FUNGAL DISEASES

J Gen Plant Pathol (2003) 69:176–185 © The Phytopathological Society of JapanDigital Object Identifier (DOI) 10.1007/s10327-002-0034-7 and Springer-Verlag Tokyo 2003

Koichi Kashimoto · Yoshinori MatsudaKazumi Matsutani · Takeshi Sameshima · Koji KakutaniTeruo Nonomura · Kiyotsugu Okada · Shin-ichi KusakariKengo Nakata · Susumu Takamatsu · Hideyoshi Toyoda

Morphological and molecular characterization for a Japanese isolate oftomato powdery mildew Oidium neolycopersici and its host range

Received: December 5, 2002 / Accepted: December 26, 2002

K. Kashimoto · Y. Matsuda · K. Matsutani · T. Sameshima ·T. Nonomura · H. Toyoda (*)Laboratory of Plant Pathology and Biotechnology, Faculty ofAgriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505,JapanTel. +81-742-43-1511; Fax +81-742-43-1155e-mail: toyoda@nara.kindai.ac.jp

K. KakutaniPharmaceutical Research and Technology Institute, Kinki University,Osaka, Japan

K. Okada · S. KusakariAgricultural, Food and Environmental Sciences Research Center ofOsaka Prefecture, Osaka, Japan

K. NakataResearch Institute, Kagome Company, Tochigi, Japan

S. TakamatsuFaculty of Bioresources, Mie University, Mie, Japan

Abstract A single conidium of tomato powdery mildewwas isolated from heavily infected leaves of tomato (cv.Moneymaker) grown in the greenhouse of Kinki Univer-sity, Nara Prefecture, Japan. It was successively multipliedso the morphological and taxonomic characteristics of thepathogen and its host range under high humidity conditionscould be analyzed. The isolate KTP-01 of the tomato pow-dery mildew optimally developed infection structures at25°C under continuous illumination of 3500lx. More than90% of the conidia germinated and developed moderatelylobed appressoria. After forming haustoria, the pathogenelongated secondary hyphae from both appressoria andconidia. The hyphae attached to leaf surfaces by severalpairs of appressoria and produced conidiophores withnoncatenated conidia. In addition to its morphological simi-larity to Oidium neolycopersici, the phylogenetic analysis(based on the sequence of internal transcribed spacer re-gions of rDNA) revealed that KTP-01 could be classifiedinto the same cluster group as O. neolycopersici. In hostrange studies, KTP-01 produced abundant conidia on thefoliage of all tomato cultivars tested and tobacco (Nicotianatabacum), and it developed faint colonies accompanied bynecrosis on leaves of potato (Solanum tuberosum), red pep-

per (Capsicum annuum), petunia (Petunia � hybrida), andeggplant (S. melongena). The pathogen did not infect otherplant species including Cucurubitaceae plants, which havebeen reported to be susceptible to some foreign isolates.Thus, the present isolate of the tomato powdery mildew wasassigned as O. neolycopersici, a pathotype different fromforeign isolates of the pathogen.

Key words Tomato powdery mildew · Oidiumneolycopersici · Phylogenetic analysis · ITS

Introduction

Powdery mildew that causes severe damage on greenhousetomatoes has been newly reported worldwide. These patho-gens produce fibrosin body-free conidia singly (Jones et al.2000; Noordeloos and Loerakker 1989; Olalla and Torés1998; Smith et al. 1997; Whipps et al. 1998), in short chains(Arredondo et al. 1996; Kiss 1996; Neshev 1993; Perneznyand Sonoda 1998; White et al. 1997), or both (Bélanger andJarvis 1994; Fletcher et al. 1988; Karasevicz and Zitter 1996;Vakalounakis and Papadakis 1992). No paper has describedthe formation of cleistothecia by these powdery mildews.Kiss et al. (2001) reported that all recent outbreaks oftomato powdery mildew outside Australia were causedby a species that formed conidia singly or occasionally inpseudochains of two to six conidia in high relative humidity.They proposed the creation of a new species, Oidiumneolycopersici, provided the Australian isolates that alwaysformed conidia in chains retained the name O. lycopersici.The powdery mildew of greenhouse tomato was also re-ported in Japan (Kiss et al. 2001; Matsuda et al. 2001; Sato1991; Uchida et al. 2001) and was shown to be similar to O.neolycopersici (Jones et al. 2001; Kiss et al. 2001).

