Hellenic Plant Protection Journal

December 2015 ISSN 1791-3691 Hellenic Plant Protection Journal

Special Issue Abstracts 16th Hellenic Phytopathological Congress

A semiannual scientifi c publication of the BENAKIBEB PHYTOPATHOLOGICAL INSTITUTE EDITORIAL POLICY The Hellenic Plant Protection Journal (HPPJ) (ISSN 1791-3691) is the scientifi c publication of the Benaki Phytopathological Institute (BPI) replacing the Annals of the Benaki Phytopathological Institute (ISSN 1790-1480) which had been published since 1935. Starting from January 2008, the Hellenic Plant Protection Journal is published semiannually, in January and July each year. HPPJ publishes scientifi c work on all aspects of plant health and plant protection referring to plant pathogens, pests, weeds, pesticides and relevant environmental and safety issues. In addition, the topics of the journal extend to aspects related to pests of public health in agricultural and urban areas. Papers submitted for publication can be either in the form of a complete research article or in the form of a suffi ciently documented short communication (including new records). Only original articles which have not been published or submitted for publication elsewhere are considered for publication in the journal. Review articles in related topics, either submitted or invited by the Editorial Board, are also published, normally one article per issue. Upon publication all articles are copyrighted by the BPI. Manuscripts should be prepared according to instructions available to authors and submitted in electronic form on line at http://www.hppj.gr. All submitted manuscripts are considered and

Hellenic Plant Protection Journal Protection Hellenic Plant published after successful completion of a review procedure by two competent referees. The content of the articles published in HPPJ refl ects the view and the offi cial position of the authors. The information and opinions contained herein have not been adopted or approved by the HPPJ Editorial Board. The HPPJ Editorial Board neither guarantees the accuracy of the information included in the published articles nor may be held responsible for the use to which information contained herein may be put. For all parties involved in the act of publishing (the author(s), the journal editor(s), the peer reviewers, and the publisher) it is necessary to agree upon standards of expected ethical behavior. HPPJ follows the ethics statements of De Gruyter journals, which are based on the Committee on Publication Ethics (COPE) Code of Conduct guidelines available at www.publicationethics.org.

EDITORIAL BOARD Editor: Dr F. Karamaouna (Pesticides Control & Phytopharmacy Department, BPI) Associate Editors: Dr A.N. Michaelakis (Entomology & Agric. Zoology Department, BPI) Dr K.M. Kasiotis (Pesticides Control & Phytopharmacy Department, BPI) Dr I. Vloutoglou (Phytopathology Department, BPI) Editorial Offi ce: M. Kitsiou (Library Department, BPI) A. Karadima (Information Technology Service, BPI)

For back issues, exchange agreements and other publications of the Institute contact the Li- brary, Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia, Attica, Greece, e-mail: [email protected]. This Journal is indexed by: AGRICOLA, CAB Abstracts-Plant Protection Database, INIST (Institute for Scientifi c and Technical Information) and SCOPUS.

The olive tree of Plato in Athens is the emblem of the Benaki Phytopathological Institute

Hellenic Plant Protection Journal also available at http://www.hppj.gr and www.bpi.gr

16th Hellenic Phytopathological Congress

Thessaloniki, Greece, October 16-18, 2012 Organizing Committee

Chairman: Professor Katis Nikolaos Aristotle University of Thessaloniki

Vice Chairman: Emeritus Professor Tzavella-Klonari Kate Aristotle University of Thessaloniki

Secretariat: Assistant Professor Karaoglanidis George Aristotle University of Thessaloniki

Treasurer: Assistant Professor Antoniou Polymnia Agricultural University of Athens

Members: Assistant Professor Lagododi Anastasia Aristotle University of Thessaloniki

Lecturer Maliogka Varvara Aristotle University of Thessaloniki

Polymerou Vaios Regional Center for Crop Protection and Quality Control of Thessaloniki

Bozoglou Kontantinos BASF Hellas SA

Veloukas Thomas Aristotle University of Thessaloniki Hellenic Plant Protection Journal, Special Issue: 1-91, 2015 DOI 10.1515/hppj-2015-0011

ABSTRACTS

Summaries of invited lectures, oral and poster presentations given at the 16th Hellenic Phytopathological Congress Thessaloniki, Greece, October 16-18, 2012

The 16th National Phytopathological Congress, organized every two years by the Hellenic Phyto- pathological Society (HPS), was held in Thessaloniki, Greece, on October 16–18, 2012. The meet- ing was attended by more than 400 participants, and 61 oral presentations, 63 posters and 7 invited lectures were presented dealing with plant diseases caused by fungi, bacteria and viruses, non-parasitic disorders, molecular diagnostics and characterization of pathogens, host-pathogen interaction, and biological, integrated and chemical disease control. In addition, one round table discussion was held on “Production of certifi ed propagative material of fruit trees: international experience and Greek legislation- Problems related to its application”. Abstracts of the invited lectures, oral presentations and posters of the congress are presented below.

MYCOLOGY

Abstracts - 16th Hellenic Phytopathological Congress 7

INVITED LECTURES

Phytophthora ramorum: an emerging pathogen of forest and ornamental plants in Europe and North America P. TSOPELAS Hellenic Agricultural Organization “Demeter”, Institute of Mediterranean Forest Ecosystems, Terma Alkmanos, GR-115 28 Athens, Greece

Phytophthora ramorum is an emerging patho- eries and parks. However, in the majority gen with a destructive impact on forest eco- of hosts P. ramorum causes less serious dis- systems of North America and Europe. It was eases, infecting mostly the leaves and the described as a new species in 2001, although young shoots. In some cases infections are has been known since 1993 to infect rhodo- not conspicuous; the plants are considered dendron and viburnum plants in nurseries of healthy and are transferred by trade, result- the Netherlands and Germany. P. ramorum ing in disease spread to new areas. P. ramo- is considered to be an exotic pathogen, in- rum is a quarantine organism in Europe, troduced separately into Europe and North North America and other areas of the world. America from an unknown region, speculat- Since 2002 EU regulations have been im- ed to be Asia. Up to the present, three clon- posed in order to prevent further spread of al lineages NA1, NA2 and EU1, have been dis- P. ramorum among ornamental plants in the tinguished by the use of molecular markers member countries. Surveys in the EU have in North America, while in Europe only the detected the pathogen in 20 of the 27 mem- latter (EU1) has been detected. The patho- ber countries. In Greece, P. ramorum was ini- gen has a large host range (more than 140 tially detected in the Phthiotida prefecture plant species) and the host-list continues to in 2010, on rhododendron plants imported expand, and includes a signifi cant number from Belgium. During 2011-2012 the patho- of forest trees and shrubs as well as many or- gen was found in three more areas of Greece namental plants. Certain hosts have shown a (Athens, Pelion and Drama), infecting nurs- high susceptibility to P. ramorum, with lethal ery plants of viburnum and camellia pro- stem and branch infections (formation of duced in the country, as well as rhododen- bleeding cankers). In California and Oregon, dron plants imported from Belgium. The hot the disease, known as “sudden oak death” and dry climatic conditions in many areas of (SOD), is lethal to certain oak species (Quer- Greece do not favour the spread of P. ramo- cus spp.) and tanoak (Notholithocarpus densi- rum; however, there are many suitable habi- fl o r u s ), having a devastating impact on forest tats for the establishment of the pathogen, ecosystems. Recently (in 2009) the pathogen including natural oak and beech forests in was found to cause signifi cant mortality in the highlands as well as maquis scrublands Japanese larch plantations in southern Eng- with evergreen oaks and other hosts of P. land. The disease can be also lethal to rho- ramorum. There is also the possibility of in- dodendron and viburnum plants in nurs- fection of cultivated plants.

