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La Cladosporiosis o también conocida enfermedad del moho de la hoja en el tomate, es causada por Fulvia fulva (Syn. Cladosporium fulvum, Passalora fulva Cooke). Este hongo en base a su filogenia y morfología se ubica en el reino Fungi, phylum Ascomycota, clase Dothideomycetes, orden Capnodiales, familia Mycosphaerellaceae. La enfermedad del moho de la hoja predomina en zonas de producción de tomate de la Argentina en las que se dan condiciones de alta humedad y temperaturas medias a altas, lo que ocurre solo en algunos lugares a campo y luego en los invernaderos. El hongo F. fulva pasa las etapas con ambientes desfavorables como la temporada fría del invierno como saprótrofo o en estado conidial o en estructuras de resistencia, microesclerocios, sobre los residuos del cultivo y/o en el suelo por un período de hasta un año. Cuando la condición es favorable los microesclerocios germinan y genera conidios, que actúan como inóculo primario. La infección de F. fulva comienza con la dispersión de los conidios que en condiciones de alta humedad se adhieren a la superficie de la hoja, produciendo sobre esta, hifas que ingresan a los tejidos de la hoja por los estomas abiertos y colonizan el espacio intercelular. El hongo desarrolla en el espacio apoplástico hasta que despliega conidióforos que aparecen en la cara abaxial de las hojas infectadas. La interacción F. fulva-tomate cumple con la relación gen a gen, en la que cada producto genético de avirulencia de patógenos (avr) es reconocido por una proteína receptora codificada por el gen de resistencia dominante (Cf) del huésped correspondiente. En los últimos tiempos se han aislado las proteínas efectoras o de avirulencia de F. fulva (Avr2, Avr4, Avr4E, Avr5 y Avr9), así como también las proteínas extracelulares (Ecp1, Ecp2, Ecp4, Ecp5 y Ecp6). F. fulva tiene, al menos potencialmente, la capacidad de sintetizar no solo una gran diversidad de proteínas efectoras que además pueden combinarse y así de esa manera se ha definido la existencia de 17 razas de F. fulva las que difieren en su capacidad para sintetizar proteínas efectoras. La base de la patogenicidad que F. fulva tiene en las plantas de tomate es el resultado de la interacción de las efectoras que interaccionan con los híbridos de tomate y cuando no son detectadas por los genes de resistencia Cf vulneran los mecanismos de defensa de la planta. Así se planteó el objetivo general de esta tesis que fue ampliar el análisis de aislados de F. fulva, evaluar los mismos a nivel de los genes de avirulencia y evaluar de esta manera el potencial desarrollo de herramientas para el manejo del moho de la hoja del tomate. En cuanto a los objetivos específicos, estos consistieron en disponer de un análisis morfo-fisiológico y molecular de los aislados de F. fulva, e identificar y caracterizar las razas del hongo que conviven en las áreas de producción muestreadas y si hay una dinámica de cambios genéticos en la población del patógeno. Otro objetivo específico fue disponer de una caracterización a nivel de los efectores de F. fulva y genes que codifican proteínas extracelulares de manera de conocer no solo que razas del hongo están presentes en la región, sino también, si el sistema se encuentra evolucionando bajo presión de selección. Otro objetivo específico fue conocer la naturaleza química de las melaninas que sintetiza F. fulva y su vía de síntesis. Por último se planteó el objetivo de conocer los metabolitos secundarios que sintetiza F. fulva, que es hemibiótrofo, y su comparación con el perfil de un patógeno necrotrófico, que eventualmente podrían interactuar con las plantas de tomate y resultar en interacciones compatibles o incompatibles. Por ello en esta tesis en primer lugar, se procedió a aislar e identificar a partir de plantas de tomate con síntomas típicos del moho de la hoja del tomate, creciendo en lotes de producción, a F. fulva. Se amplió el número de aislados del hongo y se depositaron en la colección del Centro de Investigaciones de Fitopatología CIDEFI. Esto se realizó utilizando el medio de cultivo agar-agua en el que el hongo desarrolló un pigmento púrpura. Una vez que se obtuvieron cultivos monospóricos de F. fulva en un numero adicional de 8 lo que hizo un total de 14 aislados si consideramos los obtenidos y descritos en la mencionada colección, se confirmó la identidad de los aislados amplificando y secuenciando los amplicones de las secuencias intergénicas no transcriptas que fueron 100% homólogas a la secuencia del ITS de la cepa tipo de F. fulva. Se procedió a analizar el crecimiento y la diversidad genética con marcadores ISSR y se encontró que además mostró una considerable diversidad genética que no estuvo relacionada con el cultivar de origen del aislado ni con el sitio de aislamiento. Los nuevos aislados nuevamente se clasificaron dentro de las dos razas ya descritas anteriormente 0 y 2. Así se procedió a la amplificación de los genes de avirulencia de F. fulva y a las proteínas ECP con pares de primers homólogos a las secuencias descritas de los avr y de las ECP. La obtención de amplicones del tamaño esperado y la secuencia de los mismos confirmaron la identidad de los aislados que fueron todos representantes de F. fulva. Pero además se confirmó que estas dos razas se encuentran evolucionando ya que se describieron cambios en las secuencias de los genes avr y ecp. Siendo este el primer trabajo que describe la ocurrencia de polimorfismos a nivel de las proteínas Ecp. En cuanto al análisis de los pigmentos que sintetiza F. fulva las características químicas y fotoquímicas del pigmento oscuro sintetizado por F. fulva, así como la presencia de flaviolina cuando las reductasas fúngicas son inhibidas demuestran que el hongo sintetiza melanina-DHN. Por otro lado, se demostró que el gen pks1 está implicado en la síntesis de los pigmentos oscuros, puesto que codifica para un dominio KS ya asociado a otras PKS involucradas en la síntesis de melanina-DHN. Por último, en lo que hace al perfil de metabolitos secundarios sintetizados por F. fulva, éste sintetiza y libera cuantitativamente menoscompuestos orgánicos volátiles (COVs) que S. lycopersici, hongo necrotrófico que provoca mancha gris de la hoja del tomate. F. fulva sintetizó un espectro específico y más diverso de COVs, que contrasta con la cantidad de COVs liberada por S. lycopersici. La producción de alcoholes furfurilo y compuestos derivados por F. fulva y S. lycopersici se encontró que estuvo asociado al desarrollo de síntomas en las hojas. Por otro lado, es evidente que F. fulva produce compuestos que contribuyen a la colonización de los tejidos. Estos resultados constituyen las bases para futuros estudios destinados al control de estas enfermedades en las zonas de producción de tomate de la Argentina, como el Cinturón Hortícola Platense y Corrientes., Facultad de Ciencias Agrarias y Forestales