To characterize the tomato powdery mildew in Japanfurther, we attempted to isolate single conidia from pustuleson diseased tomato leaves, determined the optimal condi-tions for colonization of tomato leaves by the pathogen, andcompared the rDNA internal transcribed spacer (ITS) se-quences between the present isolate and other powdery

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mildews of tomato. In this paper we describe successfulisolation and subsequent multiplication of single conidiaand then examine the effects of temperature and light inten-sity on germination, formation of appressoria and hausto-ria, elongation of secondary hyphae, and pustule formationby the isolate. Under optimal conditions for inoculation, weexamine the host range of the isolate using several plantspecies (15 families containing 40 species, 102 cultivars)including commercial tomato cultivars available at presentin Japan.

Materials and methods

Single conidium isolation

Developed leaves forming pustules of powdery mildewwere detached from 2-month-old plants of tomato(Lycopersicon esculentum M., cv. Moneymaker) cultivatedunder greenhouse conditions (26° � 4°C). Conidia on de-tached leaves were dusted onto leaves of 1-month-old to-mato seedlings grown in a powdery mildew-free acrylic box(45 � 45 � 30cm) whose lateral sides were covered with afine-meshed net (mesh size 3 µm). The boxes with the inocu-lated plants were placed in a growth chamber controlled at25°C under continuous illumination of a fluorescent lamp(3000 lx). After 10 days of incubation, leaves forming singlepustules were used for single conidium isolation. Using aglass needle attached to a micromanipulator, conidia thathad formed singly on conidiophores were transferred ontoleaves detached from pathogen-free tomato plants. Inocu-lated leaves were placed in a moistened petri dish and incu-bated under the conditions mentioned above. Conidia werecollected from single conidium-derived pustules and multi-plied through three cycles of inoculation and single sporeisolation. Finally, the isolate was designated KTP-01 andused in the following experiments.

Observation of fungal development

Newly formed conidia were dusted onto glass slides (ap-proximately 3000 conidia per slide) by a method previouslydescribed (Chatani et al. 1996), placed in a moistened petridish, and incubated at various temperatures (15–35°C) inthe dark or under continuous illumination (50–3500lx) by afluorescent lamp. Germinated conidia were observed bylight microscoply.

To examine conidial germination and appressorium for-mation on tomato leaves, conidia were dusted onto thetomato leaves. Five leaflets of the third leaf detached from2-month-old seedlings (cv. Moneymaker) were used for in-oculation. The conidia (100–300 spores/cm2) were dustedonto leaves and incubated for various periods at 25°C undercontinuous illumination of 3500lx. Conidia on leaves werecollected by gently applying small pieces (1 � 1cm) oftransparent adhesive tape to inoculated leaves; they werethen transferred to a glass slide with lactophenol/anilineblue to examine germination and appressorium formationby the conidia on leaves microscopically (Matsuda et al.2001). Alternatively, inoculated leaves were decolored in aboiling alcoholic lactophenol solution for 1–2min and thenstained with aniline blue by the method described previ-ously (Toyoda et al. 1978) to observe the formation of haus-toria and secondary hyphae by the pathogen. Scanningelectron microscopy was also carried out on fresh materialto complement observations made by light microscopy(Matsuda et al. 2001). Approximately 500 conidia were ob-served during each experiment, and the experiment wasrepeated three times.

Host range studies

Fifteen plant families containing 40 species (totally 102 cul-tivars) were inoculated with conidia of KTP-01 to examine

Table 1. DDBJ database references for the powdery mildew fungi used for phylogenetic analysis

Fungus Accession no. Registrants and date for registration

Erysiphe aquilegiae var. ranunculi AB015929 Takamatsu et al. 2000Erysiphe blasti AB015918 Takamatsu et al. 2000Erysiphe convolvuli AF011298 Saenz and Taylor 1999Erysiphe cruciferarum AF031283 Adam et al. 1999Erysiphe juglandis AB015928 Takamatsu 2000Erysiphe macleayae AB016048 Takamatsu et al. 2000Erysiphe platani AF011311 Saenz and Taylor 1999Erysiphe polygoni AF011307 Saenz and Taylor 1999Erysiphe pulchra var. japonica AB015924 Takamatsu et al. 2000Erysiphe weigelae AB015931 Takamatsu 2000Oidium (neo)lycopersici AF171876 Jones et al. 1999Oidium neolycopersici (Et-1) AF229019 Kiss et al. 2001Oidium neolycopersici (VPRI20724) AF229015 Kiss et al. 2001Oidium sp. (MUMH66) AB032483 Takamatsu and Kiss 2000Oidium sp. (DNA231) AB032484 Takamatsu and Kiss 2000KTP-01 AB094991 Matsuda et al. 2002