Pomegranate diseases. Remarks on Greek and global problems E.C. TJAMOS AND P.P. A NTONIOU Laboratory of Phytopathology, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

Pomegranate cultivation has rapidly expand- This presentation refers to fi ndings concern- ed in recent years in Greece and worldwide. ing diseases of pomegranate in Greece ob-

© Benaki Phytopathological Institute 8 Hellenic Plant Protection Journal - Special Issue tained from fi eld observations, laborato- lead to secondary attacks by pathogenic ry isolations and experimental studies, and fungi, we underline the impact of frost dam- from data from international literature on age and damage by rodents. Also herbicide serious diseases. toxicities and bad cultivation practices are Our Greek data are focused on fungal, non- included. parasitic and postharvest diseases with ref- Regarding most recent international data, erences to their causes, diffi culty in diag- the fungus Ceratocystis fi briata causes severe nosis, their impact and their control. The symptoms and is considered the most seri- commonest pathogens causing fungal dis- ous pathogen of pomegranate in India, Chi- eases are Eutypa / Libertella, Botryosphaeria/ na and Iran. The disease appears as yellow- Neofusicoccum, wound pathogens of non- ing and defoliation in one or more branches parasitic origin (Cytospora / Valsa, Pestalo- in a few days or after 2-3 months to reach full tiopsis, Pestalotia) and vascular wilts (Ver- wilting and necrosis. The disease is charac- ticillium dahliae or Ceratocystis fi briataor terized by grey-brown discoloration of the Ophiostoma stenoceras-sporothrix schenckii wood vessels and adjacent tissues. complex). Regarding Greek ophiostomas Another signifi cant bacterial disease of it was demonstrated that our isolates were pomegranate is caused by Xanthomonas ax- not Ceratocystis fi briatabut of the Ophiosto- onopodis pv. punicae, which has been found ma stenoceras-sporothrix schenckii complex to cause serious damage in India and threat- that showed limited pathogenicity, caus- ens to spread to other countries. ing mild symptoms on young pomegranate Finally, viral diseases or virus-like diseases plants. have been rarely reported in pomegranates. Regarding post-harvest diseases the fungi The only references relate to a mosaic virus Botrytis cinerea, Alternaria alternata, Asper- (Cucumber mosaic virus) which causes de- gillus niger and Cytospora sp. have being fre- formation of leaves, yellowing and reduced quently observed. fl owering (1984, in Yugoslavia) and Hop As for non-parasitic diseases, which often stunt viroid (HSVD) in Turkey (in 2000).

ORAL & POSTER PRESENTATIONS

Diplodia corticola: a new pathogen of oak in Greece P. TSOPELAS1, N. SOULIOTI1 and S. PALAVOUZIS2 1Hellenic Agricultural Organization “Demeter”, Institute of Mediterranean Forest Ecosystems, Terma Alkmanos, GR-115 28 Athens, Greece. 2Laboratory of Plant Pa- thology, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

The fungus Diplodia corticola (teleomorph: fecture in Thessaly, central Greece. Infect- Botryosphaeria corticola) was reported for ed trees showed symptoms of branch and the fi rst time in Greece in 2010, to infect shoot dieback, that were more intense dur- kermes oak (Quercus coccifera) in the Mes- ing the summer period. Cankers were evi- senia prefecture (Peloponnese). In the fol- dent on infected branches, while abundant lowing years, infections from this pathogen pycnidia emerged through the bark. Fungal were also observed on holm oak (Quercus isolates from infected tissue as well as from ilex) and downy oak (Quercus pubescens) in pycnidia, on malt extract agar (MEA), were the same prefecture. Infections were also initially white with dense aerial mycelium, noted on kermes oak in the neighbouring becoming dark grey to almost black with Ilia prefecture as well as in the Karditsa pre- age. Conidia formed in culture were similar

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 9 in shape and size to those formed in the pyc- rDNA and comparison with known sequenc- nidia on the cankers; cylindrical with round- es of the fungus. Two isolates of D. corticola ed ends, hyaline and unicellular, in some were used in inoculation tests on branches cases two-celled and darker upon maturity, of kermes and holm oak trees and the fun- rarely three-celled, (24-34 x 12-17 μm). The gus was re-isolated from the cankers as well identity of D. corticola was confi rmed by se- as from pycnidia formed on the inoculated quencing the ITS1 and ITS2 regions of the branches.

First report of potato wart disease caused by Synchytrium endobioticum (Schilb.) Perc. in Greece: detection, impacts and pathotype identifi cation Ι. VLOUTOGLOU1, G.C.M. VAN LEEUWEN2, Η. ELEFTHERIADIS3, Ι. SARIGKOLI3, Κ.Β. SIMOGLOU3, D. TSIROGIANNIS1 and D. GILPATHI4 1Benaki Phytopathological Institute, Department of Plant Pathology, Laboratory of Mycology, 8 St. Delta Str., GR-145 61 Kifi ssia, Athens, Greece. 2National Reference Centre, National Plant Protection Service, PO Box 9102, 6700 HC, Wageningen, The Netherlands. 3Region of Eastern Macedonia & Thrace, Regional Unit of Drama, Rural Economy & Veterinary Directorate, Department of Quality and Phytosanitary Control, Dioikitirion, GR-661 00 Drama, Greece. 4Ministry of Rural Development and Food, Directorate General of Plant Produce, Directorate of Plant Produce Protection, 150, Sygrou Avenue, GR-176 71 Athens, Greece

Potato wart disease, caused by the quaran- infested area. In October 2011, wart mate- tine fungus Synchytrium endobioticum, was rial, collected from the two infested fi elds, detected for the fi rst time in Greece in two was sent to the NPPO of the Netherlands potato (Solanum tuberosum L.) fi elds in the (National Reference Centre, Wageningen), Perithori region (Kato Nevrokopi, Region- where tests were performed for pathotype al Unit of Drama) during the 2011 offi cial identifi cation. The wart material of the two surveys. The disease exhibited typical wart fi elds was separately composted and the re- symptoms on stolons and tubers with some sulting ‘compost’ (inoculum) was used for of the latter being largely converted into Spieckermann tests with the following dif- warts. Pest identifi cation was based on the ferential potato cultivars: cv. Markies (sus- EPPO diagnostic protocol PM 7/28 (1). Phy- ceptible to all pathotypes), Producent, Del- tosanitary measures are being implement- cora, Saphir, Miriam and Belita. Based on ed in the area in compliance with the EU the resistant reaction of cvs Saphir and Be- Council Directive 69/464/EEC. The potato lita and the susceptible reaction of cvs Mar- crops grown on the infested fi elds were de- kies, Producent, Delcora, and Miriam, it was stroyed, the fi elds were designated as infest- concluded that pathotype 18(T1) is present ed and a safety zone, allowed to be plant- in both fi elds. In the present work, the im- ed only with potato cultivars resistant to the pact of the presence of S. endobioticum in pathotype present, was defi ned around the the area is also discussed.

Development of a quantitative PCR method to diff erentiate between viable and nonviable cells of plant pathogenic fungi using propidium monoazide (PMA) M.-D. TSOLAKIDOU, Ι.S. PANTELIDES, L. KANETIS and D. TSALTAS Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus

The quantifi cation of plant pathogens using ly inhibited. In this study we evaluated the DNA-based molecular tools can be mislead- suitability of PMA treatment to distinguish ing due to an inability to distinguish signals between viable and nonviable plant patho- originating from live and dead cells. Howev- gens. A PMA-qPCR combined assay was ap- er, methods that use DNA-intercalating dyes plied to viable and inactivated fungal patho- like propidium monoazide (PMA) have been gens. Cell suspensions were incubated with used to selectively remove cells with com- PMA, and then exposed to light to secure the promised cell membranes from the analysis, intercalation of PMA with the DNA of dead which can be considered to be dead. These cells, and to inactivate any unbound PMA. dyes are nearly completely cell membrane- Treated cells were extracted and the rela- impermeable and therefore can be selective ratios of live and dead cells were evalu- tively used to modify only exposed DNA ated by qPCR. After heat treatment and DNA from dead cells while leaving DNA from vi- modifi cation with PMA, all fungal species able cells intact. Once these dyes enter a tested showed an approximate 100- to 1000- cell, they bind to DNA and can be covalently fold diff erence in cell viability as estimated crosslinked to it by light exposure. PCR am- by qPCR analysis, which was consistent with plifi cation of such modifi ed DNA is strong- estimates of viability based on culturing.

Population genetic structure of Phytophthora infestans in Cyprus L. KANETIS, L. PITTAS, D. TSALTAS and N. IOANNOU Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus

A total of 521 isolates of P. infestans, the caus- has been reported in many European coun- al agent of potato late blight, were collected tries and which is characterized by high ag- in Cyprus from 2009 to 2011. The scope of the gressiveness and resistance to the fungicide present study was the genotypic character- metalaxyl-M, appeared frequently through- ization of the local population of the micro- out the sampling period. The 2011 sampling organism including mating type and DNA was concentrated in a single potato fi eld and fi ngerprinting patterns using 12 microsatel- only genotypes of 13_A2 were identifi ed. lite markers. During the fi rst two years of the Overall, the relatively low genetic variability study, when a nationwide collection took of the Cyprus populations of P. infestans indi- place, the presence of both mating types cates the absence of sexual reproduction of was documented at a ratio of approximate- the microorganism, despite the existence of ly 1:1. In 28.5% of the sampled fi elds both both mating types. Monitoring the genetic mating types coexisted, suggesting the po- background of the local population may pro- tential for sexual reproduction. In addition, vide information on the appearance of sexu- 13 genotypes of P. infestans were identifi ed, al reproduction of the pathogen, and the po- with prevailing types 13_A2, 2_A1 and 23_ tential invasion of new genotypes, primarily A1. More specifi cally, genotype 13_A2, which from seed exporting countries.

