarvis 1994, Kiss 1996, Pernezny

& Sonoda 1998) were used for these anamorphs.

The situation is even more complicated with Pseudoidium.

Reports describing this taxon on tomato came from two

continents : Europe : UK (Whipps et al. 1998), Netherlands

(Noordeloos & Loerakker 1989), Germany (Gabler, Gerlach &

Braun 1990), the Czech Republic (Lebeda & Rod 1990),

Greece (Vakalounakis & Papadakis 1992), Hungary (Milotay

& Dormanns-Simon 1997), Switzerland (Bolay 1998) and

Spain (Olalla & Tore! s 1998) ; and North America : USA (Smith,

Douglas & LaMondia 1997) and Canada (Bains, Bennypaul &

Mirza 1999).

Noordeloos & Loerakker (1989) redescribed a Pseudoidium,

infecting tomato in the Dutch glasshouses since the 1980s, as

‘O. lycopersicum Cooke & Massee emend. ’. They based this on

the oldest description of a powdery mildew on tomato

collected in southern Australia (Cooke & Massee 1888),

although this name was previously excluded from the

Erysiphales accepted by Braun (1987) who rightly regarded it

as a nomen ambiguum. Noordeloos & Loerakker (1989) were

able to find and illustrate an interpretable anamorphic mildew

from the holotype, and demonstrate that it was an Oidium

species. Unfortunately, the holotype specimen was in poor

condition and Noordeloos & Loerakker (1989) were unable to

determine the nature of the conidiogenesis of this taxon.

Although they adopted the name O. lycopersicum for the

European tomato powdery mildew fungus, they did not

provide convincing evidence that it was the same species as

the Dutch Pseudoidium anamorph. The holotype of Cooke &

Massee’s O. lycopersicum was lost after this taxonomic study

(I. Russ, pers. comm.) and there is no more original material.

Clearly, use of the name O. lycopersicum is in need of

clarification and neotypification.

Based on specimens from the UK and Czech Republic,

Whipps et al. (1998) and Lebeda & Mieslerova (1999)

suggested that recent outbreaks of powdery mildew on

tomato were caused by a single species of Pseudoidium and

that this anamorph was often misidentified as a catenate

Oidium species because its conidia clung together in ‘pseudo-

chains ’ in high relative humidity. This could explain why the

first reports of the pathogen in the UK (Fletcher et al. 1998)

were not positively identified as either a Pseudoidium or a

catenate Oidium species. The name O. lycopersicum (Whipps et

al. 1998), or its orthographically corrected form, O. lycopersici

(Lebeda & Mieslerova 1999), was recommended for this

species. However, these authors did not compare the

morphology of their local specimens with specimens from

other geographical regions. Thus, a further comprehensive

study is needed to identify the powdery mildews infecting

tomato throughout the world.

The present work combines the use of morphological and

molecular tools to identify the Oidium species infecting

tomato. Its objectives are to : (1) compare the morphological

characteristics of Oidium tomato pathogens from all over the

world, using light microscopy and SEM of both fresh

materials and herbarium specimens ; (2) determine and analyze

the rDNA ITS sequences of these specimens ; (3) study the

mating type of different isolates in vitro ; and (4) to solve the

nomenclatural problems associated with tomato powdery

mildew anamorphs.

MATERIALS AND METHODS

Powdery mildew materials

Twenty-five Oidium tomato powdery mildew isolates were

examined as fresh material (Table 1). The European, African

and Caribbean isolates have been maintained for years in

different laboratories at INRA, PPI and WAU, respectively

(Table 1). Isolates maintained at INRA were kept in Petri

dishes on surface disinfected cotyledons of Lagenaria ciceracia

cv. Minibottle on mannitol sucrose (MS) agar (mannitol,

20 g l−", sucrose, 10 g l−", agar, 7 g l−", tetracycline hydro-

Powdery mildew fungi on tomato 686

Table 1. Designation, country of origin, year of collection, source and morphological characteristics of Oidium species studied on fresh tomato leaves.

Designationa Country of origin

Year of

collection

Conidiophore

typeb

Conidia

Sourced

Length range

(and mean)

Width range

(and mean)

Germ tube

apexc

BP-P1 Hungary 1994 P 24–(33±8)–40 12–(15±2)–18 l PPI




BP-P5 (BPI 746455) Hungary 1998 P 26–(34±5)–42 13–(15±5)–18 l PPI

Et-1 (BPI 747013) France 1989 P 22–(33±5)–46 12–(14±8)–20 l INRA

Et-11 France 1990 P 26–(33±4)–42 12–(14±4)–18 l INRA

Et-21 Tanzania 1993 P 24–(33±9)–46 12–(14±9)–20 l INRA

Et-23 France 1994 P 26–(34±2)–46 12–(15±2)–20 l INRA

Et-24 France 1996 P 24–(34±4)–46 12–(15±2)–20 l INRA

Et-25 Guadeloupe 1997 P 24–(33±8)–44 12–(14±6)–18 l INRA

PV (BPI 746227) Netherlands 1999 P 24–(34±7)–44 12–(13±8)–18 l WAU

RZ Netherlands 1999 P 25–(33±8)–40 12–(13±5)–18 l WAU

DR Netherlands 1999 P 26–(34±4)–40 12–(14±6)–20 l WAU

EN Netherlands 1999 P 24–(34±8)–42 12–(13±6)–18 l WAU

JAP-1 (MUMH66) Japan 1995 P 28–(35±6)–44 13–(14±5)–20 l TPU

JAP-2 Japan 1996 P 26–(34±4)–42 12–(14±2)–18 l TPU

CZ Czech Rep. 1998 P 24–(31±6)–36 10–(13±0)–16 l WAU

UK-1 UK 1999 P 26–(35±2)–44 12–(14±8)–20 l CSL

LI (BPI 746228) NY, USA 1999 P 28–(36±2)–44 13–(14±8)–18 l CU

ITH (BPI 746229) NY, USA 1999 P 30–(36±3)–42 16–(17±8)–22 l CU

GEN (BPI 746231) NY, USA 1999 P 26–(34±8)–42 14–(16±2)–20 l CU

CAN (BPI 746230) Ontario, Canada 1999 P 28–(34±3)–38 12–(14±2)–16 l AC

MD-1 (BPI 746232) MD, USA 1999 P 26–(32±2)–38 12–(13±8)–16 l USDA

MD-2 MD, USA 1999 P 28–(33±4)–40 14–(16±0)–18 l USDA

a Accession numbers were given in parentheses for isolates deposited as herbarium specimens ; BPI¯US National Fungus Collection, Beltsville, MD;

MUMH¯Mie University Mycological Herbarium, Japan.b P¯ conidium formed singly and characteristic of Oidium subgen. Pseudoidium.c l¯ germ tube apex simple or lobed.d AC¯Agriculture and Agri-food Canada, Greenhouse and Processing Crop Research Centre, Harrow, Ontario, Canada ; CSL¯Central Science