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the host range of the pathogen. Potatoes used for the hostrange studies were grown from tubers supplied by Dr. Y.Takamatsu, Laboratory of Horticulture Science, Kinki Uni-versity. All other test plants were grown from seeds ob-tained from the producers or were available from ouruniversity collection. Test plants were placed individuallyinto 12.5-cm pots, grown in the propagation glasshouse for3–4 weeks, and transferred to the greenhouse controlled at25° � 3°C for inoculation. No powdery mildew was ob-served on plants during growth in the propagation glass-house.

For each inoculation, five test plants (two or three trueleaves for large-leaf plants such as cucurbits and five toeight true leaves for small-leaf plants such as Sesamum sp.,and primary leaves for Gramineae plants) and three tomatoplants (1-month-old tomato seedlings of Moneymaker) asindicators were placed in an acrylic cylinder. Conidia weredusted onto the leaves as described earlier. Successful in-oculation was confirmed by observing pustule expansion onthe entire surface of inoculated leaves of the indicator to-mato plants. Fungal development and the appearance ofnecrosis in inoculated leaves were recorded 7 and 14 daysafter inoculation based on the criteria of Whipps et al.(1998). Inoculation was conducted in the greenhouses ofKinki University and the Agricultural, Food, and Environ-mental Sciences Research Center of Osaka Prefectureduring March, April, May, June, November, and December2002.

Internal transcribed spaces sequence determination

For internal transcribed spaces (ITS) determination,conidia were collected from KTP-01-inoculated plants 7days after inoculation, and fugal DNA was isolated fromfresh conidia by the method of Walsh et al. (1991). Conidiawere added to 50 µl of 5% Chelex (Bio-Rad, Tokyo, Japan)and incubated at 56°C for several hours. After mixing vigor-ously, the extract was heated in a boiling water bath for8min. The extract was mixed vigorously again and thencentrifuged at 15000g for 5min. The supernatant was usedas template DNA.

The polymerase chain reaction (PCR) (iCycler, Bio-Rad) was carried out using the ITS4 and ITS5 primersdesigned by White et al. (1990) to cover 5.8S rDNA and itsboth-side intergenic spacer regions. PCR was performedaccording to the protocols described previously (Hirata andTakamatsu 1996): 30 cycles for denaturing (95°C, 30s), an-nealing (52°C, 30s) and extension (72°C, 30s) followed by afinal extension cycle of 7min at 72°C. The amplified DNAwas electrophoresed in 1.5% agarose gel, and the DNAband was excised from the gel and inserted in a pGEM-Teasy vector for sequencing (Promega, Madison, WI, USA).The nucleotide sequence of the amplified region was deter-mined using Big Dye Terminator Cycle Seqencing ReadyReaction Kit from Applied Biosystems (Tokyo, Japan) onan ABI Prism 310 Genetic Analyzer (Perkin Elmer AppliedBiosystems, Tokyo, Japan) in the Pharmaceutical Researchand Technology Institute, Kinki University, Japan.

Genetyx-Mac software (Version 11.2; Sequence analysissoftware package; Genetyx, Tokyo, Japan) was used forsequence analysis, and BLAST software was accessedthrough the National Center for Biotechnology Informa-tion (NCBI).

Phylogenetic analysis

The ITS sequence data for the powdery mildews formingnoncatenated conidia containing some isolates of O.lycopersici were obtained from the DNA Data Bank ofJapan (DDBJ) database (Table 1). The sequence datanewly obtained from KTP-01 were combined and used forthe phylogenetic analysis. The sequences were initiallyaligned using the DNA Space Version 3.5 (Hitachi SoftwareEngineering, Yokohama, Japan). The alignment was thenvisually refined with a word processing program using color-coded nucleotides. The alignment is available upon request(ymatsuda@nara.kindai.ac.jp). For the parsimony analysis,we used the maximum-parsimony (MP) method with a heu-ristic search using PAUP Version 3.1.1. This search wasrepeated 100 times with different random starting points,using the stepwise addition option to increase the likelihoodof finding the most parsimonious tree. All sites were treatedas unordered and unweighted, with gaps treated as missingdata. The branch-swapping algorithm was Tree BisectionReconnection (TBR), the MULPARS option was in effect,and zero-length branches were collapsed. The strength ofthe internal branches from the resulting trees was testedby bootstrap analysis using 1000 replications (Felsenstein1985).