Classifi cation of Cretan Verticillium dahliae isolates to races and their virulence characterization in diff erential hosts A.E. POMPODAKI1, E.A. MARKAKIS1 and E.K. LIGOXIGAKIS2 1Laboratory of Plant Pathology, School of Agricultural Technology, Technological Educational Institute of Crete, Stavromenos, GR-710 04 Heraklion, Crete, Greece. 2Laboratory of Plant Pathology, Plant Protection Institute of Heraklion, Hellenic Agri- cultural Organization “Demeter”, Mesa Katsampas, GR-710 03 Heraklion, Crete, Greece

Race classifi cation and host range pathoge- (3.2 %). The pathogenicity of isolates on the nicity determination of 32 Verticillium dahl- aforementioned fi ve hosts was investigat- iae isolates, originating from 19 plant spe- ed on the basis of external symptoms and cies from 8 diff erent botanical families, were by calculating the relative areas under dis- carried out. The physiological races of iso- ease progress curves (relative AUDPC). Re- lates were identifi ed using the two diff er- sults showed that eggplant was the most ential tomato cultivars Belladona (suscepti- susceptible, followed by turnip and toma- ble to both races 1 and 2 of V. dahliae) and to cv. Belladona, while sweet pepper and to- Ace 55VF (resistant to race 1, susceptible to mato cv. Ace 55VF were less susceptible to race 2 of V. dahliae). Among these isolates, the isolates used. The pathogenicity of iso- 14 were characterized as race 2 (43.8 %), 12 lates varied from highly to mildly virulent race 1 (37.5 %) and 6 nonpathogenic (18.7 on eggplant and turnip whilst on Belladona, %) on tomato. The host range pathogenici- Ace 55VF and sweet pepper it varied from ty of isolates was determined using four dif- highly virulent to nonpathogenic. Belladona ferential hosts (eggplant, turnip, tomato (Ve- exhibited a similar level of susceptibility to ) and sweet pepper). Among the isolates, 5 race 1 and 2 of V. dahliae, but was more sus- were pathogenic to both eggplant and tur- ceptible than Ace 55VF to race 2. Interest- nip (15.6 %), 21 to eggplant, turnip and to- ingly, the isolates originating from eggplant mato (65.6 %), 5 to eggplant, turnip, tomato were clearly more virulent than those orig- and sweet pepper (15.6 %) and 1 was patho- inating from tomato and black nightshade genic to eggplant, turnip and sweet pepper on all solanaceous plants tested.

Phytopathological characterization, morphology, genetics and molecular diff erentiation combined in an integrated population study of Verticillium dahliae I.A. PAPAIOANNOU1, E.K. LIGOXIGAKIS2, D.J. VAKALOUNAKIS2, E.A. MARKAKIS3 and M.A. TYPAS1 1Department of Genetics and Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, GR-157 01 Athens, Greece. 2Laboratory of Plant Pathology, Plant Protection Institute, National Agricultural Research Foundation, GR-710 03 Heraklion, Crete, Greece. 3Laboratory of Plant Pathology, School of Agricultural Technology, Technological Educational Institute of Crete, GR-710 04 Heraklion, Crete, Greece

Understanding the genetic diversity of V. recorded. The tomato-sweet pepper patho- dahliae populations and an early recording type was morphologically distinct from the of their pathogenic profi le are essential for others, while conidial length and pigment disease management. An extended V. dahli- intensity were discriminatory parameters ae population mainly originating from Crete, among VCGs 2A, 2B and 4B. The PCR-based Greece, was characterized in terms of patho- molecular marker Tr1/Tr2 was reliable in race genicity/virulence, morphology/physiology, prediction among tomato-pathogenic iso- vegetative compatibility and mating type. lates, except for members of VCG 4B, while Tomato race 2 has supplanted race 1 and the application of markers Tm5/Tm7 and 35- was more virulent on a susceptible toma- 1/35-2 was highly successful in distinguish- to cultivar than race 1. Pathotypes of all iso- ing tomato pathotypes. E10 marker was re- lates were determined using four diff erential lated to VCG 2B. A SNP in the ITS2 region, hosts (tomato, eggplant, sweet pepper and and two novel molecular markers, M1 and turnip). All isolates from Crete fell into VCG M2, proved useful for the fast and accurate subgroups 2A, 2B and 4B, while a remark- determination of major VCGs 2A, 2B and 4B, ably high incidence of bridging isolates was and can be used for high-throughput popu-

© Benaki Phytopathological Institute 12 Hellenic Plant Protection Journal - Special Issue lation analyses in future studies. The mating probably does not control heterokaryon in- type was unrelated to VCG classifi cation and compatibility in V. dahliae.

Interspecifi c variability and virulence of Monilinia spp. isolates in stone fruits A. PAPAVASILIOU, M. VOLAKAKI, S. TESTEBASIS and G.S. KARAOGLANIDIS Aristotle University of Thessaloniki, Faculty of Agriculture, Plant Pathology Laboratory, GR-541 24 Thessaloniki, Greece

Brown rot is the one of the most important detected, M. laxa and M. fructicola, with fre- diseases of stone fruits (Prunus spp.) world- quencies of 59 and 41%, respectively. Spe- wide. The causal pathogens of brown rot cifi cally, M. fructicola was more common on include mainly four species of Monilinia : fruits (89%) and M. laxa was found in equal M. fructigena, Μ. fructicola, Μ. laxa and M. frequency in fl owers (49.5%) and in fruits polystroma. Until recently M. fructigena and (50.5%). The second objective of this study M. laxa were thought to be the main agents was to compare the aggressiveness of the of brown rot in Europe. However, in recent two species and among isolates of the same years, M. fructicola, a quarantine pathogen species that were collected from the two dif- in the European Union, has been reported ferent stages of infection; fl owering and rip- in many countries of Europe although its ening of fruits. Generally, the pathogenicity presence had not been confi rmed in Greece. of M. fructicola was found to be signifi cantly The fi rst objective of this study was to de- higher than that of Μ. laxa on the wounded tect the causal pathogens of brown rot in fruits of cherry and plum, with no signifi cant 4 stone fruits crops (peach, cherry, apricot diff erence detected on the wounded fruits and plum) in Greece. For this purpose, dur- of peach and apricot. Moreover, no signifi - ing 2011 and 2012, 1434 Monilinia spp. iso- cant variation in levels of aggressiveness lates were collected from two diff erent geo- among isolates from the two diff erent stag- graphical regions: Central Macedonia and es of infection was detected. This represents Thessaly. The sampling was conducted dur- the fi rst report of M. fructicola in Greece. The ing 2 phenological stages: blooming and wide dispersal of this pathogen and its high fruit ripening. All isolates were identifi ed at prevalence necessitates further research on a species level, by morphological character- the genetic variability of the fungus, its sen- ization and molecular identifi cation based sitivity to fungicides, and on the epidemiol- on the size of the intron of the cytochrome ogy of the disease in Greece. b gene of Monilinia spp. Two species were

Characterization of Rhizoctonia solani from potato in Cyprus L. KANETIS, D. TSIMOURIS, D. TSALTAS and Ν. ΙOANNOU Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus

Eighty isolates of Rhizoctonia solani were vealed that most of the isolates belonged collected from sclerotia formed on the sur- to AG-3 (92,8%), while the rest belonged to face of potato tubers during 2011. Isolates the subgroups AG-4-HG-I (2,8%) and AG-4- were assigned to the anastomosis group HG-II (4,4%), respectively. Optimum growth (AG) using molecular primers (ITS-1 and ITS- rates for the AG-4-HG-I and AG-4-HG-II iso- 4) from the ribosomal DNA. Phylogenetic lates were at 30°C, while for the AG-3 iso- analysis of the obtained PCR products re- lates was at 20°C. Sensitivity of the select-

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 13 ed isolates to the fungicide pencycuron was aggressiveness. The most susceptible plant tested in vitro (EC50 values 0.013-0.222 μg/ was barley for group AG-4-HG-I, melon and ml). Furthermore, the pathogenicity and ag- lettuce for group AG-4-HG-II and melon for gressiveness of 30 isolates was evaluated AG-3 isolates. In contrast, the least suscep- in vitro on seedlings of barley, lettuce, mel- tible were vetch and melon for groups AG- on, vetch and wheat. The aforementioned 4-HG-I, AG-4-HG-II and AG-3, respectively. seedlings were chosen based on rotation The results of the present study could sup- systems routinely followed by local farmers. port the development of integrated man- The AG-4-HG-II isolates were the most ag- agement programmes for potato infesta- gressive in all plant species studied, while tions by R. solani in Cyprus. the AG-3 group showed the lowest levels of

Characterization of new genetic regulators of the Arabidopsis thaliana innate immune system with homology to programmed cell death genes in mammals S.D. KOUNTOURI1, M.D. KAMINIARIS1, J.D.G. JONES2 and D.I. TSITSIGIANNIS1 1Agricultural University of Athens, Laboratory of Plant Pathology, Department of Crop Science, 75 Iera Odos Str., GR-118 55 Athens, Greece. 2The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom

Programmed cell death (PCD) is a process ducing Factor) gene and the DAP (Death As- that normally takes place during develop- sociated Protein) genes that play a crucial ment and defence of multicellular organisms. role in mammalian PCD. In animals, AIF and Research in recent years has demonstrated DAP genes are associated with diseases re- the existence of common biochemical path- lated to increased apoptotic events such as ways of PCD among plant, animal and micro- infection with HIV, neurodegeneration, and bial cells, and a possible link to serious diseas- heart attacks. The genetic model A. thaliana es like cancer and degenerative diseases in has fi ve diff erent putative AIF-like proteins humans. The plant hypersensitive response and 8 DAP like proteins with regions similar (HR), a form of PCD with many common char- to “Death Domains”. We will present the role acteristics with mammalian apoptosis, is as- of At-AIF genes in plant-host interactions, in sociated with the rapid death of host cells the activation of the plant innate immune triggered during the entrance of the patho- system, and in plant resistance or susceptibil- gen into plant tissues. Two interesting fami- ity to the pathogens Verticillium dahliae, Hy- lies of genes likely involved in the activation aloperonospora arabidopsidis, Alternaria bras- of the plant defence system are the ortho- sicicola, Fusarium oxysporum f.sp. raphani, and logues of the mammalian AIF (Apoptosis In- Pseudomonas syringae pv. tomato.