Die Samtfleckenkrankheit, verursacht durch den pilzlichen Erreger Fulvia fulva (syn. Passalora fulva) stellt im ökologischen Tomaten-Anbau ein zunehmendes Problem dar, da bisherige, monogen bedingte Sortenresistenzen durch veränderte Pathotypen des Pilzes durchbrochen werden. Ziel des Projekts ist es daher, in dem Pathosystem Tomate-Fulvia fulva erstens eine neue, auf das System angepasste Züchtungsstrategie (F4-Kreuzungsnachkommen) zu erproben, zweitens durch klassische Kreuzungszüchtung neue Resistenzquellen zu erschließen und zu validieren und drittens die Pathotypenverteilung und -dynamik deutschlandweit zu erfassen. Beim Projektpartner Oldendorfer Saatzucht (Kultursaat e. V.) standen zahlreiche Tomatenlinien mit interessanten Eigenschaften für die Züchtungsarbeit zur Verfügung. Diese Linien wurden im Rahmen des Projektes züchterisch weiterentwickelt und die Eigenschaften jeder neuen Nachkommensgeneration in Resistenzversuchen unter standardisierten Bedingungen am JKI überprüft. Dabei kamen Fulvia fulva-Isolate aus Frankreich (MATREF; Ff:0, Ff:2.4.5) und eigene Isolate von den zwei Züchterstandorten (Ff:1.2.5 aus Holste, Ff:2.3.6.9 bei Überlingen) zum Einsatz. Im Ergebnis der vierjährigen Arbeiten konnten mehrere Linien mit vielversprechenden Resistenzeigenschaften auch gegenüber den neuen aggressiven Ff-Pathotypen selektiert werden. Um handelsfähige Sorten mit guten Produkt- und Resistenzeigenschaften zu erhalten, ist jedoch weitere Züchtungsarbeit erforderlich. Um die Dynamik der Rassenentwicklung des Pathogens sowie dessen regionale Verbreitung erfassen zu können, wurde das Vorkommen verschiedener (neuer) Pathotypen von F. fulva in Deutschland über mehrere Jahre erfasst. Bereits mit den vorliegenden Daten kann eine aktuelle räumliche Verteilung der unterschiedlichen Pathotypen dargestellt werden. Daraus ergeben sich wichtige Hinweise für die Züchtung und die Praxis wie die Relevanz bestimmter Resistenzgene oder auch die Nutzung von Sorten mit einem dem Pathotyp am Anbauort entsprechenden Resistenzspektrum. Fazit: Die erzielten Ergebnisse leisten einen wichtigen Beitrag zur Sicherung der Ertragsstabilität im ökologischen Tomaten-Anbau. Es wurden für die Entwicklung der neuen Züchtungsstrategie deutliche Fortschritte erreicht, auf denen zukünftig aufgebaut werden kann. Mit dieser Strategie können längerfristig gegenüber der Samtfleckenkrankheit widerstandfähige Tomatensorten entwickelt werden. Außerdem wurde ein aktuelles Bild der Ff-Pathotypenverteilung in Deutschland gewonnen, das der Züchtung und den Anbauern wertvolle Hinweise für den Umgang mit der Krankheit in der Praxis gibt. Das Projekt wird durch das Bundesministerium für Ernährung und Landwirtschaft aufgrund eines Beschlusses des Deutschen Bundestages im Rahmen des Bundesprogramms Ökologischer Landbau und andere Formen nachhaltiger Landwirtschaft gefördert.

Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during colonisation of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre-penetration phase and during conidiation at the end of the infection cycle, but they are repressed during the biotrophic phase of tomato colonization. It was suggested that tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, while retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over-produce cladofulvin during the biotrophic growth phase. Non-producers infected tomato similar to wild type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over-producers caused strong necrosis and desiccation of tomato leaves, which in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low temperature stress. Overall this study demonstrates that repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, while its production is important for survival outside its host.

S.G., J.C., C.H.M. and P.J.G.M.d.W. were financially supported by a grant from the Royal Netherlands Academy of Sciences.; Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during the colonization of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre-penetration phase and during conidiation at the end of the infection cycle, but are repressed during the biotrophic phase of tomato colonization. It has been suggested that the tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, whilst retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over-produce cladofulvin during the biotrophic growth phase. Non-producers infected tomato in a similar manner to the wild-type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over-producers caused strong necrosis and desiccation of tomato leaves, which, in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low-temperature stress. Overall, this study demonstrates that the repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, whereas its production is important for survival outside its host.

Fulvia fulva (syn. Cladosporium fulvum, Mycosphaerellaceae) is a dematiaceous fungus that causes tomato leaf mould. It is characterized by its biotrophic lifestyle and the synthesis of the bianthraquinone secondary metabolite, cladofulvin. The aim of the study was to characterize the dark pigment photochemically synthesized by F. fulva and to elucidate its biochemical pathway. We isolated a black pigment from in vitro cultures of the fungus. We determined the pigment to be 1,8-dihydroxynaphthalene (DHN)-melanin based on its chemical and photochemical characteristics, as well as the presence of flaviolin, when fungal reductases were inhibited by tricyclazole. Furthermore, the pks1 gene involved in pigment synthesis has a KS domain already associated with DHN-melanin. Our findings support the relevance of studying melanization in F. fulva., Centro de Investigaciones de Fitopatología, Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas, Instituto de Fisiología Vegetal, Instituto de Botánica "Dr. Carlos Spegazzini"