Results and discussion

In 1998 an outbreak of powdery mildew was first detected incommercial tomato cultivars grown in greenhouses of KinkiUniversity (Nara Prefecture, Japan). The disease occurredevery year thereafter, being especially severely in young

Table 2. Formation of appressoria and primary haustoria by tomatopowdery mildew KTP-01 on tomato leaflets

Leaflets Formation (%)a

Appressoria Haustoria

Upper 91.0 � 3.4 84.9 � 1.6Middle

Left side 91.8 � 3.9 86.1 � 3.4Right side 92.0 � 1.9 85.7 � 2.2

LowerLeft side 93.0 � 3.7 82.8 � 3.0Right side 92.1 � 4.1 83.9 � 3.9

a Conidia (100–300 spores/cm2) were dusted onto leaflets of the thirdleaves. The rates of appressorium and haustorium formation wereestimated by the formulas: (appressorium-forming conidia/totalconidia) � 100 and (haustorium-forming conidia/appressorium-forming conidia) � 100, respectively. More than 500 conidia wereobserved in each experiment. Data were given as means and standarddeviation of three replications

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seedlings. The pathogen vigorously expanded to cause dam-age in tomato plants in the greenhouse and in the field whenchemical control was insufficient. Our previous work(Matsuda et al. 2001) revealed that commercial tomato cul-tivars available in Japan were highly susceptible to naturalinfection with the powdery mildew. Similarly, the diseaseoccurred severely in greenhouse- and field-grown tomatoesin several European countries (Burgerjon et al. 1990;Fletcher et al. 1988; Jones et al. 2000, 2001; Kiss 1996;Neshev 1993; Noordeloos and Loerakker 1989; Olalla andTorés 1998; Vakalounakis and Papadakis 1992; Whipps etal. 1998), Canada (Bélanger and Jarvis 1994), and America(Arredondo et al. 1996; Karasevicz and Zitter 1996;Pernezny and Sonoda 1998; Smith et al. 1997; White et al.1997). Although some investigators (Ciccarese et al. 1998;Lindhout et al. 1994; Mieslerová et al. 2000; Neshev 1993)screened Lycopersicon species resistant to the powdery mil-dew, precise identification of the pathogen is essential toestablish an efficient breeding program that includes resis-tance to tomato powdery mildew. Nevertheless, taxonomiccharacterization of the pathogens has been complicatedbecause of no success in detecting a sexual stage(cleistothecia). This led to incorrectly naming the newpowdery mildew pathogen on tomato plants: O.lycopersicum (Lindhout et al. 1994; Noordeloos andLoerakker 1989; Whipps et al. 1998), Oidium sp.(Arredondo et al. 1996; Neshev 1993), and Erysiphe sp.(Bélanger and Jarvis 1994; Burgerjon et al. 1990; Fletcher etal. 1988; Karasevicz and Zitter 1996; Kiss 1996; Olalla andTorés 1998; Pernezny and Sonoda 1998; Smith et al. 1997;Vakalounakis and Papadakis 1992; White et al. 1997). Inaccordance with the International Code of Botanical Lit-erature, Mieslerová and Lebeda (1999) re-registered O.lycopersici for O. lycopersicum; and some reports(Ciccarese et al. 1998; Jones et al. 2000; Mieslerováet al. 2000) used the name O. lycopersici for the powderymildew pathogen of tomato. To solve this complicated situ-ation for taxonomic identification of the tomato powdery

mildews, Kiss et al. (2001) carried out morphological andphylogenetic analyses of the isolates obtained worldwideand assigned a new species, O. neolycopersici, as the patho-gen that produces noncatenated conidia. In the presentstudy, we examined whether the pathogen isolated in ourlaboratory is identical to the new species by analyzing itsmorphological and ITS data.