Laboratory, York, UK; CU¯Cornell University, Department of Plant Pathology, NY; INRA¯ INRA, Unite! de Pathologie Ve! ge! tale, Montfavet, France ;

PPI¯ Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary ; TPU¯College of Technology, Toyama Prefectural University, Japan ;

USDA¯USDA Systematic Botany & Mycology Laboratory, Beltsville, MD; WAU¯Wageningen Agricultural University, Department of Plant Breeding,

The Netherlands.

chloride, 25 mg l−") as described by Bardin et al. (1997) for

Podosphaera [Sphaerotheca] fuliginea (Schlecht. : Fr.) U. Braun &

S. Takamatsu 2000. They were transferred to fresh cotyledons

every 4 weeks. Isolates maintained at PPI and WAU were kept

on isolated tomato plants in the greenhouse and transferred to

healthy plants when necessary. All the North American

isolates were collected in commercial greenhouses in 1999 and

maintained on isolated tomato plants or detached tomato

leaves in Petri dishes for only 2 months at USDA SBML.

Twenty-nine herbarium specimens originating from 12

countries spanning five continents were also studied (Table 2).

In order to minimize the risk of spread of any powdery

mildew during this study, only herbarium specimens were

sent from one continent to another for examination. When

living isolates were sent under licence from one European

country to another, they were kept in complete isolation and

destroyed after examination. A total of three tomato powdery

mildew specimens were used for polymerase chain reaction

(PCR) amplifications and DNA sequencing (Table 3). Ad-

ditionally, four powdery mildew specimens infecting eggplant,

tobacco and zucchini were also sequenced (Table 3).

Light microscopy of fresh and herbarium material

Hyphae, conidiophores and conidia of fresh materials were

stripped off the leaf surfaces with clear adhesive tape,

mounted on a microscope slide with the fungal mycelium

uppermost and examined either dry, or in water, or in cotton

blue in lactophenol or in 2% potassium hydroxide using light

microscopy with or without Nomarski interference contrast

optics. Herbarium materials were rehydrated before exam-

ination by boiling a small piece of infected leaf, with the fungal

mycelium downwards, in a drop of lactic acid on a slide as

described by Shin (1988) and Shin & La (1993). After boiling,

the rehydrated mycelium was scraped off the leaf and

mounted either in lactic acid or in cotton blue in lactic acid for

light microscopy using Nomarski interference contrast optics.

The following information was noted during the examination

of both fresh and herbarium specimens : size and shape of

conidia, presence or absence of fibrosin bodies in fresh

materials, nature of conidiogenesis, characteristics of the

conidiophore, e.g. size and shape of foot cell, position of the

basal septum, shape and position of hyphal appressoria,


Table 2. Designation, country of origin, year of collection and morphological characteristics of Oidium species on tomato leaves from herbaria.

Morphological studies were done on rehydrated specimens.

Designationa Country of origin

Year of

collection

Conidiophore

typeb

Conidia

Hyphal

appressoriad

Length range

(and mean)

(µm)

Width range

(and mean)

(µm)

Germ

tube

apexc

Oidium sp.

BPI 746225 Switzerland 1995 P 26–(31±2)–38 12–(14±4)–16 l l

BPI 746226 Switzerland 1995 P 28–(32±2)–36 12–(14±6)–16 l l

BPI 746456 CT, USA 1998 P 28–(33±4)–40 14–(16±2)–18 l l

BPI 746457 CT, USA 1998 P 26–(33±8)–42 14–(16±3)–18 l l

BPI 746433 CA, USA 1999 P 25–(32±6)–38 12–(15±4)–18 l l

IMI 24738 Malaysia 1947 P 26–(31±5)–36 12–(12±6)–16 l l

IMI 37809 Malaysia 1949 P 28–(31±3)–36 12–(12±3)–16 l l

IMI 79299a North Borneo 1959 P 28–(30±4)–34 12–(13±8)–15 l l

IMI 106600 Hong Kong 1964 P 22–(31±2)–38 10–(12±5)–14 l l

IMI 114186 Nepal 1965 P 26–(34±2)–38 14–(15±5)–16 l l

IMI 119565 Hong Kong 1966 P 28–(32±2)–36 12–(13±2)–16 l l

IMI 126385 Nepal 1966 P 24–(29±4)–36 12–(12±4)–14 – l

IMI 130346 Nepal 1967 P 24–(30±2)–36 12–(12±4)–15 l l

IMI 184157 Hong Kong 1974 P 22–(30±9)–37 12–(12±3)–14 l

IMI 206541 India 1976 P 26–(30±6)–37 10–(12±5)–14 – l

IMI 266709 Thailand 1981 P 28–(33±2)–42 12–(14±1)–18 l l

IMI 274829 India 1983 P 26–(33±5)–40 12–(14±2)–16 l l

IMI 316606 India 1987 P 28–(33±9)–40 10–(13±8)–16 – l

VPRI 19847 Victoria, Australia 1994 R 22–(26±4)–32 12–(14±5)–17 s n

DAR 35762 Victoria, Australia 1980 R 24–(26±4)–32 13–(14±2)–15 s n



DAR 70008 Tasmania, Australia 1994 R 22–(25±8)–30 12–(13±5)–16 s n

DAR 71625 South Australia, Australia 1996 R 23–(27±1)–32 12–(14±6)–17 s n

Erysiphe orontii e

IMI 335048 UK 1989 P 22–(30±3)–36 11–(12±3)–14 l l

IMI 358049 Hong Kong 1993 P 28–(33±8)–38 13–(13±9)–15 l l

IMI 361479 Venezuela 1993 P 26–(29±5)–36 12–(12±7)–14 l l

E. polyphaga e

IMI 291441 Bhutan 1984 P 26–(28±7)–35 12–(12±4)–16 l l

IMI 313145 UK 1987 P 24–(31±3)–36 12–(12±6)–15 l l

a The names of the specimens listed here were assigned by their suppliers ; BPI¯US National Fungus Collection, Beltsville, MD; IMI¯CABI Bioscience,

UK Centre (Egham), Surrey, UK; DAR¯ Plant Pathology Herbarium, Orange Agricultural Institute, Australia ; VPRI¯ Plant Disease Herbarium, Institute for

Horticultural Development, Victoria, Australia.b P¯ conidia formed singly and characteristic of Oidium subgen. Pseudoidium ; R¯ conidia maturing in a chain and characteristic of O. subgen. Reticuloidium.c l¯ germ tube apex simple or lobed ; s¯ germ tube apex simple ; –¯ not determined in the absence of germinating conidia.d l¯ appressoria nipple-shaped to lobed or multilobed ; n¯ appressoria nipple-shaped.e This species is now referred to Golovinomyces orontii (Braun 1999).

position of germ tubes of conidia, when found, and shape of

appressoria on germ tubes of conidia ; 25 conidia were

measured for each specimen examined.