The role of ethylene perception in plant resistance against Fusarium oxysporum I.S. PANTELIDES1,2, S. PAPPA1, M. KARGAKIS1, S E. TJAMOS1 and E.J. PAPLOMATAS1 1Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece. 2Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, CY-3603 Lemesos, Cyprus

The plant fungal pathogen Fusarium oxyspo- es more than 100 formae specialis. The aim rum is the causal agent of root rot or wilt dis- of the present research was to provide an in- eases in several plant species and compris- sight into the host plant-microbe molecular

© Benaki Phytopathological Institute 14 Hellenic Plant Protection Journal - Special Issue interactions. For this purpose, the respons- ent mutants. Furthermore, the expression of es of Arabidopsis thaliana mutant plants im- the defence related genes PR1, PR2, PR3, PR4, paired in known pathogen response path- PR5 and PDF1.2. was examined using qPCR. ways were used to explore the components It was revealed that the genes PR1, PR2 and of defence against Fusarium oxysporum f.sp. PR5 were overexpressed in etr1-1 plants com- raphani. It was observed that etr1-1 plants pared to Col-0 plants, indicating their role in were the most resistant among the diff er- plant defence mechanisms.

Investigation of the role of VdSteA G protein coupled pheromone receptor in the virulence and biology of the vascular wilt pathogen Verticillium dahliae Ι.Α. STRINGLIS1,2, I. KALAITZOGLOU1, Ε.J. PAPLOMATAS1 and D.Ι. TSITSIGIANNIS1 1Agricultural University of Athens, Department of Crop Science, Laboratory of Plant Pathology, 75 Iera Odos Str., GR-118 55 Athens, Greece. 2Current Address: Utrecht University, Department of Biology, Plant Microbe Interactions, Padualaan 8, 3584 CH, Utrecht, The Netherlands

V. dahliae is a soil-borne fungus causing wilt used for alignment comparison with the ge- diseases in several hosts. The particular bi- nome of V. dahliae in order to detect poten- ology of this fungus complicates its treat- tial GPCRs. Seven diff erent groups of GPCRs ment through conventional methods. Thus, emerged from the phylogenetic analysis, the study of genes implicated in the inter- varying in sensing diff erent environmental actions of V. dahliae with its hosts is neces- signals. Agrobacterium mediated disruption sary to unravel the pathogenicity or viru- of a pheromone GPCR (named as VdSteA) in lence mechanisms and to discover putative two wild type races, 70V and 25V of V. dahli- novel methods to control the disease. G Pro- ae was performed in order to study the role tein Coupled Receptors (GPCRs) represent of this receptor in virulence and morpholo- the largest family of transmembrane recep- gy. 70V and 25V ΔVdSteA mutants displayed tors consisting of seven transmembrane do- a reduction in virulence in eggplant and to- mains. GPCRs are critical factors in regulat- mato plants and 70V ΔVdSteA mutants exhib- ing the morphogenesis, defence, mating, ited increased microsclerotia formation and infection and virulence in various organ- conidiation when compared to their corre- isms. Protein sequences of characterized sponding wild types. Both ΔVdSteA mutants GPCRs of the well-studied fungi Aspergil- exhibited higher conidial germination rates lus nidulans and Magnaporthe grisea were compared to wild types.

Study of the role of the secondary metabolite regulatory gene VdLaeA in the virulence and biology of the phytopathogenic fungus Verticillium dahliae Α.Μ. GIANNAKOPOULOU1,2, Α.Α. GKATZOUNI1 and D.Ι. ΤSITSIGIANNIS1 1Agricultural University of Athens, Department of Crop Science, Laboratory of Phytopathology, 75 Iera Odos Str., GR-118 55 Athens, Greece. 2Current Address: John Innes Centre, The Sainsbury Laboratory, Norwich, UK

Fungal secondary metabolites are com- and pharmaceutical interest. Previous stud- pounds with high degree of specialization ies have shown that the phytopathogenic that fulfi l various roles in toxin production, fungus V. dahliae produces phytotoxins and sporulation processes and the biosynthe- other molecules that induce the process of sis of substances of special biotechnological programmed cell death or other forms of

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 15 host resistance. The exact nature, of these fungus. Pathogenicity experiments in the compounds in V. dahliae, however, remains greenhouse revealed that the transformed unknown. In Aspergillus, the global regulator ΔVdlaeA strains resulted in signifi cantly re- of secondary metabolism laeA encodes a nu- duced disease levels in eggplants, toma- clear protein that is required for the expres- toes and Arabidopsis thaliana hosts. ΔVdlaeA sion of secondary metabolite genes, while strains also showed alteration in the rates its presence is considered indispensable for and morphology of germinating conidia, in mycotoxin, antibiotic and mycelial pigment mycelial development, and in microsclerotia biosynthesis. BLAST analysis of the V. dahli- formation. The study of the regulatory gene ae genome with the laeA gene of A. nidulans VdlaeA may contribute to a broader under- led to the discovery of a homologous gene standing of the molecular mechanisms by that was named VdlaeA. VdlaeA was delet- which secondary metabolites are produced, ed in V. dahliae in order to clarify whether and more specifi cally to the investigation of products of secondary metabolism play any its role in V. dahliae virulence. role in the virulence and physiology of this

Molecular identifi cation of and ochratoxin Α production by Aspergillus Section Nigri isolates from vineyards in Cyprus Ι.S. PANTELIDES, Ε. ARISTEIDOU, D. TSALTAS and Ν. IOANNOU Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus

The aim of this study was to investigate the calmodulin gene. The PCR products were se- infection of Cyprus vineyards with black quenced and the sequences obtained were aspergilli (Aspergillus section Nigri), the mo- compared to those in the NCBI database. It lecular identifi cation and the evaluation of was found that 148 isolates were identifi ed ochratoxin A (OTA) production of the iso- as A. tubingensis (92%), 12 as A. niger (7.4%) lated aspergilli. The mycotoxin ΟΤΑ is con- and 1 as A. carbonarius (0.6%). Liquid chro- sidered to be nephrotoxic, teratogenic, matography analysis revealed that 17 of the immunosuppressive, carcinogenic and neu- 161 isolates were toxigenic; 16 of the A. tub- rotoxic. Initially, black aspergilli were isolat- ingensis (1.3 pg/mg-1.96 ng/mg) and the A. ed from grapes of the varieties ‘Marathef- carbonarius (1.43-1.68 ng/mg). This study tiko’ and ‘Cabernet Sauvignon’ during 2010, confi rmed the infection of Cyprus vineyards that originated from four areas of the Limas- with black aspergilli and showed A. tubin- sol district, one of the main wine-produc- gensis to be the predominant species. More- ing regions of Cyprus. In total 161 isolates over, 10.8% of these isolates were toxigenic. (18%) were selected based on the macro- The population synthesis seems to be diff er- scopic characteristics of black aspergilla. ent compared to other Mediterranean coun- DNA from each isolate was used in PCR reac- tries as only a single isolate was identifi ed as tions with primers that amplifi ed part of the A. carbonarius.