First, we isolated single conidia from pustules on tomatoleaves, transferred them onto pathogen-free tomato leavesand then selected the isolate (KTP-01) that produced largepustules with abundant conidiophores. After repeating theisolation procedure three times, the pathogen was multi-plied for the subsequent experiments. Figure 1A showsgermination by the conidia on a glass slide in the dark.Germination was initiated at 2–3h and reached a maximum7h after incubation. The conidial germination of KTP-01was highest at 25°C. During the entire incubation period(9h), most conidia continuously elongated germ tubes.Figure 1B shows the change in the rate of germinatedconidia of KTP-01 at 25°C under various light intensities.Germination was promoted with increased light intensity,and the highest rate (97.8%) was achieved by illuminationof 3500 lx. Much higher light intensity was not tested be-cause of the limited capacity of the growth chamber that weused.

Table 2 shows the formation of appressoria and primaryhaustoria by the conidia dusted onto five leaflets of the thirdleaves. More than 90% of the conidia produced appressoria,and more than 80% of the appressorium-forming conidiadeveloped primary haustoria underneath the appressoria.There was not a considerable difference in the formationof appressoria or haustoria among the leaflets of the thirdleaves. Thus, the powdery mildew successfully developedthe infection structures on the third leaves under the post-inoculation conditions (25°C, 3500 lx) used here. These re-sults suggest that this condition is optimal for the powderymildew to establish a high infection rate on tomatoleaves.

Fig. 1. Effects of temperature (A)and light intensity (B) on germina-tion by conidia of the tomatopowdery mildew KTP-01. Conidiawere dusted onto a glass slide andincubated in a moistened petri dishin the dark. Numbers in A and Brepresent temperature (°C) and lightintensity (lx) used for incubation,respectively. More than 500 conidiawere observed in each experiment.Data are the means and standarddeviations of five replications

A B

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Under the present conditions, the conidia were dustedonto third leaves of tomato, and their morphologicalchanges were monitored periodically (Fig. 2). Conidialgermination started at 2h and reached a maximum 7–8hafter inoculation. The development of moderately lobedappressoria (Fig. 3A) was initially detected at 5h, and theirformation was highest 22–24h after inoculation. Nonlobedhaustoria were observed underneath the appressoria 12hafter inoculation (Figs. 2A, 3B). After the primary hausto-ria were formed, secondary hyphae developed from theconidia and then from the primary appressoria (Fig. 3C).Finally, six or seven hyphae were produced from theconidia after 7 days of inoculation (Fig. 2B). Each hyphawas attached to the leaf surface by several pairs of hyphalappressoria (Fig. 3D). Secondary haustoria were formedunderneath one of paired hyphal appressoria. Figure 3Eshows imprinted profiles of peeled secondary hyphae andpaired appressoria left on the leaf surface, where the pen-etration pore was detected in one of the paired appressoria.The pathogen produced 40–60 conidiophores (per pustule)with noncatenated conidia (Fig. 3F), and the pustules cov-ered the entire surface of inoculated leaflets 7 days afterinoculation. The size and shape of the conidia and conidio-phores were in complete agreement with the data reportedpreviously (Matsuda et al. 2001).

Additionally, the fully expanded leaflets of the upperleaves (fourth to tenth leaves) were similarly inoculated toexamine differences in sensitivity to the powdery mildewamong different compound leaves. The pathogen producedpustules that entirely covered the leaves 7 days after inocu-lation (data not shown), indicating that there was no differ-ence in susceptibility among these leaves. Thus, the presentstudy optimized the laboratory conditions for inoculation oftomato plants by KTP-01.

In conclusion, the germination was sensitive to tempera-ture and light intensity and was completely inhibited byhigh temperatures (�35°C). A light intensity of 3500lx wassufficient to obtain considerably higher rates of develop-ment of fungal infection structures (appressoria, haustoria,secondary hyphae, conidiophores, conidia), and the rates

were comparable to those of the pathogen on inoculatedtomato plants grown in a temperature-controlled green-house (data not shown). In our previous work, the occur-rence of powdery mildew on greenhouse-grown tomatoeswas shown to be more frequent during low-temperatureseasons (spring and autumn) but was rare during summerin Nara District (Matsuda et al. 2001). This may reflectinhibited germination by the pathogen under the high-temperature conditions of summer.