Leaf mold symptoms were found on commercial tomato cultivars carrying the Cf-9, a resistance gene to leaf mold pathogen Fulvia fulva in 2012 at Buyeo, Chungnam in Korea. Fifteen-fungal isolates were obtained from four Cf-9 cultivars of tomato including ‘Cutie’, ‘Dotaerangdia’, ‘Unicorn’ and ‘Rapito’. Due to their same morphological appearances and colony color, nine isolates were selected and identified as F. fulva based on molecular analysis of the internal transcribed spacer rDNA sequence. Pathogenicity of the 15 isolates on five commercial cultivars carrying Cf-4, Cf-5, and Cf-9 were tested. All the isolates showed strong pathogenicity on Cf-9 cultivars, ‘Cutie’ and ‘Dotaerangdia’, and Cf-5 cultivar, ‘Yoyocaptain’. In contrast, on Cf-4 cultivar, ‘Superdotaerang’, five isolates were virulent and the other isolates were not. In addition, two fungal isolates, infecting Cf-9 cultivar and non-infecting Cf-4 cultivar, were selected and their pathogenicity was tested on 17 commercial cultivars reported as tomato having Cf-9 resistance gene. Among them, 15 cultivars were susceptible and 2 cultivars were resistant. It is likely that the two cultivars include other resistance gene. To our knowledge, this is the first report on the occurrence of Cf-9 infecting F. fulfva strains in Korea.

von Kerstin Wagner Mit englischer Zusammenfassung Universität für Bodenkultur Wien, Univ., Masterarbeit, 2016

In order to develop a environmentally friendly control protocol for managing tomato leaf mold disease in the field, we employed bacteria- and fungi-based commercially available microbial preparations. The field experiment was conducted from April to July in 2010. Average incidence rates tomato leaf mold caused by Fulvia fulva were 13.1% at the two plastic houses located in Jangsung, Jeonnam area. Initially 11 microbial preparations were tested for antifungal activity against F. fulva in vitro. Among them, 7 selected preparations showed to be inhibited the mycelial growth of the fungal pathogen over 50%. Four microbes suppressed disease incidence as much 50% under greenhouse condition. Eventually in the field two microbial products including Bacillus subtilis GB-0365 and B. subtilis KB-401 respectively were showed control value up to 71.8% for four times sprays from 20 days to 70 days after transplanting. Furthermore, the control value of three times spray program demonstrated 79.3%. Efficacy of the three and four spray programs was more effective than that of non-spray control treatment. Our results indicated that adjustment of application method of commercially available microbial preparation could be used to control a target plant disease as an effective and efficient crop protection system for organic farming.

This study was performed to develop a formulation using an antagonistic bacterium Bacillus amyloliquefaciens A-2 to control tomato leaf mold caused by Fulvia fulva. B. amyloliquefaciens A-2 was grown in a medium with rice oil and mixed with various carrier and additives. One of the formulations, A2-MP, showed the best disease control value among the tested formulations. The disease control value of A2-MP at 100-fold and 500-fold diluted treatment was not significantly different from that of chemical fungicide triflumizole in a growth chamber. Although disease control effect was decreased by serial diluted treatment of the prepared A2-MP, 1,000-fold diluted treatment of A2-MP still showed high disease control value of 72.0%. For the green house experiments, the disease control values of A2-MP was indicated as 79.4% which is similar to that of chemical fungicide, triflumizole showing 79.6%. When the disease control activity of the formulation A2-MP was compared in tomato production conditions, disease control values of 100-fold diluted A2-MP and 3,000 fold diluted triflumizole exhibited 60%, 81.6%, respectively. The disease control efficiency by A-2MP was 73% of the disease control value of chemical fungicide. The formulation A-2MP maintained the stable bacterial viability and disease control activity when stored at . This result suggested that A-2MP develped from B. amyloliquefaciens A-2 could be used to control tomato leaf mold.

Chitosan has an antifungal activity and is widely used for control of various plant disease and plants growth in the field in Korea. Disease control efficacy of two preparations (SH-1, SH-2) of mixtures of high and low (chitooligosaccharide) molecular weight chitosan compounds against tomato leaf mold caused by Fulvia fulva was investigated under plastic greenhouse conditions. Both SH-1 and SH-2 formulations displayed potent disease control activity in two experiments. The protective activity of both preparations was comparable to synthetic thiophanate-M. The persistence activity of the formulations was sustained until 21 days after application. Effective concentration of the chtosan compounds for disease control was 1,200 mg a.i./L. In pot tests, chitosan preparations, at a concentration of 600 mg a.i./L, promoted plants growth. These results indicate that the chitosan preparations have a potential as an eco-friendly natural fungicide for the control of tomato leaf mold and plant growth regulator.

Tomato leaf mold caused by Passalora fulva was found on two tomato varieties carrying the Cf-9 gene in Japan, in 2007. The isolates obtained from Chiba and Fukushima were identified as race 4.9.11, and those from Gunma were races 4.9 or 4.9.11. This is the first report in Japan of tomato leaf mold caused by P. fulva strains that can overcome the Cf-9 gene.