Scanning electron microscopy (SEM)

Surface patterns of conidia in specimens selected after the

examination of all the materials under the light microscope

were studied under the SEM in the UK (at Central Science

Laboratory, MAFF). Specimens were prepared for SEM as

described by Cook et al. (1997). Two fresh isolates maintained

in the laboratory (BP-P4 and UK-1) and five herbarium

specimens (DAR 35762, DAR 35763, DAR 35764, DAR

70008 and DAR 71625) were used in this study. For the fresh

isolates, a piece of leaf lamina ca 0±5 cm square was excised,

mounted on a cryo stub with Tissue Tek and gold coated with

cryoscopy in a Philips XL20 SEM fitted with a lanthium

hexaboride head and a cryo-sputter chamber. Observations

were done at relatively low kV and a magnification up to

¬12000. In order to reduce the risk of spread of the catenate

Oidium species on tomato, found only in Australia during this

study, only herbarium specimens were sent to the UK for

examination. This dried material did not need to be frozen. It

was simply mounted with double sided clear adhesive tape on

an aluminium stub and gold coated without cryoscopy before

transfer to the SEM. This had the advantage of a simpler

technique unaffected by ice crystals and with a reduced risk of

ionisation, the disadvantage being the fact that the primary

patterns were inevitably partially obscured by superimposed

wrinkling patterns. Nevertheless, the wrinkling patterns are

also very useful for identification (Cook et al. 1997).

DNA extraction, PCR amplification and DNA sequencing

Isolation of DNA from fresh fungal specimens was performed

using the 5% Chelex method (Walsh, Metzger & Higuchi


Table 3. Designations, host plants, countries of origin and database accession numbers of the powdery mildew rDNA ITS sequences used in this study.

Fungus Host plant Designation and source Country of origin Database accession number

Arthrocladiella mougeotii Lycium chinense MUMH135a Japan AB022380

Erysiphe aquilegiae var. ranunculi Cimifuga simplex TPU495a Japan AB015929

E. convolvuli Convolvulus arvensis UC1512307a UK AF011298

E. cruciferarum Arabidopsis thaliana UEA1b UK AF031283

E. macleayae Macleaya cordata TPU1873a Japan AB016048

E. polygoni Polygonum arenastrum UC1512295a USA AF011307

E. weigelae Weigela hortensis TPU1669a Japan AB015931

E. blasti e Lindera umbellata MUMH2a Japan AB015918

E. juglandis e Pterocarpa rhoifolia TPU1745a Japan AB015928

E. platani e Platanus racemosa UC1512309a USA AF011311

E. pulchra var. japonica e Cornus controversa MUMH90a Japan AB015924

Golovinomyces biocellatus f Monarda fistulosa DNAc USA AF011291

G. cichoracearum f,g Arabidopsis thaliana UCSC1a USA AF031282

G. cichoracearum d,f,g Cucurbita pepo Ecr-3b France AF229017

G. cichoracearum d,f,g C. pepo Ecr-1b France F229016

G. cichoracearum f Eupatorium chinense MUMH37a Japan AB000934

G. magnicellulatus f Phlox paniculata DNAc USA AF011303

G. orontii f Agastache pallida var. pallida UC1512314a USA AF011304

G. orontii f Arabidopsis thaliana MGHb USA AF009176

G. orontii f Delphinium belladonna UC1512293a USA AF011305

G. orontii d,f Nicotiana tabacum BP-1TOBb Hungary AF229013

G. orontii f N. tabacum L.I.b Japan AB022413

G. sordidus f Plantago major DNAc USA AF011309

Oidium longipes d Solanum melongena VPRI 22141a Switzerland AF250777

O. lycopersici d Lycopersicon esculentum VPRI 19847a Australia AF229021

O. neolycopersici d L. esculentum Et-1b France AF229019

O. neolycopersici d L. esculentum VPRI 20724a The Netherlands AF229015

a Herbarium specimens at the following Herbaria : MUMH¯Mie University Mycological Herbarium, Japan ; TPU¯Toyama Prefectural University

Herbarium, Japan ; UC¯University of California Herbarium, CA; VPRI¯ Plant Disease Herbarium, Institute for Horticultural Development, Victoria,

Australia.b Isolate maintained on living plants or detached leaves.c DNA extracted from fresh material, no voucher specimen deposited.d New ITS sequence obtained for this study.e Genus formerly known as Microsphaera.f Genus formerly known as Erysiphe.g These isolates cannot belong to G. cichoracearum, because this species is resticted to the host family Compositae (Braun 1987). Therefore, they probably

belong to G. orontii.

1991) with modifications as described in Saenz & Taylor

(1999). This extract was autoclaved for 20 min, and centrifuged

for 10 min at 15000 g. Serial dilutions of 1}10 and 1}100 of

the supernatant were then used as template DNA for

polymerase chain reaction (PCR) amplifications. DNA from

herbarium specimens was isolated by placing scrapings of

mycelium and conidiophores into a 5% Chelex solution

containing 0±001% Triton X100, and incubating in a water

bath for 30 min at 94 °C. A 1 µl aliquot from this mixture was

used as the template.

For DNA from fresh specimens, PCR amplifications utilised

the ITS5 and ITS4 primers pairs (White et al. 1990). The

amplified fragment spanned the entire ITS region. Each

reaction was subjected to 40 thermal cycles with the following

parameters : denaturation at 94° for 1 min, annealing at 53°for 1 min, extension at 72° for 1 min. The ITS region from the

herbarium specimens was amplified using the following two

primers, PMITS1 (5-TCGGACTGGCCTCAGGGAGA-3) and

PMITS2 (5-TCACTCGCCGTTACTGAGGT-3). These pri-

mers have been shown to have some specificity for the ITS

region of powdery mildews (Cunnington & Takamatsu,

unpubl.). In these reactions, 6% Tween 20 was used as a PCR

enhancer (Demeke & Adams 1992) and the thermal cycling

times were 1 min at 94°, 1 min 65° and 2 min 72°, for 35

cycles.

Amplified products were electrophoresed on a 2% agarose

gel and then cleaned using QIAquick spin columns (Qiagen,

Chatsworth, CA). DNA was resuspended into about 30 µl of

sterile water. Ethidium bromide-stained DNA PCR products

were visually compared to standards in order to optimise the

concentration for cycle sequencing. Some specimens were

cycle sequenced using an ABI Prism BigDye Terminator Cycle

Sequencing Kit (Perkin–Elmer, Foster City, CA) and were read

by the DNA Sequencing Facility, Monash University, Victoria,

Australia. Other PCR purified amplicons were sent to the

DNA Sequencing Facility, Center for Agricultural Biotech-

nology, at the University of Maryland, for cycle sequencing,

polyacrylamide gel electrophoresis, and data collection.