The host range of the tomato powdery mildews has beenwidely studied using various plant species (Arredondo et al.1996; Burgerjon et al. 1990; Fletcher et al. 1988; Kiss 1996;Olalla and Torés 1998; Whipps et al. 1998) including wildLycopersicon species (Lindhout et al. 1994; Mieslerováet al. 2000; Neshev 1993). Although the pathogens heavilyinfected all tomato varieties or cultivars tested, their infec-tivity for various plant species was not always identicalamong the workers: Eggplant (Solanum melongena) andpotato (S. tuberosum) were susceptible hosts (Burgerjonet al. 1990; Fletcher et al. 1988; Whipps et al. 1998). Tobacco(Nicotiana tabacum) was both a susceptible host(Arredondo et al. 1996; Burgerjon et al. 1990; Fletcher et al.1988; Whipps et al. 1998) and a resistant host (Kiss 1996).Cucumber (Cucumis sativus) also was both a susceptiblehost (Burgerjon et al. 1990; Whipps et al. 1998) and a resis-tant host (Fletcher et al. 1988; Kiss 1996; Olalla and Torés1998).

In our host range studies, KTP-01 infected and sporu-lated on the foliage of tobacco (N. tabacum) and all tomatocultivars tested without causing necrosis in inoculatedleaves (Table 3). The necrotic reaction was induced inhaustorium-harboring epidermal cells and their neighbor-ing cells of inoculated leaves of potato, eggplant, red pepper(Capsicum annuum), and petunia (Petunia � hybrida)(Fig. 4), although Fletcher et al. (1988) reported that petu-nia was highly sensitive to their isolate. This necrotic reac-tion effectively decreased the fungal growth (Mieslerováand Lebeda 1999). KTP-01 did not infect leaves of otherplant species, especially showing no growth on leaves ofCucurbitaceae plants, contrary to the results of Burgerjon

Fig. 2. Time course of morphologicalchanges by KTP-01 inoculated ontotomato leaves. A Germination (a),appressorium formation (b), andhaustorium formation (c). BProduction of conidial hyphae. Morethan 500 conidia were observed ineach experiment. Data are the meansand standard deviations of threereplications

A B

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Table 3. Host range tests for the tomato powdery mildew KTP-01

Family and species Common name Cultivar Producera Necrosisb Susceptibilityc

7 14 7 14

AmaranthaceaeGomphrena globosa Globe amaranth Sennichiko TA – – 0 0

ChenopodiaceaeSpinacia oleracea Spinach Lead SA – – 0 0

CompositaeArctium lappa Edible burdock Salada Musume TA – – 0 0Bellis perennis English daisy Daisy TA – – 0 0Callistephus chinensis Chinese aster Large Flowered Mix TA – – 0 0Chrysanthemum coronarium Garland chrysanthemum Kabuharityuuba TA – – 0 0Cosmos bipinnatus Cosmos Tyoujizaki cosmos TA – – 0 0Helianthus annuus Sunflower Sunrich lemon TA – – 0 0Lactuca sativa Lettuce Summer green SA – – 0 0

Tima santyu SA – – 0 0Convolvulaceae

Ipomoea batatas Sweet potato Benihayato KU – – 0 0Ipomoea nil Morning glory Scarlet Ohara TA – – 0 0Ipomoea tricolor Morning glory Heavenly Blue TA – – 0 0

CruciferaeBrassica campestris Chingensai Chingensai SA – – 0 0

Komatsuna Rakuten TA – – 0 0Shirona Daibansei Shirona TA – – 0 0

Brassica oleracea Broccoli Ryokkayasai AT – – 0 0Cabbage Shikidori TA – – 0 0

Eruca sativa Rocket-salada Rocket SA – – 0 0Raphanus sativus Radish Cherry Mate TO – – 0 0

CucurbitaceaeCitrullus lanatus Watermelon Fujihikari HA – – 0 0

Takahikari HA – – 0 0SA-75 HA – – 0 0

Cucumis melo Melon Earle’s Clio HA – – 0 0Yellow King HA – – 0 0

Cucumis sativus Cucumber Sagami hanpakufushinari TA – – 0 0Suzunarisuyou TA – – 0 0

Cucurbita maxima Pumpkin Kuriebisu TA – – 0 0Shintosa HA – – 0 0

Cucurbita moschata Pumpkin Kagayaki HA – – 0 0Kotsumanankin HA – – 0 0

GramineaeHordeum vulgare Barley Goseshikoku KU – – 0 0

Kobinkatagi KU – – 0 0Triticum aestivum Wheat Norin yon-gou KU – – 0 0Zea mays Maize Peter corn 235 AT – – 0 0