Les cultures de tomate peuvent être attaquées par de nombreux agents pathogènes et ravageurs, aussi bien aériens que telluriques. Suivant le type de culture (en plein champ, sous abris froids ou chauffés), l’importance relative de ces maladies et ravageurs est très différente.

eingereicht von Philipp Lammer Zsfassung in engl. Sprache Wien, Univ. für Bodenkultur, Masterarb., 2014

Leaf mold symptoms were found on tomato varieties carrying the Cf-9 resistance gene against Passalora fulva, the causal agent of leaf mold, in Japan in 2008. Disease symptoms and morphological characteristics of the isolates were similar to those of P. fulva. After inoculating a set of tomato differentials with the isolates, all isolates were identified as race 2.9 of P. fulva, previously unreported.

Races of 131 isolates of tomato leaf mold fungus, Fulvia fulva, collected from 30 prefectures of Japan during 1997-1998 were identified. Four races, race 0, 2, 2.4 and 2.4.11 were detected. Race 2 was predominant with 113 isolates identified as this race. All four races were found in Kanto district, but only race 2 was detected in Chugoku, Shikoku and Kyushu districts. Race 0 and 2 were thought to be identical to the known races in Japan. However, race 2.4 and 2.4.11 had a virulence pattern different from the known races in Japan.

Raziskava je potekala v letih 2006 in 2007 na Univerzitetnem kmetijskem centru Fakultete za kmetijstvo in biosistemske vede. Vključevala je več genetsko in morfološko različnih sort in njihovih križancev paradižnika, s ciljem, da bi preverili odpornost na nekatere najpomembnejše bolezni, analizirali ključne agromorfološke lastnosti in ocenili primernost posameznih genotipov za kmetijsko pridelavo. Poskus je bil nastavljen po standardni zasnovi naključnih blokov v štirih ponovitvah. Med kvantitativnimi lastnostmi je bila največja variabilnost ugotovljena pri višini rastline in dolžini peclja ploda. Velika variabilnost je bila ugotovljena tudi pri nekaterih kvalitativnih lastnostih kot so oblika, barva in čvrstost ploda. Najbolj čvrste korelacijske povezave so bile ugotovljene med dimenzijami plodov. V letu 2006 je bila ugotovljena dokaj močna okužba z žametno pegavostjo (Fulvia fulva), leto kasneje pa je prevladovala krompirjeva plesen na paradižniku (Phytophthora infestans). Leta 2006 je bila ugotovljena tudi prisotnost črne listne pegavosti (Alternaria solani) na plodovih. Raziskava je pokazala, da so bili nekateri križanci visoko kakovostni in bi jih bilo smiselno vključiti v žlahtnjiteljski program, ali pa jih še nekaj generacij samooplojevati, da bi dosegli želeno homozigotnost in jih nato testirati kot potencialne nove sorte. The research was carried out during 2006 and 2007 at the University Agricultural Centre of the Faculty of Agriculture and Life Sciences. It included a number of genetically and morphologically different varieties of tomato and their hybrids, with the aim to test resistance against some of the most important diseases, to analyse key agro-morphologic characteristics and assess suitability of individual genotypes for agricultural production. The experiment was based on randomised complete block design, in four replications. In terms of quantitative characteristics, the largest variability was found in relation to the height of plants and length of the fruit stalk. Significant variation was also found in terms of qualitative characteristics such as fruit shape, colour and firmness, whereas the strongest correlation was noted between different dimensions of the fruit. Rather strong infection with leaf mold (Fulvia fulva) was noted in 2006, while a year later, late blight (Phytophthora infestans) was the predominant disease. Early blight (Alternaria solani) was frequently observed on fruits in 2006. Research showed that some hybrids were of high quality and it would make sense to include these in breeding programmes or else continue with their self-pollination for a number of future generations in order to achieve the desired homozigosity, and subsequently test them as potential new varieties.