Primers ITS1, ITS2, ITS3, ITS4, and ITS5, were used to

sequence the ITS region (White et al. 1990).

Data analysis

Two data sets were created for phylogenetic analysis, one

consisting of the Pseudoidium species and the other consisting

of the catenate Oidium species. The powdery mildews used in


this study are listed in Table 3. Newly obtained sequences

were combined with selected sequences from Saenz & Taylor

(1999), Takamatsu et al. (1999), and Mori et al. (2000). The

alignments are available upon request (gsaenz!unm.edu). All

gaps were included in the analysis. The data sets were

analysed using the parsimony and neighbour-joining methods

found in PAUP* (version 4±0b3a ; Swofford 1999). Parsimony

analysis was conducted using a heuristic search using the Tree

Bisection Reconnection (TBR) branch swapping option and

employing the random stepwise addition of sequences option

beginning with a random seed number and 100 replications.

MaxTrees was set to auto-increase, saving all optimal trees

and swapping on all trees. Distance analysis was performed

using the neighbour-joining method. Pairwise distances

between taxa was determined by using both the Kimura two-

parameter (K2P ; Kimura 1980) model for nucleotide substi-

tutions, and the Hasegawa-Kishino-Yano (HKY85; Hasegawa

et al. 1985) model for nucleotide substitutions and unequal

base frequencies. Branch support was determined by bootstrap

analysis (Felsenstein 1985) using 1000 replications. Decay

indices or Bremer support (Bremer 1988) was determined by

Autodecay version 4.0 (Eriksson 1998). Maximum likelihood

analyses were conducted using Phylip 3.572 (Felsenstein

1993). The transition}transversion (ts}tv) ratios for each data

set were estimated using PAUP*. These ts}tv ratio numbers

were then used in the Phylip maximum likelihood (DNAML)

analyses. The DNAML settings included searching for the

best tree, using global rearrangements, randomising the input

order, and jumbling ten times.

Crossing different isolates in vitro

Six different monoconidial tomato powdery mildew isolates

(Et1, Et11, Et22, Et24, Et25, BP-P1), maintained in vitro at

INRA (Table 1), were paired with each other in all possible

combinations on surface disinfested Lagenaria ciceracia

cotyledons on MS agar as described in Bardin et al. (1997) for

cucumber powdery mildew isolates. Cotyledons were ex-

amined 3–4 weeks after inoculation for cleistothecia pro-

duction.

RESULTS

Microscopic observations

Microscopic examination of tomato powdery mildew patho-

gens clearly showed that all the European, North American,

African and Caribbean isolates, maintained alive in different

laboratories, and also all the herbarium specimens obtained

from Europe, North and South America and Asia were

anamorphs of Pseudoidium because conidia always matured

singly on the conidiophores (Fig. 1). Surprisingly, all the

herbarium specimens obtained from Australia represented

another anamorph where conidia clearly matured in chains

and thus belonged to catenate Oidium species (Fig. 2). Striking

differences were also observed in the shape and size of both

conidia and germ tubes of conidia, structure of the

conidiophores and shape of hyphal appressoria of these two

tomato powdery mildew anamorphs (Tables 1–2).

1 2

3 4

Figs 1–4. Light microscope micrographs of Oidium spp. removed

from herbarium specimens of tomato after rehydration in lactic acid.

Fig. 1. Conidiophore of an Oidium species (O. subgen. Pseudoidium)

here re-described as O. neolycopersici, collected in Malaysia in 1947

(IMI 24738). Bar¯ 30 µm. Fig. 2. Conidiophores of a catenate

Oidium species (O. subgen. Reticuloidium), here neotypified as

O. lycopersici, collected in South Australia in 1980 (DAR 35763).

Bar¯ 40 µm. Fig. 3. Germinating conidium of O. neolycopersici from

1964 (IMI 106600). Bar¯ 10 µm. Fig. 4. Germinating conidia of

O. lycopersici from 1994 (DAR 70008). Bar¯ 30 µm.

All the anamorphs of Pseudoidium studied were characterised

by ellipsoid-ovoid or doliform conidia with mean conidial

lengths generally greater than 30 µm and germ tubes

terminating in lobed, or rarely simple, apices (Fig. 3). In

contrast, conidia of all the Australian catenate Oidium species

were smaller, with mean conidial lengths less than 30 µm.

These conidia were cylindrical, with one, or sometimes two,

germ tubes terminating in simple apices (Fig. 4). Conidial

lengths and widths for all the specimens are shown in Tables

1–2.

Differences were also observed in the structure and

dimensions of the conidiophores. First, in the catenate species

the basal septum of the foot cells was always displaced at a

distance of 7–27 µm from the point of branching. In

Pseudoidium species, such displacement of the basal septa was

not observed. Second, the total length of the conidiophores in

Pseudoidium species measured 58–115 µm (mean : 91±5 µm).

They consisted of 2–3 cells attached to a 29–74 µm (mean :

42±4 µm) long foot-cell that was either cylindrical or sometimes

inflated in the middle and constricted at the base. Conidia

were always produced singly, but sometimes remained

attached to each other in high relative humidity forming

‘pseudo-chains ’ of 2–6 conidia. In catenate anamorphs, the


5 6

87

Figs 5–8. Scanning electron micrographs of herbarium specimens of O. (subgen. Reticuloidium) lycopersici, from Australia. Bars¯ 5 µm.

Fig. 5. Conidia in a chain. Fig. 6. Fibrillar pattern on end wall of conidium. Fig. 7. Conidium with a polygonal, reticulate pattern of

wrinkling of outer wall. Fig. 8. Simple, unlobed appressoria (indicated by arrows) on hypha.

total length of the conidiophores was greater, 150–210 µm

(mean : 185±7 µm), measured from the point of branching, with

a long, 100–145 µm (mean : 124±4 µm), foot-cell followed by

2–4 shorter cells. Conidia were produced in real chains

consisting of 3–7 conidium initials and mature conidia.

Hyphal appressoria were also different. In the Pseudoidium

anamorphs, they were as expected, simple to lobed or even

multi-lobed, opposite or spread along the hyphae. They

contrasted with the inconspicuous or nipple-shaped appres-

soria situated singly or in sequence in the Australian catenate

anamorphs.

Scanning electron microscopy

Cook et al. (1997) have shown that herbarium specimens,

though not as informative as fresh material, can reveal useful

information under the SEM. For the Australian herbarium

material, the SEM study revealed long conidial chains (Fig. 5)

whose septa showed a fibrillar pattern (Fig. 6) and whose

outer walls bore polygonal}reticulate wrinkling patterns (Fig.