LabiataeOcimum basilicum Sweet basil Sweet Basil TO – – 0 0

LeguminosaePhaseolus vulgaris Kidney bean Kurotanesanjyakuoonaga TA – – 0 0

Topcrop TA – – 0 0Pisum sativum Pea Oosayaendou AT – – 0 0

MalvaceaeAbelmoschus esculentus Okra Early five TA – – 0 0

PedaliaceaeSesamum indicum Sesame Shiro Goma TA – – 0 0

PolygonaceaeFagopyrum esculentum Buckwheat Shinshu Soba TO – – 0 0

SolanaceaeCapsicum annuum Pepper Shishitou SA – – 0 0

Red pepper Tougarasi SA � � 0 1Sweet pepper Ace TA – – 0 0

Kyoumidori TA – – 0 0Kyounami TA – – 0 0

Lycopersicon esculentum Tomato Big Fukuju MA – – 3 3Caroltrio AT – – 3 3Chibikko MA – – 3 3Coco TA – – 3 3Corona MA – – 3 3Firstpower SA – – 3 3Fukuju Ni-gou TA – – 3 3Hakko MA – – 3 3

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Table 3. Continued

Family and species Common name Cultivar Producera Necrosisb Susceptibilityc

7 14 7 14

Hikari Fukuju MA – – 3 3Hokin Fukuju TO – – 3 3Home Momotaro TA – – 3 3House Momotaro TA – – 3 3King Fukuju MA – – 3 3Kiomaru MA – – 3 3Kyoryokuminori MA – – 3 3Kyouryoku Beiju TA – – 3 3Kyouryoku Beiju Ni-gou TA – – 3 3LS-89 SA – – 3 3Marryroad SA – – 3 3Minicarol AT – – 3 3Momotaro TA – – 3 3Momotaro Eight TA – – 3 3Moneymaker KU – – 3 3Multi First TA – – 3 3Odoriko SA – – 3 3Ojyu TA – – 3 3Pepe TA – – 3 3Petit TA – – 3 3Pico TA – – 3 3Ponderosa TA – – 3 3Puti Eru TO – – 3 3Red Pear TA – – 3 3Saturn TA – – 3 3Sekaiichi MA – – 3 3Shubi MA – – 3 3Sopy MA – – 3 3Sugerlamp AT – – 3 3Toyofuku SA – – 3 3Toyomasa SA – – 3 3Twinkle Sweet MA – – 3 3Yellow Pear TA – – 3 3Yellow Pico TA – – 3 3Yubi MA – – 3 3Yuho AT – – 3 3Zuiei SA – – 3 3

Nicotiana tabacum Tobacco Bright yellow KU – – 3 3Nicotiana benthamiana Tobacco KU – – 0 0Petunia � hybrida Petunia Baccarat Mix SA � � 1 2Solanum melongena Eggplant Karehen NA � � 1 2

Senryou Ni-gou TA � � 1 2Solanum muricatum Pepino Pepinokun TA – – 0 0Solanum tuberosum Potato Danshakuimo KU � � 0 1

May Queen KU � � 0 1Umbelliferae

Daucus carota Carrot Benikalochin KY – – 0 0ViolaceaeViola � wittrockiana Garden pansy Bingo Clear Yellow TA – – 0 0

a TA, Takii seeds, Kyoto, Japan; SA, Sakata seeds, Yokohama, Japan; AT, Atariya Noen, Chiba, Japan; TO, Tohoku seeds, Utsunomiya, Japan;HA, Hagiwara Nojyo, Nara, Japan; MA, Marutane, Kyoto, Japan; KY, Kyowa seeds, Tokyo, Japan; NA, Nanto Seeds, Nara, Japan; KU, KinkiUniversity seed collectionb By 7 and 14 days after inoculation. �, necrosis present as a result of inoculation with KTP-01; –, no necrosisc 0, no conidiophore production; 1, limited conidiophore production (�30 conidiophores per inoculation site); 2, higher level of conidiophoreproduction (�30 conidiophores per inoculation site) with limited spread of fungal colony; 3, highest conidiophore production with extensivespread of the colony

et al. (1990) and Whipps et al. (1998). On the other hand,sunflower (Helianthus annuus) was susceptible to the iso-late of Burgerjon et al. (1990) but not to KTP-01 or theisolate of Whipps et al. (1998). Thus, the host range datawere not identical among the pathogens tested. This mayreflect the existence of different pathotypes, as pointed outby Jones et al. (2001).