Fulvia fulva, the agent of leaf mould, is known to be a major parasite of tomato leaves in greenhouses. Its development is characterized by a biotrophic phase followed by a necrotrophic phase. Hansfordia pulvinata is a hyperparasite of this pathogen, but its destructive action is restricted by its inability to colonize F. fulva during the biotrophic phase, before lesion formation. An integrated system to optimize the mycoparasitic activity of H. pulvinata was studied. The hyperparasite was tolerant to fosetyl-Al in situ. At a minimal concentration of 500 mg/L, the fungicide inhibited F. fulva spore germination after spraying on tomato leaves but did not have any effect during the necrotrophic phase. Thus, fosetyl-Al, which prevented reinfection, and the destructive hyperparasite H. pulvinata had complementary effects. Key words: Hansfordia pulvinata, fosetyl-Al, Fulvia fulva, integrated control, leaf mould, tomato.

Propolis etanol ekstraktının (PEE) 7 farklı konsantrasyonunun (0.0, 0.03, 0.06, 0.25, 0.5, ve 1 ppm) Verticillium dahliae Kleb., Fulvia fulva (Cooke) Cif., ve Penicillium digitatum Sacc. ’a karşı antifungal etkileri in vitro koşullarda araştırılmıştır. Propolis, farklı konsantrasyonlarda PDA besi ortamına karıştırılmış veya tekli olarak kullanılmıştır. Deneme sonucunda, PEE’nin artan konsantrasyonlarında, denemeye alınan fungusların miselyal gelişiminde azalma ortaya çıktığı belirlenmiştir. Propolisin V. dahliae üzerine etkinliği 1.0 ve 0.5 ppm konsantrasyonlarında sırasıyla % 84.8 ve % 83.3 olurken 0.06 ve 0.03 ppm’de % 2.1 ve % 33.9 olarak saptanmıştır. Propolis ekstraktı, en yüksek konsantrasyonda Fulvia fulva’ya karşı % 80.1 oranında etkili bulunmuştur. Propolisin en düşük konsantrasyonu (0.03 ppm), % 19.3 oranında etkisiz olmuştur. Propolisin etanol ekstraktının P. digitatum üzerine etkisi, 1. 0 ppm ve 0. 5 ppm konsantrasyonlarında sırasıyla % 86.2 ve % 79.3 olarak saptanmıştır. The antifungal effects of 7 concentrations (0.0, 0.03, 0.06, 0.125, 0.25, 0.5, and 1 ppm) of propolis ethanol extract (PEE) against Verticillium dahliae Kleb., Fulvia fulva (Cooke) Cif., and Penicillium digitatum Sacc. was investigated in vitro. Propolis was mixed alone or in combination with potato dextrose agar (PDA) medium at various concentrations. The results indicated that the mycelial growth of the tested fungi decreased with each increase in PEE concentrations. The effectiveness of the propolis on V. dahliae was 84.8 % and 83.3 % at 1.0 ppm and 0.5 ppm concentrations, while it was 52.1 % and 33.9 % at 0.06 ppm and 0.03 ppm, respectively. Propolis extract had an effect 80.1 % at the highest concentration against Fulvia fulva. The lowest concentration of propolis (0.03 ppm) was ineffective with 19.3 %. The effect of ethanol extract of propolis on Penicillium digitatum was 86.2 % and 79.3 % at 1.0 ppm and 0.5 ppm concentrations, respectively. The lowest concentration of propolis (0.03 ppm) had an effect 29.4 %.

Leaf mold of tomato was found on cv. Momotaro-fight in 2003 in Ehime Prefecture. The symptoms were marginal indefinite yellowing on the upper leaf surface, and downy, gray to brown sporulation on the lower surface underneath the spots. The symptoms and morphology were the same as seen with Passalora fulva. The fungal isolates were identified as races 2.4, 2.4.11, 4, and 4.11 by inoculation tests. Races 4 and 4.11 have never before been found in Japan.