Genetic and molecular characterization and evaluation of Greek non- toxigenic isolations of the fungi Aspergillus as potential biocontrol agents against afl atoxins M. GEORGIADOU1, S.P. AGORITSIS2, Κ. VICHOU2, G. VARDOUNIOTIS2, S. YANNIOTIS1, Ε. PAPLOMATAS2, P. J. C OTTY3 and D.I. TSITSIGIANNIS2 1Agricultural University of Athens, Department of Food Science & Technology, Labo- ratory of Food Engineering, 75 Iera Odos Str., GR-118 55 Athens, Greece. 2Agricultural

University of Athens, Department of Crop Science, Laboratory of Plant Pathology, 75 Iera Odos Str., GR-118 55 Athens, Greece. 3Department of Plant Pathology, USDA, Agricultural Research Service, Tucson, AZ 85721, USA

One of the most effi cient strategies to re- results, the Greek strains were grouped in duce the afl atoxin levels at pre-harvest lev- 20 diff erent vegetative compatibility groups el in several crops is the application of na- (VCGs), whereas 65% of the total number of tive biological control agents in the fi eld. isolates grouped to the same VCG. Next, afl a- One of the mechanisms of this technique is toxin and cyclopionic acid (CPA) gene clusters based on the competitive exclusion of the were investigated for possible indels using a afl atoxigenic fungi Aspergillus fl avus and A. multiplex set of primers with the PCR meth- parasiticus by applying non-toxigenic micro- od. The results showed indels in 7 Aspergil- organisms such as bacteria, yeasts or non- lus strains either in afl atoxin or in CPA gene toxigenic Aspergillus strains. The aim of the cluster. Also, the effi cacy of representative present study was the evaluation of 136 non- strains from diff erent VCGs in afl atoxin re- toxigenic strains of the genus Aspergillus, that duction was evaluated with in vitro competi- were isolated from pistachios of the typical tion tests in autoclaved maize kernels. Com- Greek variety “Aegina” collected from sever- petition tests showed that treatments with 2 al orchards in Greece. The major goal is to re- non-toxigenic strains from diff erent VCG reduce afl atoxin contamination from the fi eld sulted in 80% afl atoxin reduction compared by selecting and applying the most suitable to control (toxigenic strain alone). The above non-toxigenic strains. For that purpose, the results demonstrate that some of the isolat- Greek isolates were DNA-characterized and ed Greek non-toxigenic Aspergillus strains grouped with the method of Simple Se- can be used as potential biocontrol agents quence Repeats (SSRs) or Microsatellites us- in pre-harvest stages in several afl atoxin sus- ing a multiple set of primers. Based on SSRs ceptible crops in Greece.

Molecular characterization of the cyp51, mdr and afl R genes and the eff ect of DMI resistance mechanisms on fi tness parameters and afl atoxin production in Aspergillus parasiticus E.G. DOUKAS1, A.N. MARKOGLOU1, J.G. VONTAS2 and B.N. ZIOGAS1 1Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos Str., GR- 118 55 Athens, Greece. 2Faculty of Applied Biology and Biotechnology, Department of Biology, University of Crete, GR-714 09 Heraklion, Greece

Aspergillus parasiticus mutant strains resis- gene, a pathway transcriptional regulato- tant to DMIs were isolated at a high muta- ry gene in afl atoxin biosynthesis, showed tion frequency after UV-mutagenesis and that this gene was not expressed in R2 mu- selection on media containing fl usilazole. tant strains tested. Study of fi tness-deter- Two diff erent resistant phenotypes, char- mining parameters showed that most fl usi- acterized as R1 and R2 on the basis of their lazole-resistant mutant strains had mycelial afl atoxigenic ability, were identifi ed. All R1 growth rate, sporulation and spore germi- mutant strains produced afl atoxins at con- nation rates lower that the sensitive strain. centrations signifi cantly higher (up to 3-fold) Cross-resistance studies with other fungi- than the wild-type parent strain on yeast ex- cides showed that all R1 mutant strains were tract sucrose medium, whereas the majori- also resistant to the DMIs imazalil and tebu- ty of the mutant strains (R2 phenotype) had conazole, but retained their parental sensi- lost their afl atoxigenic ability. Real-time PCR tivity to fungicides aff ecting other metabolic analysis of the expression levels of the afl R pathways and/or cellular processes. Contrary

to the above, all R2 mutant strains exhibited pression of the cyp51A gene were found in a low to moderate multi-drug resistance to the R2 mutant strains tested. Real-time PCR DMIs and to several other fungicide class- analysis showed high levels (up to 25-fold es. Two diff erent homologous genes, cyp51A higher) of the mdr transcript in all R2 mutant and cyp51B, encoding C-14 alpha sterol dem- strains tested. This is the fi rst report describ- ethylase (Cyp51) and an mdr gene encoding ing the existence of two cyp51 genes and a an ATP-binding cassette protein were cloned potential mdr gene coding for an ATP bind- and characterized. Sequence comparison of ing cassette protein in A. parasiticus. These cyp51A gene revealed an amino acid substitu- results also indicate that multiple biochem- tion from glycine (GGG) to tryptophan (TGG) ical mechanisms, including target-site modi- at position 54 (G54W) in two out of three of fi cation due to mutation of the cyp51A gene,

R1 mutant strains. Analysis of deduced amino cyp51A overexpression, and the function of acid sequence of cyp51B showed that no mu- an ABC transporter protein, are responsible tations were associated with DMI resistance. for DMI-resistance in A. parasiticus. Our fi nd- Study of the transcription levels of cyp51A ings suggest that A. parasiticus has the ge- showed that this gene was over-expressed in netic and biochemical potential to give rise the third afl atoxigenic mutant strain. Neither to highly afl atoxigenic DMI-resistant isolates amino acid substitutions within nor overex- in the fi eld.

Identifi cation and determination of antibiotic susceptibility of cultured enterobacterial fl ora from leafy vegetables in Cyprus I. ANASTASIOU, E. SAVVA and D. TSALTAS Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus

In the last few years, the consumption of tion, within the ranges of 6.31-7.85 and 5.86- raw leafy vegetables has increased due to 6.7 log cfu/g respectively. More than 95% of their well-documented nutritional value. the total microfl ora and 87% of Enterobac- The contamination of these crops by mi- teriaceae identifi ed were resistant to ampi- crobes originating from soil, manure and cillin. The rates of resistance to cefotaxime handler microfl ora is of increasing interest (33.41%) and gentamicin (17.35%) for Enter- from a public health perspective. In addi- obacteriaceae were lower, but for the total tion to the microbial load, the antibiotic re- microfl ora resistance to the latter antibiot- sistance of these microorganisms as a result ics was higher (83.33% and 76.58% respec- of antibiotic usage in agriculture and oth- tively). Rucola and spinach produced pos- er related fi elds it is of increasing concern. itive results for Escherichia coli, with rucola The aim of this study was to identify and being the only sample found to be contam- enumerate the enterobacterial microfl o- inated with isolates resistant to cefotaxime ra on samples of leafy vegetables (lettuce, (0.43 log cfu/g). The samples also had similar rucola, spinach, purslane) in Cyprus, and to population numbers for yeasts and moulds evaluate the levels of resistance of these mi- (4.04-5.67 log cfu/g). The current study croorganisms to the antibiotics ampicillin, shows that leafy vegetables can be a signif- cefotaxime, gentamicin and vancomycin. icant source of pathogenic microorganisms, The results showed high levels of total mi- including antibiotic resistant isolates. crofl ora and Enterobacteriaceae contamina-

Eff ect of Alternaria leaf-spot on the content of antioxidant compounds in infected kiwi orhards

A. AINALIDOU1, K. KARAMANOLI1, G. KARAOGLANIDIS3, U. MENKISSOGLU-SPIROUDI2 and G. DIAMANTIDIS1 1Laboratory of Agricultural Chemistry, Faculty of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece. 2Pesticide Science Labarotary, Faculty of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece. 3Laboratory of Phytopathology, Faculty of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece

Alternaria alternata can infect kiwi leaves active ingredient, for chlorfenuron. Total an- causing necrotic lesions and premature de- tioxidant capacity was evaluated with FRAP foliation that may lead to complete yield and DPPH techniques, while the chromato- losses. The objective of this study was to graphic profi le of phenolic compounds and evaluate the changes in content of kiwi fruit the ascorbic acid concentration were deter- antioxidant constituents (phenolic com- mined by liquid chromatography (HPLC). pounds and ascorbic acid) after the infection According to the results the presence of the of vines by Alternaria alternata. Furthermore fungus caused the induction of biosynthesis the vines were treated with CPPU (forchlo- of phenolic compounds and the reduction rfenuron), a growth regulator agent of the of ascorbic acid concentration to untreat- synthetic cytokinins group, which is wide- ed with CPPU fruits. In contrast, the applica- ly used in kiwi fruit orchards for both, in- tion with CPPU caused an increase of ascor- crease of productivity and improvement of bic acid concentration in all kiwi fruits up to product quality. CPPU was applied to both 50%. The total antioxidant capacity of fruits healthy and infected vines in order to study was strong correlated to ascorbic acid con- the combined eff ect of the fungus and of the centration.