7). Simple, inconspicuous unlobed appressoria were found

with difficulty on the hyphae (Fig. 8). These features are

diagnostic for one of the catenate Oidium species described as

O. subgen. Reticuloidium (Cook et al. 1997).

For the Pseudoidium species, two fresh isolates, one from

Hungary, BP-P4, and the other from England, UK-1, were

examined under the SEM. In contrast to the Australian

pathogens, the appressoria were conspicuous and moderately

lobed (Fig. 9). A close examination of the nature of the

conidiogenesis confirmed that conidia matured singly in these

fungi (Fig. 10), as also revealed by light microscopy (Fig. 1).

However, conidia produced singly sometimes remained

attached to each other forming pseudo-chains consisting of

2–3 (Fig. 11), rarely 6 or more conidia. This was also observed

under the light microscope in all the fresh materials studied.

The outer walls of turgid conidia were smooth to somewhat

scaly (Fig. 12), and the septa had a fibrillar pattern (Fig. 13).

When wrinkled, outer walls developed an angular}rectangular

pattern (Fig. 14). Apart from a somewhat smoother outer wall

than usual, this set of patterns is sufficiently diagnostic for O.

subgen. Pseudoidium (Cook et al. 1997, Braun 1999), now the

sole anamorph for the redescribed holomorph genus Erysiphe

sensu stricto (Braun et al. 2001, Braun & Takamatsu 2000).

Thus, the SEM study confirmed that the non-catenate and

catenate anamorphs seen under the light microscope clearly

represent two different Oidium species which can infect

tomato. All the European, Asian, American and African

specimens studied fell into the anamorph genus O. subgen.

Pseudoidium, whereas all the Australian specimens studied fell

into the genus O. subgen. Reticuloidium (a more precise taxon

than the heterogeneous ‘euoidium ’).

Molecular phylogenetic studies

A total of three rDNA ITS sequences of powdery mildew

anamorphs infecting tomato were determined. The data of the


9 10

1112

1413

Figs 9–14. Scanning electron micrographs of fresh specimens of O. (subgen. Pseudoidium) neolycopersici from Europe. Bars¯ 5 µm. Fig.

9. Lobed appressorium on hypha. Fig. 10. Conidium attached to a conidiophore. Fig. 11. Conidia in pseudochains. Fig. 12. Turgid

conidium with scaly outer wall. Fig. 13. Fibrillar pattern on end wall of conidium. Fig. 14. Conidium with an angular, rectangular

pattern of wrinkling on outer wall.

fungi sequenced for this study and their accession numbers are

listed in Table 3. These sequences were initially analysed with

ITS sequences from the studies of Takamatsu et al. (1998,

1999), Saenz & Taylor (1999), and Mori et al. (2000). After a

preliminary analysis (data not shown), powdery mildew taxa

showing close relationships with the tomato powdery mildew

anamorphs sequenced were selected for further, more detailed

analyses.

Oidium subgenus Pseudoidium analysis

The Pseudoidium species data set included twelve taxa, two

of which were tomato pathogens. The data set consisted of

695 alignable characters, 73 characters were variable and

57 characters were phylogenetically informative. All three

methods of analyses, parsimony, neighbour-joining, and

maximum likelihood, yielded trees with very similar topo-

logies. We chose Erysiphe [sect. Microsphaera] juglandis as an

outgroup for all analyses, based upon the results of Takamatsu

et al. (1999). Each method yielded trees with two clades, the

E. aquilegiae clade and the E. [sect. Microsphaera] platani clade,

while each method differed on the branching order of three

taxa : E. [sect. Microsphaera] pulchra, E. [sect. Microsphaera]

blasti, and E. weigelae. The parsimony search yielded three


Erysiphe polygoni ex Polygonum, United States

1

E. convolvuli ex Convolvulus, United States

E. cruciferarum ex Arabidopsis, United Kingdom

Erysiphe* platani ex Platanus, United States

E. weigelae ex Weigela, Japan

E. aquilegiae ex Cimifuga, Japan

E. macleayae ex Macleaya, Japan

Oidium neolycopersici ex Lycopersicon, France

O. neolycopersici ex Lycopersicon, The Netherlands

E.* blasti ex Lindera, Japan

E.* pulchra ex Cornus, Japan

E.* juglandis ex Pterocarpa, Japan

5 NucleotideSubstitutions

2

479

2

69

2

8

100

286

1

Fig. 15. Phylogenetic analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA gene for 12 Pseudoidium anamorphs.

The tree is one of three most parsimonious trees, which was found using a heuristic search employing the random stepwise addition

option of PAUP* phylogenetic analysis (Swofford 1999). The tree also has the highest likelihood of the three most parsimonious trees.

Percentages of parsimony analysis of 1000 bootstrapped data sets supporting specific branches are given below the branches. Branches

& 95% are strongly supported and are shown in boldface (Felsenstein 1985). Bootstrap values below 50% are not shown. Decay

indices or Bremer support (Bremer 1988) is shown above the branches. Erysiphe *juglandis was used to root the tree. Powdery mildews

on tomato are shown in boldface. The numbers adjacent to the right of the tree correspond to clades discussed in the text : 1¯ the E.

platani clade, 2¯ the Erysiphe aquilegiae clade. Taxa marked with *, indicate genera formerly known as Microsphaera.

equally most parsimonious trees (MPT). The trees were 200

steps in length with a consistency index (CI) of 0±770, a

retention index (RI) of 0±620, and a rescaled consistency index

(RC) of 0±477 (Farris 1989). The three trees were the result of

the inclusive branching order of E. polygoni and E. blasti. Two

supported clades resulted from the bootstrap analysis, the

E. aquilegiae clade was strongly supported (100%), and the

E. platani clade received minor support (69%) (Fig. 15). The E.

aquilegiae clade (Fig. 15) consisted of E. aquilegiae, E. macleayae,

and the two O. subgen. Pseudoidium tomato powdery mildews,

one specimen from France and the other from Netherlands.

The two Pseudoidium isolates from tomato were well supported

(86%) as sister taxa, and were nested within the E. aquilegiae

clade. The E. platani clade consisted of E. platani, E. cruciferarum,

E. convolvuli, and E. polygoni. Decay analysis, or Bremer

support, gave strong support to the E. aquilegiae clade (8),

while the E. platani clade received minor support (2). The clade

containing the two Pseudoidium isolates from tomato received

minor support (2). Both neighbour-joining analyses, utilising

either the K2P or the HKY85 models of nucleotide

substitutions, yielded identical trees (trees not shown).