Tomato (Solanum lycopersicum L.) is one of the most commonlyused vegetables worldwide. The production of organic tomatoes,grown without using any chemicals, has continuously increased inKorea since consumers started to demand organic foods. In organictomato greenhouses in Iksan and Jeonju, Korea, a change wasobserved in a tomato leaf mold ( Passalora fulva , syn. Fulvia fulva )in spring, 2009. The dark brown lesions of this mold changed intowhite to pale grayish ones, by growth of an overgrowing fungus(Fig. 1A). On some old leaves, the leaf mold lesions were largelydestroyed and covered with the white fungus (Fig. 1 B & C). It wasalso observed that young colonies of leaf mold were suppressed bythe white fungus, and further expansion of the lesions was arrested.The overgrowing white fungus was microscopically examinedfrom representative samples, which are housed at Korea University(KUS-F24001, F24017). Conidiophores were erect, straight, smooth,variable in length, reaching up to 500 µm, 2-4 µm wide, branchedrepeatedly at the upper part, pale olivaceous brown (Fig. 1D).Conidiogenous cells were integrated, terminal, cylindrical, sub-hyaline, denticulate, 7-15×2-3.5 µm (Fig. 1E & F). Conidia weresolitary, globose, smooth, aseptate, subhyaline, 4-6µm, with aninconspicuous hilum (Fig. 1G). Based on these morphologicalcharacteristics, the fungus was identified as Hansfordia pulvinata(Berk. & M. A. Curtis) S. Hughes (Saccardo, 1886).To confirm the identity of the fungus, the ITS rDNA region of anisolate obtained from KUS-F24001 was amplified and sequenced.A resulting sequence of the isolate was deposited in GenBank(HM060587). A BLAST search indicated that the ITS sequenceshared 98% similaritywith a sequence of H. pulvinata (AY908993). Monoconidial isolates of H. pulvinata were successfully cultur-ed, forming velvety grayish colony with abundant conidia on PDA(Fig. 1H). The isolates were deposited in Korean AgriculturalCulture Collection, Suwon, Korea (KACC44498 and 44502). Amycoparasitic test was carried out using detached leaves from 1-month-old tomato plants infected with P. fulva . An inoculum (ca. 10

A procedure for producing high yields and high regeneration frequencies of protoplasts from Fulvia fulva is described. This procedure was devised by systematic trials of various parameters, such as culture age and osmotic support, which are known to affect protoplast yield and regeneration. Mycelium from liquid cultures of 24–48 h, incubated with Novozym 234 in buffered 1.0 M MgSO4, gave the best conditions for protoplast release. Regeneration frequencies up to 50% were obtained with a complete medium containing 0.8 M sucrose for osmotic support.

Races ofCladosporium fulvum, which can overcome the resistance of the genes Cf2, Cf4, Cf5, Cf8, Cf9 and Cf11 have appeared in the Netherlands, France and Poland. Known isolates from the Netherlands and France and three new isolates from Poland have been investigated for the presence of virulence genes using a set of genotypes carrying resistance genes Cf2 to Cf11.Several Dutch isolates of races, earlier designated as 2.4, 2.4.5 and 2.4.5.9, were found to break down the resistance gene Cf11. These races must therefore be designated as 2.4.11, 2.4.5.11 and 2.4.5.9.11 respectively. In the new Polish isolates virulence genes, overcoming the resistance genes Cf2, Cf4, Cf8, Cf9 and Cf11 were found. Since all races able to grow on genotypes with Cf4, could also grow on genotypes carrying Cf8, it was impossible to discriminate between the genes Cf4 and Cf8. These Polish isolates were designated as races 4.11, 2.4.11 and 2.4.9.11. The consequences of the occurrence of these races for tomato breeding are discussed. Fysio's vanCladosporium fulvum, die de resistentie-genen Cf2, Cf4, Cf5, Cf8, Cf9 en Cf11 kunnen doorbreken, zijn in Nederland, Frankrijk en Polen opgetreden. Met behulp van een groep genotypen, die de resistentie genen Cf2 tot en met Cf11 dragen, zijn Nederlandse, Franse en enkele nieuwe Poolse isolaten onderzocht op de aanwezigheid van virulentiegenen. Enkele Nederlandse isolaten, eerder aangeduid met 2.4, 2.4.5. en 2.4.5.9, bleken het resistentie-gen Cf11 te kunnen doorbreken. Deze moeten daarom aangeduid worden als respectievelijk 2.4.11, 2.4.5.11 en 2.4.5.9.11. In de nieuwe Poolse isolaten werd virulentie gevonden voor Cf2, Cf4, Cf8, Cf9 en Cf11. Alle fysio's die op genotypen met Cf4 konden groeien, groeiden ook op genotypen met Cf8. Daarom kon geen ondersheid gemaakt worden tussen Cf4 en Cf8. De Poolse isolaten behoren tot de fysio's 4.11, 2.4.11 en 2.4.9.11. De gevolgen van het voorkomen van deze fysio's voor de tomateveredeling worden besproken.