Fungal diseases of sunfl ower in R.U. Drama in period 2010-2011 A.I. ANASTASIADIS Hellenic Agricultural Organization “Demeter”, Center “Demeter” Drama, 5th km. Drama-Thessaloniki, GR-661 00 Drama, Greece

The diseases aff ecting sunfl ower in the Re- rate cases in 2010 and 2011, respectively. gional Unit of Drama throughout the dura- Of the fungal diseases that attack the stem tion of the growing seasons 2010-2011 were and root system (Macrophomina, Phoma, recorded. During this two-year period there Sclerotinia and Phomopsis), Macrophomina were incidences of Downy Mildew, Alter- caused the most extensive damage. In dry naria, Septoria, Powdery Mildew, Sclerotin- sandy fi elds, and in combination with dry ia, Macrophomina, Phoma and Phomopsis. and warm conditions, the percentage of in- Systemic mildew, considered the most se- fected plants reached in some cases 90%. In rious disease of sunfl ower worldwide, ap- 2011 incidences of Phoma, with maximum peared in 2011, with the most highly infect- infection rates of 50%-60% was observed, ed fi eld showing a 15-20% infection rate, while incidences of Sclerotinia Wilt (rotting while in 2011, a fi eld was observed with a of the stem base) and Phomopsis were ob- secondary mildew infection. Septoria, conserved in individual plants. When sunfl ow- sidered to be the most important of the fo- ers are grown in the same fi eld for 2 or more liar diseases (which include Alternaria, Sep- consecutive years, the incidence of disease toria, and Powdery mildew), caused severe is more common, particularly that caused by infections in 2011, while Alternaria and Pow- Septoria and Phoma. dery Mildew were observed in two sepa-

Inactivation of the VdVeA (Velvet A) gene in Verticillium dahliae and investigation of its role in the physiology and pathogenicity of the fungus A. ANTONIADI and D.I. TSITSIGIANNIS Agricultural University of Athens, Department of Crop Science, Laboratory of Plant Pathology, 75 Iera Odos Str., GR-118 55 Athens, Greece

Verticillium dahliae is a soilborne plant Velvet complex, in association with a third pathogenic fungus causing the syndrome of protein, VelB. The aim of this study was to wilt diseases and posing a signifi cant threat elucidate the role of the orthologous gene to several annual and perennial crops world- of A. nidulans, VdVeA, in the virulence and wide. The inability of common management morphology of the fungus V. dahliae. A gene methods to control V. dahliae has led to the replacement strategy was applied by incor- investigation of molecular mechanisms that porating two genomic sequences of about might regulate its virulence. Studies have 1000 bp before the start and stop codons re- previously shown that the fungus V. dahliae spectively, in the binary vector pGKO2. Be- produces phytotoxins and other secondary tween these two regions the geneticin cas- metabolites that induce cell death or oth- sette was subcloned in order to replace the er forms of host defence. It has been found VdVeAgene after transformation. The inac- that in several species of Fusarium spp. and tivation construct of VdVeA was introduced Aspergillus spp. the gene veA encodes a pro- into various strains of V. dahliae using the Ti tein that can regulate the fungal secondary plasmid of Agrobacterium tumefaciens via a metabolism, induce the diff erentiation of double recombination event. The eff ect of fungal development in relation to light, and inactivation of the VdVeA gene on the phys- regulate reproduction and pathogenicity. iology and pathogenicity of the fungus was Along with the VeA, a second protein called explored in in vitro experiments and in plan- LaeA forms a nuclear complex called the ta virulence assays in several hosts.

Fast and accurate identifi cation of Fusarium oxysporum formae speciales complex using High Resolution Melting (HRM) analysis I. GANOPOULOS1,2, P. MADESIS2 and A. TSAFTARIS1,2 1Department of Genetics and Plant Breeding, School of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece. 2Institute of Applied Biosciences, CERTH, GR-570 01 Thermi, Thessaloniki, Greece

The fungus Fusarium oxysporum consists universal internal transcribed spacer (ITS) by a number of diff erent strains which are primers] coupled with high-resolution melt- grouped together in to groups called for- ing (HRM) analysis for the identifi cation and mae speciales making F. oxysporum a highly discrimination of F. oxysporum formae spe- complex species. This complexity is respon- ciales complex. The melting curve analysis sible for the timely and diffi cult discrimi- of the ITS amplicons succeeded in specifi - nate of the diff erent Fusarium formae spe- cally classify all isolates into seven F. oxyspo- cials which is performed via biochemical rum formae speciales and generated sev- or phenotypic methods. Thus it is of para- en distinct HRM curve profi les. The smallest mount importance to develop novel, rapid, DNA sequence diff erence recognized in this and simple to perform identifi cation meth- study was one nucleotide. We conclude that ods. Herein, we describe the development based on the mentioned results HRM curve of a novel real-time PCR based assay [using analysis of Fusarium ITS sequences is a sim-

© Benaki Phytopathological Institute 20 Hellenic Plant Protection Journal - Special Issue ple, quick, and reproducible method which an unlabeled probe and the full-length am- allows both the identifi cation of seven F. ox- plicon. Finally, the completion of both reac- ysporum formae speciales and at the same tion and analysis in a closed tube saves time time their screening for variants. Our geno- by eliminating the separate steps and re- typing assay uses the combined information duces the risk of contamination. of simultaneously acquired HRM data from

Molecular and phytopathological investigation of the role of the global regulator of secondary metabolism AcLaeA in the mycotoxigenic fungus Aspergillus carbonarius M. ILIADI and D.I. TSITSIGIANNIS Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

The fi lamentous fungus Aspergillus car- virulence and ochratoxinA production by A. bonarius is considered one of the main fungi carbonarius in grapes by deleting this gene responsible for sour rot in grapes and for the from the genome of two wild types of the production of the carcinogenic mycotoxin fungus. Using PCR with specifi c primers, two ochratoxin A. Recently, a novel gene named genome sequences located about 1000 bp laeA that operates as a global regulator of before the start and stop codon of AclaeA, secondary metabolism was discovered in respectively, were amplifi ed and subcloned several species of Aspergillus and Fusarium. into the vector pBluescript. Between these Inactivation of the laeA gene leads to dis- two regions the geneticin cassette was sub- ruption of mycotoxin biosynthesis. The gene cloned in order to replace the AclaeA gene laeA encodes a nuclear methyltransferase after transformation. The AclaeA deletion protein that is required for the expression of construct was transferred to the binary vec- secondary metabolite genes, while its pres- tor pGKO2 and then introduced using the Ti ence is considered indispensable for myco- plasmid of Agrobacterium tumefaciens via a toxin, antibiotic and mycelial pigment bio- double recombination event, into two wild synthesis. BLAST analysis of the genome of type strains of A. carbonarius. The evalua- A. carbonarius with the laeA gene of A. nid- tion data on the morphological character- ulans resulted in the identifi cation of an or- istics and virulence experiments in red and thologous gene named AclaeA. The goal of white grape varieties of ΔAclaeA mutants is this study was to investigate the role of the presented. regulatory gene AclaeA in the physiology,

First report of Diaporthe neotheicola as pathogen causing shoot blight in kiwifruit TH. THOMIDIS and E. EXADAKTYLOU Alexander Technological Education Institute of Thessaloniki, Department of Crop Production, GR-574 00 Sindos, Thessaloniki, Greece

Kiwifruit is an important crop in Greece. Al- found to be wilted and blighted in many or- though it is considered to be a crop with few chards of Episkopi, Naoussa (Imathia Prefec- pests and diseases, new diseases have been ture, Greece). Close examination of these identifi ed in the last few years. shoots revealed distinct dark cankers. Fun- In June 2009, shoots of the kiwifruit plants gal isolations were made on acidifi ed potato (cvs “Hayward” and “Tsechelidis”) were dextrose agar, and the species responsible

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 21 was identifi ed based on ITS-5.8S rDNA-ITS2 pathogen was reisolated from the artifi cially region and Elongation Factor 1-alpha by the inoculated shoots and fruits. CBS Fungal Biodiversity Centre, Identifi ca- In laboratory experiments, the rate of my- tion Service (Utrecht, Netherlands) as Dia- celial growth of D. neotheicola in vitro was porthe neotheicola A.J.L. Phillips & J.M. San- reduced as the temperature was increased tos. from 25 to 30°C, and also when decreased Koch’s postulates were fulfi lled by the artifi - from 20 to 10°C. Growth was totally inhibit- cial inoculation of 20 segments of 1-year-old ed at 35 and 2-4°C. woody shoots of the kiwifruit cultivar “Hay- This study is the fi rst report of the occur- ward”, and 20 fruits. Shoots and fruits inocu- rence of a shoot blight and canker disease lated with agar discs without mycelium were of kiwifruit in Greece caused by the fungus used as control. Identical disease symptoms D. neotheicola. were observed in the inoculated shoots. The

Metabolomic analysis of Verticillium dahliae races 1 and 2 V. KOUROUSIAS1, C. KALLONIATI2, S.E. TJAMOS1, E. FLEMETAKIS2 and E.J. PAPLOMATAS1 1Laboratory of Plant Pathology, 2Laboratory of Molecular Biology, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