Bootstrap support indices for each neighbour-joining analysis

were also nearly identical, and the overall support for the

E. platani clade and the E. aquilegiae clade, including internal

branches, increased as compared to the parsimony bootstrap

indices. The E. aquilegiae clade was again strongly supported

(100%), while the support for the E. platani clade increased

from 69% (parsimony) to 87%. The two Pseudoidium isolates

from tomato were again well supported (87%) as sister taxa,

while E. macleayae was strongly supported (99%) as a close

relative. The maximum likelihood analysis yielded a tree very

similar to the parsimony and neighbour joining analyses, in

that both the E. aquilegiae and E. platani clades appeared.

Catenate Oidium (O. subgenus Reticuloidium) analysis

The catenate Oidium data set contained fifteen taxa, including

a tomato powdery mildew specimen from Australia. The data


Oidium longipes ex Solanum “eggplant”, Switzerland 1

Golovinomyces* magnicellulatus ex Phlox, United States

Arthrocladiella mougeotii ex Lycium, Japan

G.* sordidus ex Plantago, United States

5 Nucleotide changes per1000 positions

2

3

O. lycopersici ex Lycopersicon tomato, Australia

G.* orontii ex Delphinium, United States

G.* orontii ex Agastache, United States

G.* orontii ex Nicotiana tobacco, Hungary

G.* cichoracearum ex Arabidopsis, United States

G.* orontii ex Nicotiana tobacco, Japan

G.* cichoracearum ex Cucurbita “zucchini”, France

G.* orontii ex Arabidopsis, United States

G.* cichoracearum ex Cucurbita “zucchini”, France

G.* cichoracearum ex Eupatorium, Japan

84

54

100

100

100

86

100

53

96

65

94

63

G.* biocellatus ex Monarda, United States

Fig. 16. Neighbour joining analysis of the ITS region of the ribosomal RNA gene for 15 catenate Oidium species belonging to

subgenus Reticuloidium. The neighbour joining tree was found using the Kimura 2-parameter model for nucleotide substitutions. Branch

lengths correspond to pairwise distances between taxa. Branch support was determined by 1000 bootstrapped data sets, shown above

the branches. Branches¯ 95% are shown in bold. Arthrocladiella mougeotii was used to root the tree. Powdery mildews on tomato are

shown in boldface. The numbers adjacent to the right of the tree correspond to clades discussed in the text : 1¯ the Golovinomyces*

magnicellulatus clade, 2¯ the G.* biocellatus clade, 3¯ the G.* orontii clade. Taxa marked with *, indicate genera formerly known as

Erysiphe. Note that the three sequences labelled G. cichoracearum in clade 3 are more likely to have come from isolates of G. orontii (see

Table 3).

set consisted of 629 alignable characters, 42 characters were

variable and 59 characters were phylogenetically informative.

All three methods of analyses, parsimony, neighbor-joining,

and maximum likelihood, yielded trees with nearly identical

topologies. We chose Arthrocladiella mougeotii (anamorph :

O. subgen. Graciloidium) as an outgroup for all analyses, based

upon the results of Mori et al. (2000). Each method yielded

trees with three clades (containing anamorphs of O. subgen.

Reticuloidium species) : the Golovinomyces magnicellulatus clade

(note that Golovinomyces was formally referred to as Erysiphe),

the G. biocellatus clade, and the G. orontii clade. The G.

magnicellulatus clade consisted of G. magnicellulatus, and two

solanaceous Oidium species : O. longipes from eggplant in

Switzerland, and O. lycopersici from tomato in Australia. The

G. biocellatus clade consisted of G. biocellatus and two G. orontii

isolates. The G. orontii clade consisted of three G. orontii

isolates, three G. cichoracearum isolates, believed to be G.

orontii also, and a G. sordidus isolate. The parsimony search

yielded 18 equally most parsimonious trees (MPT). The trees

were 147 steps in length with a consistency index (CI) of

0±816, a retention index (RI) of 0±827, and a rescaled

consistency index (RC) of 0±675. The 18 trees were the result

of the inclusive branching order of the seven taxa in the G.

orontii clade (trees not shown). Three supported clades

resulted from the bootstrap analysis, the G. magnicellulatus

clade received moderate support (78%). The two solanaceous

Oidium species within the G. magnicellulatus clade were

strongly supported as sister taxa (100%). The G. biocellatus

clade was strongly supported (100%). The G. orontii clade

was well supported (94%). However, the branching order of

these closely related powdery mildews was inclusive. Both

neighbour-joining analyses, utilising either the K2P or the

HKY85 models of nucleotide substitutions, yielded identical

trees (Fig. 16). Bootstrap support indices for each neighbour-

joining analysis were also nearly identical, and the overall

support for the three clades, including internal branches,


increased as compared to the parsimony bootstrap indices.

Using the K2P distance model, the bootstrap support for the

G. magnicellulatus clade increased from 77% (parsimony) to

84%, and again the two solanaceous Oidium species were

strongly supported (100%) as sister taxa. The G. biocellatus

clade was again strongly supported (100%). The support for

the G. orontii clade increased from 93% (parsimony) to 100%,

with the branching order in the K2P tree nearly duplicating

the branching order in the MPT with the highest likelihood.

The maximum likelihood analysis yielded a tree with topology

nearly identical to the parsimony and neighbour joining

analyses (tree not shown).

Crossing different isolates in vitro

No cleistothecia or cleistothecium initials were produced in

any pairs of the Pseudoidium isolates crossed in vitro.

Cleistothecia have never been reported for either the

O. subgen. Pseudoidium anamorphs infecting tomato (Whipps

et al. 1998) or O. subgen. Reticuloidium (Price 1981, Wicks &

Clare 1981).

DISCUSSION

To the best of our knowledge, this study includes all the

known tomato powdery mildew material, available either as

herbarium specimens deposited in international collections or

as living isolates maintained on tomato in different labora-

tories. Their identity was investigated using morphological

characteristics, SEM conidial surface patterns and molecular

analysis. The results show that, in spite of the many reports

of catenate Oidium species being involved, all the recent

outbreaks of tomato powdery mildew reported from outside

Australia were caused by a single anamorph belonging to

Oidium subgen. Pseudoidium. In contrast, the causal agent of

the Australian tomato powdery mildew was always an

O. subgen. Reticuloidium species which curiously was not found

on any tomato outside Australia.

Despite an editorial policy requiring the deposition of

herbarium specimens (Rossman 1985), none of the authors

reporting catenate Oidium species as causal agents of tomato

powdery mildew outside Australia deposited any voucher

specimens in international collections. Reports of such

anamorphs from outside Australia could not be investigated

such as those from Japan (Abiko 1978), Switzerland (Corbaz

1990, 1993), Bulgaria (Neshev 1993), Canada (Be! langer &

Jarvis 1994), India (Kumar et al. 1995), the USA (Arredondo et

al. 1996, Karasevitz & Zitter 1996, White et al. 1997, Pernezny

& Sonoda 1998), etc. In order to overcome the lack of

specimens, we obtained fresh or herbarium specimens from

many geographical regions (see Tables 1–2) and found only

Pseudoidium anamorphs in them. Moreover, we have re-

examined the tomato powdery mildew pathogens reported as

catenate Oidium species from Hungary (Kiss 1996) and the UK

(Cook et al. 1997) and found only Pseudoidium anamorphs.