SUMMARY Fumigation for 1 hr in a 50 cu. ft (1.42 cu. m) chamber with a rate eqmvalent to 0.75 lb potassium permanganate/3 pt formaldehyde/ 6,000 cu. ft (0.34 kg/1.70 1./169.90 cu. m) killed spores of Botrytis cinerea, Fulvia fulva and Fusarium culmorum, though in a series of 1 hr fumigations based on this rate the mycelium of B. cinerea was not killed. Fumigations for 24 hr in a 50 cu. ft (1.42 cu. m) chamber and a commercial glasshouse with 0.75 lb potassium permanganate/3 pt formaldehyde/6,000 cu. ft (0.34 kg/1.70 1./169.90 cu. m) and 0.8 lb/ 1.25pt/6,000cu. ft (0.36 kg/0.71 1./169–90 cu. m) killed both spores and mycelium of B. cinerea.

In an allelic test it was proven that the genes Cf4 and Cf8 for resistance toC. fulvum in tomato are undistinguishable, confirming a recent suggestion that Cf8 does nott provide a novel source for resistance toC. fulvum.

A description is provided for Fulvia fulva . Information is included on the disease caused by the organism, its transmission, geographical distribution, and hosts. HOSTS: Lycopersicon esculentum . DISEASE. Leaf mould of tomato is a major disease of this crop. The first symptoms are pale chlorotic spots (margins indefinite) on the upper leaf surface. Sporulation, on the lower surface beneath the spots, is downy, light grey, becoming buff to tawny brown or olive green. Defoliation may occur. Infection of blossoms and fruits is much less important. There was an interval of c . 6 weeks between the incidence of severe leaf colonization (50% leaf area) and decreases in yield (48, 1982). GEOGRAPHICAL DISTRIBUTION: Worldwide (CMI Map 77, ed. 5, 1972). TRANSMISSION: By air-dispersed conidia; these were considered to be viable for 9-12 months under adverse conditions (18, 142). Seed contamination may occur.

Leaf mould of tomato caused by Cladosporium fulvum (syn. Fulvia fulva) has proved to be a useful system for studying mechanisms of host specificity. There are several important advantages to this host-pathogen combination. First, it is a gene-for-gene interaction for which there is available a good collection of C. fulvum races and tomato cultivars with one or a combination of genes for resistance. Some cultivars are near-isogenic, and as well, several races can be considered near-isogenic because of their origin [1,2]. Second, resistance occurs just after stomatal penetration and is rapid and localized [3]. Third, the fungus grows as a biotroph in a strictly intercellular manner [4], a feature which permits the use of intercellular wash fluids in some studies [2,5,6,7] and which also allows for direct contact between races in a doubly inoculated plant. This latter feature was important in this study.

VanCladosporium fulvum, de veroorzaker van bladvlekkenziekte van tomaat, zijn talrijke fysio's bekend. Van tomaterassen met resistentiegen Cf5 werd, zowel bij materiaal uit Bulgarije als uit Frankrijk, een nieuw fysio, 2.5, geisoleerd. Dit bezit naast het noodzakelijke virulentiegen 5 ook het overbodige gen 2. Dit is weer een voorbeeld van afwezigheid van ‘stabilising selection’.

Cladosporium fulvum staat bekend om zijn vermogen zich snel aan te passen aan nieuw geintroduceerde resistentiegenen. Die laatste jaren bleven tomaterassen met ‘C5 resistentie’ in de praktijk resistent. Deze rassen zijn zelfs resistent tegen het complexe fysio 2.4.5 door het bezit van Cf9 of een ander resistentie-gen. Een fysio 2.4.5.9 trad weliswaar op bij proeven op het IVT (Wageningen), maar werd nooit in de praktijk gevonden. In 1985 werd in Frankrijk het ras Rianto F1 in een praktijkteelt aangetast. Een isolaat vanC. fulvum van dit C5-resistente ras bleek fysio 2.5.9 te zijn. Een deel van de thans bij onderzoek gebruikte resistentiebronnen, zoals Bulgaria no. 1, Japan 74S en Japan 76S, is resistent voor dit nieuwe fysio. Het optreden van dit nieuwe fysio onderstreept de wenselijkheid ten minste twee ‘nieuwe’ resistentiegenen in nieuwe rassen te gebruiken ter verbetering van de duurzaamheid van de resistentie.