The technological advancements in analyti- that the race 2 secreted a higher (by 84%) cal chemistry and computing of the last de- number of metabolites than race 1 in both cade have facilitated study of the metabo- media. Furthermore, the number of the se- lomic profi le of plants and microorganisms creted metabolites of race 1 and 2 that were under diff erent treatments. The aim of the common in both media was 20% and 37%, present study was to investigate the metab- respectively. Among the diff erent metabo- olomic profi le of Verticillium dahliae races 1 lites that were identifi ed, were substances and 2 grown in two diff erent growth media with established roles, either as elicitors or that simulate either xylem or microsclero- eff ectors, in plant-microbe interactions. tia germination conditions. It was observed

Pathogen identifi cation and incidence of pre- and post-harvest fruit rots of pomegranate in Greece S.C. KONSTANTINOU and G.S. KARAOGLANIDIS Laboratory of Plant Pathology, Aristotle University of Thessaloniki, School of Agriculture, GR-541 24 Thessaloniki, Greece

In recent years, the cultivation of pomegran- orchards and storage rooms in several re- ate has increased considerably, particularly gions of N. Greece. Fungal identifi cation at in northern Greece. Fruit rots, along with a genus level was based on colony appear- other physiological disorders contribute to ance and morphological features of fruiting quantitative yield losses, qualitative dete- bodies and spores. In total 5 and 3 pre- and rioration, and restriction of fruit storability. post-harvest pathogens were identifi ed, re- The goal of the present study was to iden- spectively. It was found that Aspergillus spp. tify and measure the incidence of fungal (45.9%) and Penicillium spp. (40.0%) were the pathogens causing pre- and post-harvest main causal agents of pre-harvest fruit rots. decay in pomegranate fruit. The sampling In contrast, post-harvest rots were caused of diseased fruit was conducted during 2011 mainly by Botrytis cinerea (70.8%) and Pili- (September to December) in a number of diella granati (15.4%). Aspergillus spp. iso-

© Benaki Phytopathological Institute 22 Hellenic Plant Protection Journal - Special Issue lates were identifi ed at a species level as A. lates obtained from fruit with post-harvest niger (65.5%) or A. tubingensis (34.5%) using rot were all identifi ed as P. adametzioides. To the Restriction Fragment Length Polymor- the best of our knowledge, this is the fi rst re- phism (RFLP) technique. Penicillium spp. iso- port of P. adametzioides and P. brevicompac- lates were identifi ed at a species level after tum causing pre- or post-harvest fruit rot of sequencing of the internal transcribed spac- pomegranate worldwide. These results, to- er (ITS) regions of ribosomal DNA (rDNA). gether with those anticipated from new The Penicillium spp. isolates obtained from samplings conducted during 2012, will pro- fruit with pre-harvest rots were identifi ed as vide signifi cant and useful information for either P. adametzioides or P. brevicompactum the implementation of successful fruit-rot at similar frequencies. Penicillium spp. iso- control measures.

Susceptibility of cultivated solanaceous plants and olive to Verticillium dahliae isolated from a new host, the weed Solanum elaeagnifolium A.L. LAGOPODI1, J. KASHEFI2, H. NIKOLAIDIS1 and N. FRYDAS1 1Plant Pathology Laboratory, School of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece. 2USDA ARS European Biological Control Laboratories, Thessaloniki, Greece

The rapidly expanding weed Solanum elae- per cv. P13, as well as on olive cv. Chalkidi- agnifolium (commonly ‘germanos’) is con- kis, Amfi ssis and Koroneiki. The results considered to constitute one of the most per- fi rmed the pathogenicity of the fungus on sistent problems in the fi elds of Greece and the weed, eggplant, pepper, and olive. This southern Europe. The fungus Verticillium is the fi rst report worldwide that the inva- dahliae is a diffi cult to control soil-borne sive in Europe weed species S. elaeagnifoli- fungus with global distribution, that was re- um is a host for V. dahliae. There is an urgent cently isolated from S. elaeagnifolium plants need to fi nd successful means to control this exhibiting wilt symptoms. In the present re- weed, as not only can it lead to the exclu- search the pathogenicity of V. dahliae, isolat- sion of cultivated species from fi elds, but it ed from germanos was studied on the weed can also increase the levels of fungal inocu- itself, on eggplant cv. Langada, on pep- lum in the soil.

First report of palm rot disease of Phoenix spp. caused by Neodeightonia phoenicum in Greece E.K. LIGOXIGAKIS1, E.A. MARKAKIS2, I.A. PAPAIOANNOU3 and M.A. TYPAS3 1Laboratory of Plant Pathology, Plant Protection Institute, National Agricultural Research Foundation, GR-710 03 Heraklion, Crete, Greece. 2Laboratory of Plant Pathology, School of Agricultural Technology, Technological Educational Institute of Crete, GR-710 04 Heraklion, Crete, Greece. 3Department of Genetics and Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, GR-157 01 Athens, Greece

In July 2007, a severe palm rot disease resem- extending along the leaf base and rachis. bling Diplodia disease was observed on Phoe- Decay and premature death of leaves was nix dactylifera in Heraklion (Crete, Greece). followed by terminal bud necrosis, and leaf Similar symptoms were later observed on P. blight and stalk rot were also observed. A fi l- canariensis. Initial pale elongated spots were amentous fungus was consistently isolated gradually converted to dark brown streaks from leaf base necrotic lesions; according

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 23 to morphological (macroscopic and micro- P. canariensis, P. theophrasti and Washing- scopic examination), molecular (sequenc- tonia fi lifera was demonstrated by artifi cial ing of the ITS1-5.8S-ITS2 region, together wound-inoculation experiments. While in- with parts of the fl anking 18S and 28S rRNA fections of N. phoenicum with P. dactylifera genes, and BLAST search), and phylogenetic are common worldwide, to the best of our (including representatives of relative fungal knowledge this is the fi rst report of such an genera) analyses performed, the pathogen infection in Greece. The disease may be fa- was identifi ed as Neodeightonia phoenicum voured by the pruning of older leaves dur- A. J. L. Phillips & Crous (syn. Diplodia phoe- ing early Spring and the widespread occur- nicum), formerly also known as Macropho- rence of the red palm weevil Rhynchophorus ma phoenicum and Strionemadiplodia phoe- ferrugineus in Greece. nicum. The pathogenicity of the fungus to

First report worldwide of leaf spot disease of Phoenix theophrasti caused by Paraconiothyrium variabile E.K. LIGOXIGAKIS1, I.A. PAPAIOANNOU2, E.A. MARKAKIS3 and M.A. TYPAS2 1Laboratory of Plant Pathology, Plant Protection Institute, National Agricultural Research Foundation, GR-710 03 Heraklion, Crete, Greece. 2Department of Genetics and Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, GR-157 01 Athens, Greece. 3Laboratory of Plant Pathology, School of Agricultural Technology, Technological Educational Institute of Crete, GR-710 04 Heraklion, Crete, Greece

A severe leaf spot disease of Phoenix theo- logical (microscopic and macroscopic exam- phrasti was observed in Heraklion (Crete, ination), molecular (sequencing of the ITS1- Greece) in the Spring of 2011. Typical symp- 5.8S-ITS2 region, together with parts of the toms of the leaves included initially small, fl anking 18S and 28S rRNA genes, and BLAST round-ovoid brown spots, later changing search), and phylogenetic (including repre- into streaks (average dimensions 7.3 x 3.3 sentatives of relative fungal genera) analy- mm), surrounded by dark brown rings. Final- ses performed, the pathogen was identi- ly, the expanding streaks often coalesced to fi ed as Paraconiothyrium variabile Riccioni, the production of enlarged necrotic lesions Damm, Verkley & Crous. The pathogenicity resulting in leaf blights. Symptoms were of the fungus was demonstrated in artifi cial also detected on petioles and leaf bases. Ex- spraying-inoculation tests on seedlings of P. tended spotting and blighting resulted in theophrasti. P. variabile has so far been isolat- leaf death and unthrifty appearance of ined from various woody host plants in South fected trees. A fi lamentous fungus was con- Africa, Italy, Turkey, China and New Zealand. sistently isolated from the periphery of the This is the fi rst report worldwide of P. varia- characteristic lesions. According to morpho- bile naturally infecting a palm species.