Similarly, Bolay (1998) reported that only a Pseudoidium and

never a catenate Oidium species has been found on tomato in

Switzerland, contrary to Corbaz (1990, 1993) who reported

E. cichoracearum. As suggested by Whipps et al. (1998) and

Lebeda & Mieslerova (1999), authors reporting catenate

Oidium species outside Australia may have been misled by the

pseudo-chains of a Pseudoidium produced in high relative

humidity.

Molecular phylogenetic studies completely corroborated

the morphological data. Recent phylogenetic analysis of

powdery mildew ITS sequence data (Saenz & Taylor 1999,

Mori et al. 2000) fully supported the revised anamorphic

taxonomy of Cook et al. (1997), and further drew attention to

the importance of the anamorphic state in determining the

evolutionary relationships within the Erysiphaceae. Preliminary

analysis of the three tomato powdery mildew specimens

clearly showed that the Australian catenate Oidium species

was not phylogenetically related to the two isolates of a

Pseudoidium. Based on these results, two data sets were created

to analyze each phylogenetic lineage separately.

Phylogenetic analyses of the Pseudoidium isolates, including

the two from tomato, resulted in two supported clades : the

E. aquilegiae clade and the E. [sect. M.] platani clade (Fig. 15).

The two specimens of Pseudoidium from tomato, collected in

France and The Netherlands, grouped as sister taxa within the

E. aquilegiae clade as their rDNA ITS sequences were nearly

identical ; this was also reported by Jones et al. (2000). Another

molecular study analysing the amplified fragment length

polymorphism (AFLP) in five Pseudoidium isolates collected

from tomato in three different European countries revealed

only minor genetic differences among them (Huang, Lindhout

& Niks 1998). These results suggest that there is little genetic

variability among the Pseudoidium anamorphs causing the

recent outbreaks of tomato powdery mildew world-wide.

Phylogenetic analyses of the catenate Oidium species

resulted in three supported clades : the G. magnicellulatus clade,

the G. biocellatus clade, and the G. orontii clade (Fig. 16). The

Australian O. lycopersici grouped strongly with another

solanaceous powdery mildew, O. longipes, within the G.

magnicellulatus clade. These results confirm that the anamorph

of the Australian tomato powdery mildew belongs to O.

subgen. Reticuloidium. The unexpectedly close phylogenetic

relationship between the Australian O. lycopersici on tomato

and the Swiss O. longipes on eggplant might be reflected by

similarities in their morphology. Both have very long

conidiophores with displaced basal septa. However, it is

the foot cells of the conidiophore that are very long in

O. lycopersici and the second and}or the third cells that are

exceptionally long in O. longipes. According to Noordeloos &

Loerakker (1989), O. longipes cannot infect tomato.

In order to examine the phylogenetic relationship between

G. orontii infecting tobacco world-wide and the Australian

catenate Oidium species on tomato, Japanese and Hungarian

tobacco powdery mildew ITS sequences were included in our

phylogenetic analysis (Table 3). As expected from the

differences between their morphology, the tobacco and the

Australian tomato powdery mildew fungi grouped in two

different clades (Fig. 16). Tobacco was repeatedly reported to

be infected with tomato powdery mildew in host range

studies (e.g. Fletcher et al. 1988, Corbaz 1990, 1993, Smith et

al. 1997, Huang et al. 2000), but Whipps et al. (1998)

suggested that these findings might have resulted from a

contamination of tobacco with G. orontii. In our trials in a


greenhouse and a climate chamber no cross-infection was

observed between G. orontii on tobacco and the Pseudoidium

species on tomato (Kiss 1996).

That the Australian tomato powdery mildew anamorph

belongs to sect. Reticuloidium, and not to sect. Pseudoidium,

raises nomenclatural problems. The Pseudoidium species was

redescribed as O. lycopersicum by Noordeloos & Loerakker

(1989) based on the original species description (Cooke &

Massee 1888), and the now lost holotype, the only type

specimen available for this name. Without being able to

determine the nature of the conidiogenesis, Noordeloos &

Loerakker (1989) considered that the Australian O. lycopersicum

was a Pseudoidium species compatible with the tomato

pathogen found in Dutch greenhouses in the eighties. In

contrast, our studies indicated that only Reticuloidium

anamorphs occur on tomato in southern Australia. Comparison

of the morphology of the Australian tomato powdery mildew

fungus with the illustration of Noordeloos & Loerakker (1989)

shows several points of comparison. Firstly, the conidiophores

are rather long and have strongly displaced basal septa in both

cases. Secondly, the mycelial appresoria are clustered, and

essentially unlobed. Finally, the type locality of O. lycopersicum

falls within south-eastern Australia, from which only a

Reticuloidium is known on tomato. This is considered sufficient

grounds to redescribe and neotypify Cooke & Massee’s

species below, at the same time correcting its orthography to

O. lycopersici. The neotype is chosen partly because it was

collected within 200 km of the lost holotype in the state of

Victoria, and also because it is the specimen sequenced in the

present study.

Oidium lycopersici Cooke & Massee, Grevillea 16 : 114(1888).

Colonies occurring on the abaxial and adaxial surfaces of

leaves, on petioles, stems and calyces of tomato. Mycelium

either conspicuous and white in some specimens, or extremely

sparse and associated with a hypersensitive necrotic spot in

other specimens. Hyphae straight to undulate, 4–6 µm wide,

hyphal cells 51–66 µm long. Mycelial appressoria well

developed, simple nipple-shaped or weakly lobed, single or

aggregated in serial clusters of up to 8 appressoria.

Conidiophores inserted medianly on the conidiophore mother

cell, moderately to extremely long, basal septum displaced by

15–45 µm, conidiophore stipe consisting of a single long cell

95–180 µm long, occasionally two cells, the basal cell usually

longer than 80 µm, stipe attenuated downwards, 9–10 µm

wide at the basal septum, 10–12 µm wide at the apical septum.

Conidia borne in chains of 3–5 conidia, edge line sinuate,

conidia lacking fibrosin bodies, elliptical to doliform to sub-

cylindrical, falling into two distinct classes, the majority of

conidia small, 25–32¬14–16 µm (in lactic acid mounts), mean

less than 30 µm long, but with a small proportion of much

larger conidia ranging from 35–45 µm long (believed to be

‘double conidia ’ in which a delimiting septum has failed to

form – such conidia germinate with germ-tubes from the

middle as well as the ends). Small conidia in water mounts

from fresh specimens are of similar length but up to 22 µm

wide and broadly ellipsoid. Conidia in lactic acid mounts from

dried herbarium specimens (Shin & La 1993) narrower

(14–16 µm wide) and sub-cylindrical. Primary germ tubes on

host tissue and glass arising from the shoulder, germ tubes

simple, clavate, 23–30¬6–8 µm, basal septum displaced by

2–8 µm. Secondary germination arising either from the body

of the conidium or from the base of the primary germ tube,

below the basal septum.