The present study indicated that the morphological char-

acteristics of KTP-01 were similar to those of tomato pow-dery mildew reported previously (Jones et al. 2000, 2001;Kiss et al. 2001; Whipps et al. 1998). However, its host rangewas not always the same as reported in earlier reports.

To analyze the similarity of KTP-01 further, molecularcharacterization was done by analyzing the phylogeneticrelation based on the ITS of rDNA. It has been recognizedthat there is a good correlation between the morphological

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Fig. 3. Light and scanning electron micrographs of tomato powderymildew KTP-01 developing on tomato leaves. A Moderately lobedappressorium 10 h after inoculation. B Nonlobed primary haustorium(arrow) underneath the primary appressorium 12 h after inoculation. CConidium (cd) forming appressoria (ap) and secondary hyphae fromconidium (cd-h) and appressorium (ap-h). The conidia were movedfrom inoculated leaves with an adhesive tape 24 h after inoculation. DPair of appressoria (arrows) underneath secondary hypha 76 h afterinoculation. E Imprint of paired hyphal appressoria left on the leafsurface by peeling off the superficial hyphae. Note the formation of thepenetration pore underneath the left-side appressorium. F Mycelialcolony producing abundant conidiophores with noncatenated conidia 7days after inoculation. Bars 10 µm (A–C); 2 µm (D, E); and 25 µm in (F)

classification system for the powdery mildews described byCook et al. (1997) and the rDNA sequencing-based classifi-cation developed by Saenz and Taylor (1999). Moreover,phylogenetic data of the transcribed spacer regions of ribo-somal DNA (rDNA) have been collected to determine thetaxonomic position of powdery mildew pathogens (Moriet al. 2000; Takamatsu et al. 1998, 1999) including O.neolycopersici (Jones et al. 2000; Kiss et al. 2001). Thepresent phylogenetic analysis (Fig. 5) revealed that KTP-01could be classified into the same cluster group assignedfor O. neolycopersici by Jones et al. (2000) and Kiss et al.(2001). Moreover, the nucleotide sequences of 5.8S rDNAand both ITS regions of KTP-01 were completely identi-cal with those of O. neolycopersici (Et-1) and Oidium sp.

Japanese isolates (DNA231 and MUMH66). Judging fromthese results, we determined that KTP-01 is a Japaneseisolate of O. neolycopersici.

Recently, the tomato powdery mildews morphologicallysimilar to KTP-01 were reported in several districts of Japan(Kiss et al. 2001; Matsuda et al. 2001; Sato 1991; Uchidaet al. 2001). In our preliminary research, tomato powderymildew forming noncatenated conidia was observed onfield- and greenhouse-grown tomatoes at the experimentalfarms of our university (Eniwa, Hokkaido, Japan) in 1999and 2000 and the Kagome Research Institute (Nasu-gun,Tochigi, Japan) in 2002, as well as on tomatoes in farmers’greenhouses in Hiroshima in 1999 (S. Matsuura, personalcommunication), in Osaka in 1999 and 2000, and in ShigaPrefecture in 2000 and 2001. These pathogens heavilyinfected commercial tomato cultivars. Because this typeof tomato powdery mildew has spread widely in Japan, itis urgent that an ecological survey of the pathogen becarried out and efficient measures to control the diseaseestablished.

Fig. 4. Necrosis in haustorium-harboring epidermal cells and theirperipheral cells of potato (A) and red pepper leaves (B) inoculatedwith tomato powdery mildew KTP-01 (14 days after inoculation). Bars50 µm

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Kiss L, Cook RTA, Saenz GS, Cunnington JH, Takamatsu S, Pascoe I,Bardin M, Nicot PC, Sato, Y, Rossman AY (2001) Identification oftwo powdery mildew fungi, Oidium neolycopersici sp. nov. and O.lycopersici, infecting tomato in different parts of the world. MycolRes 105:684–697

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Acknowledgments This work was supported in part by a Grant-in-Aid(12660050) from the Ministry of Education, Culture, Sports, Science,and Technology of Japan. We express our deepest thanks to professorDr. Y. Sato, Toyama Prefectural University, for his kind and valuablesuggestion on taxonomic analysis of the powdery mildew pathogendescribed in the present study.

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