Typus : Australia : ‘Upper Yarra ’, (K-holotypus lost fide curator,

pers comm.) ; Victoria : Timmering, on L. esculentum cv. ‘Paycetter ’

leaves, 1994, I. Pascoe (VPRI 19847-neotypus hic designatus).

Habitat : On living leaves of Lycopersicon esculentum in

greenhouses, also in the field.

Distribution : Australia.

Molecular data : The nuclear rDNA ITS sequence of the

neotype is deposited in GenBank as AF229021.

Additional specimens examined : see Table 2. Morphological studies

were also carried out on over 50 additional specimens (deposited at

VPRI) collected from south eastern Australia (Pascoe, unpubl.).

Oidium neolycopersici L. Kiss, sp. nov.

Etym. : neo-, new; and from the host Lycopersicon esculentum.

Mycelium album, supra, interdum infra folia in statu vivo, rare in

caulibus crescens. Appressoria lobata vel multilobata, opposita vel

effusa dissita. Conidiophora 58–(91±5)–115 µm longa, cellulae basis

cylindricae, 29–(42±4)–74 µm longae. Conidia 22–(33±5)–46¬10–

(13±7)–20 µm, ellipsoidea-ovoidea vel doliiformia, singula aut si

humida 2–6 in pseudocatenas, sine corpore fibrosina.

Mycelium white, thin, covering the upper and occasionally the

lower surfaces of the leaves, and also the stems. Hyphae

hyaline, septate, 4–8 µm wide, appressoria distinct, lobed to

multi-lobed, rarely nipple-shaped, opposite or dispersed.

Conidiophores erect, 58–115 µm (mean : 91±5 µm) long, foot-

cells cylindrical or sometimes inflated in the middle and

constricted at the base, 29–74 µm (mean : 42±4 µm) long,

followed by 1–2 shorter cells or a single cell of about the same

length. Conidia produced singly, or, in high relative humidity,

in pseudo-chains of 2–6 conidia, 22–46 (mean : 33±5)¬10–20

(mean : 13±7) µm, ellipsoid-ovoid or doliform, without fibrosin

bodies, germ tubes arising from an end or side of the

conidium, substraight or curved, apex enlarged, lobed, rarely

simple, germ tubes increasing in width from base to top.

Typus : France : Aigue Mortes, on Lycopersicon esculentum, 1989,

P. C.Nicot Et1 (BPI 747013-holotypus, HAL-isotypus). Living material

maintained at INRA, Unite! de Pathologie Ve! ge! tale, Avignon, France.

Habitat : On living leaves and stems of Lycopersicon

esculentum in greenhouses, also in the field.

Distribution : Nearly circumglobal wherever tomatoes are

grown.

Molecular data : The nuclear rDNA ITS sequence of isolate

Et1 was deposited in GenBank under the accession number

AF229019.

Additional specimens examined : See Tables 1–2.

This widespread new species has long been confused with the

geographically restricted O. ‘ lycopersicum ’ Cooke & Massee


1888 (corrected to O. lycopersici by Lebeda & Mieslerova

1999). At present, no species name exists for the Pseudoidium

anamorph infecting tomato and so we have provided a formal

description and new name here. Although most data in the

recent phytopathological literature suggested that O. neolyco-

persici had arisen fairly recently on tomato (e.g. Whipps et al.

1998), our data show that this is not the case. We have studied

herbarium specimens collected since 1947 (Table 1) which can

now be clearly identified as O. neolycopersici (Fig. 1). So, this

species has been infecting tomato for a long time, at least in

Asia, the origin of all the herbarium specimens older than

50 yr. Unfortunately, we could not obtain any European

herbarium specimens collected before the first European

outbreaks of tomato powdery mildew (Blancard 1988, Fletcher

et al. 1988), although the disease was repeatedly reported in

the mycological literature since 1900. So, we could not

determine if the pathogens reported as anamorphs of Erysiphe

spp. infecting tomato in different European countries (Salmon

1900, Jaczewski 1927, Blumer 1933, 1967, Hammarlund 1945)

were identical with O. neolycopersici. Nor could we obtain any

specimens of Oidium on tomato reported from Sri Lanka

(Petch 1922), Peru (Abbott 1931) and Argentina (Godoy

1939).

Oidium anamorphs on tomato, although poorly identified in

the older literature, sometimes caused severe problems in

different parts of the world long before the recent outbreaks

(e.g. Godoy 1939). However, there is only one doubtful

record on tomato seedlings (Farr et al. 1989) in North America

before the 1990s (Be! langer & Jarvis 1994) suggesting that O.

neolycopersici, now widespread in both the USA and Canada,

was introduced to North America only very recently.

This study has successfully brought together supporting

evidence from entirely separate areas of expertise, i.e. those

involving classical morphological, molecular and SEM tech-

niques. Thus, the joint use of these three methods is now

highly recommended for the precise identification of any

newly discovered powdery mildew anamorphs.

ACKNOWLEDGEMENTS

The authors are grateful to the following scientists for providing fungal

materials : Adrien Bolay, Raymond Cerkauskas, Mike Davis, David M.

Gadoury, Cai-Cheng Huang, David Kalb, Margaret T. McGrath, Nina

Shishkoff, and Victoria L. Smith. The curators of IMI and DAR, John C. David

and Michael Priest, respectively, are also acknowledged for the loan of

materials. We are especially indebted to Uwe Braun for helpful suggestions

on the taxonomic aspects. We also thank Jim Plaskowitz for assistance in

preparing Figs 1–4. The studies of L. Kiss were partly supported by a

Fulbright fellowship and a Ja! nos Bolyai fellowship and a grant (OTKA

F032931) of the Hungarian Scientific Research Fund and those of R. T. A.

Cook by the Plant Health Divison of the Ministry of Agriculture, Fisheries

and Food, UK. G. S. Saenz wishes to thank Laura Salter for advice with

Maximum Likelihood analysis, and Donald O. Natvig and John W. Taylor, for

access to computers and phylogenetic programs. Much of the morphological

study of over 50 specimens of O. lycopersici was carried out by Siva Sivapalan

and Vyrna Beilharz (Institute for Horticultural Development, Knoxfield,

Australia).

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Corresponding Editor : D. T. Mitchell

Identification of two powdery mildew fungi, Oidium neolycopersici sp. nov. and O. lycopersici,...

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