First report of pink rot of Phoenix and Washingtonia palm species caused by Nalanthamala vermoesenii in Greece E.K. LIGOXIGAKIS1, I.A. PAPAIOANNOU2, E.A. MARKAKIS3, E.E. FRAGKOULI4 and M.A. TYPAS2 1Laboratory of Plant Pathology, Plant Protection Institute, National Agricultural Research Foundation, GR-710 03 Heraklion, Crete, Greece. 2Department of Genetics and Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, GR-157 01 Athens, Greece. 3Laboratory of Plant Pathology, School of Agricultural Technology, Technological Educational Institute of

Crete, GR-710 04 Heraklion, Crete, Greece. 4Direction of Environmental, Agricultural Development and Trading, Department of Research and Horticulture Preservation of Municipality of Heraklion

Pink rot of Phoenix and Washingtonia spe- tive fungal genera) analyses performed, the cies has become common in Heraklion pathogen was identifi ed as Nalanthamala (Crete, Greece) in the last four years. The vermoesenii (Biourge) Schroers (syn. Penicil- disease is characterized by leaf spots, chlo- lium vermoesenii, Gliocladium vermoesenii). rosis and necrosis, rachis spots and decay, Artifi cial wound-inoculation tests on P. ca- sheath and trunk rot, decline, and eventu- nariensis, P. theophrasti and W. fi lifera dem- ally death of infected plants, and a pinkish- onstrated the pathogenicity of the fungus orange layer on the surfaces of infected tis- to all three palm species, with W. fi lifera be- sues. A fi lamentous fungus was consistently ing the most susceptible. N. vermoesenii has isolated from the petiole tissues of diseased a worldwide distribution and has frequently leaves and from the pinkish-orange char- been documented on several palm species. acteristic overlay. According to morpho- To the best of our knowledge, this is the fi rst logical (macroscopic and microscopic ex- report of a palm disease caused by N. vermo- amination), molecular (sequencing of the esenii in Greece. It may be favoured by the ITS1-5.8S-ITS2 region and part of the 18S pruning of older leaves during early Spring rRNA gene, and BLAST search) and phylo- and the occurrence of the red palm weevil genetic (including representatives of rela- Rhynchophorus ferrugineus.

Characterization and distribution of mating type genes in the Dothistroma Needle Blight pathogens Z.G. NAKOPOULOU1, C. PERLEROU2 and S. DIAMANDIS1 1Hellenic Agricultural Organisation “Demeter”, Forest Research Institute, GR-570 06 Vassilika, Thessaloniki, Greece. 2Forest Service of Thessaloniki, Karatasou 1, GR-546 26 Thessaloniki, Greece

Dothistroma needle blight, also known as The results of this study showed that all 23 red band needle blight, is one of the most isolates were characterized as D. septospo- important diseases of Pinus spp. The disease rum (Dorog) M. Mopelet, in an agreement is caused by two very similar Dothistroma with the global distribution of the species. spp., D. septosporum and D. pini. A total of 23 In contrast, none of the isolates was iden- isolates from the area of Xanthi, N.E. Greece, tifi ed as D. pini, which had, until recently, were assayed in order to identify the oc- been reported only in the USA, Russia and currence of Dothistroma spp. Four diff erent Ukraine. Recently, and for the fi rst time, the molecular techniques were applied in this occurrence of both species was reported in study including i) sequencing of the rDNA the same area in France. Subsequently, the ITS region ii) a species-specifi c ITS-RFLP two species have been reported to occur in technique iii) mating type primers and iv) Hungary, at the same location, on the same a diagnostic microsatellite marker Doth_A. trees and even infecting the same needles.

Distribution of mating types of Cryphonectria parasitica in Greece Z.G. NAKOPOULOU1, C. PERLEROU2, A. DROUZAS3, A. MITSAKAKI4 and S. DIAMAMDIS1 1Hellenic Agricultural Organisation “Demeter”, Forest Research Institute, GR-570 06 Vassilika, Thessaloniki, Greece. 2Forest Service of Thessaloniki, Karatasou 1, GR-546 26 Thessaloniki, Greece. 3Laboratory of Systematic Botany and Phytogeography, School

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 25 of Biology, Aristotle University of Thessaloniki, P.O Box:104, GR-541 24, Thessaloniki, Greece. 4Forest Service of Corfu, Alikes Potamou, GR-491 00 Corfu, Greece

The fungus Cryphonectria parasitica, the (MAT1-2). The study of the sexual compati- causal agent of chestnut blight, was ob- bility in our populations showed that the id- served in Greece for the fi rst time in 1963 iomorph MAT1-1 was present in most iso- (Biris 1964) and particularly in Zagora, Mt. lates (92.6%). In contrast, only one isolate Pelion. Examination of the mating type phe- (7.35%) was found to represent the MAT1-2 notype of C. parasitica isolates can reveal phenotype. This mating type distribution is the population structure and the presence, in agreement with the existing data for the or lack, of sexual reproduction. In 707 viru- occurrence of only one mating type and lent and hypovirulent isolates, fungal ge- the lack of sexual reproduction. The results nomic DNA was extracted after growth on of our study favour the application of bio- PDA dishes covered with cellophane sheets. logical control measures against chestnut Polymerase chain reaction was carried out blight, a technique already in use for man- and the size of the PCR products was de- agement of the disease in Greece. termined to be 1,649 kb (MAT1-1) or 594 bp

Necrosis of young almond trees infected by Botryosphaeria dothidea A.I. RUMBOU1 and I.X. RUMBOS2 1Center for Research and Technology, Thessaly (CERETETH), Volos, Greece. 2Hellenic Agricultural Organization “Demeter”, Plant Protection Institute of Volos, Greece

Several species of fruit trees have been revealed a brown discoloration of the wood ported to be infected by fungi of the ge- with a well defi ned line between the ne- nus Botryosphaeria. Amongst these are ap- crotic and healthy tissues. From the root- ple, peach, pistachio, kiwifruit, olive and stock new vigorous shoots had often devel- walnut. Over the period May-June 2011 oped. The species Botryosphaeria dothidea an unusual outbreak of the fungus was re- (Moug.: Fr.) Ces. & De Not was isolated from corded at four new commercial almond (cv. all trees examined. The fungus produced Ferragnes) plantations planted early in the hyaline, aseptate, fusiform conidia, 23-29 same year in the region of Thessaly, Central x 4.5-5 μm in size, belonging to Neofusic- Greece (in Tirnavos and New Anchialos). The occum mediterraneum Crous, M.J. Wingf. & percentage of dead trees was between 40- A.J.L. Phillips (anamorph). The pathogenici- 70% and the propagative material originat- ty of the obtained isolates was proved by ined from two diff erent nurseries in Thessa- oculating young almond trees cv. Ferragnes ly and North Greece. Infected trees showed grown in the fi eld. All isolates proved to be initially a yellowing discoloration of the pathogenic. Re-isolations made from discol- leaves and gradually died during the sum- ored wood yielded the same fungus. This is mer. All the infections were associated with the fi rst report of infection of almond trees the pruning wound at the point of the union in commercial plantations by the fungus B. of rootstock/scion. Transverse and longitu- dothidea, worldwide. dinal sections through aff ected wood re-

Leaf spots induced in Ilex aquifolium by Alternaria alternata G.T. TZIROS, C. PERLEROU and S. DIAMANDIS Hellenic Agricultural Organization ‘’Demeter’’ (Ex NAGREF), Forest Research Institute, GR-570 06 Vassilika, Thessaloniki, Greece

A foliar disease of holly (Ilex aquifolium L.) ia were obpyriform, with a conical or cylin- was observed in 2009 in a natural ecosys- drical beak, ovoid or ellipsoidal measuring, tem in the region of Thessaloniki (Northern 7.7 – 27.4 x 5.6 -15.0 μm (average 16.3 x 8.8 Greece, Central Macedonia, Mount Hortia- μm), showing 1 to 5 transverse septa and 0 tis). Symptoms consisted of circular, some- to 3 longitudinal septa. These data, togeth- times irregularly shaped, brown necrotic er with molecular characterization using the spots with yellow border, 3 to 7 mm in di- specifi c ribosomal internal transcribed spac- ameter. On some leaves several spots co- er region (rDNA-ITS) confi rmed the identi- alesced to form large necrotic areas, cover- ty of the fungus as Alternaria alternata (Fr.) ing approximately half of the leaf surface. Keissl. A pathogenicity test was conduct- Older lesions sometimes appeared black- ed on detached, wounded and unwound- ish brown as sporulation occurred on the le- ed, healthy I. aquifolium leaves. Ten days af- sions. The percentage of diseased leaves of ter inoculation, leaf spots similar to the ones the particular population was approximate- observed in the fi eld developed on all the ly 30%. Single spore cultures on potato car- inoculated points (wounded leaves) and on rot medium (PCA) gave rise to initially white half of the unwounded leaves, while control colonies which turned to grayish-black lat- leaves remained symptomless. A. alterna- er due to abundant sporulation. Conidio- ta was reisolated from artifi cially inoculated phores were short, septate, branched or leaves confi rming Koch’s postulates. To our unbranched, and green to brown. Mature knowledge, this is the fi rst report of A. alter- conidia were produced in long, single or nata causing leaf spot on holly in Greece. more often in branched chains. The conid-

Abstracts - 16th Hellenic Phytopathological Congress 29

ORAL & POSTER PRESENTATIONS

Potato blackleg in Greece caused by Dickeya sp. biovar 3 (D. solani) M.G. PAGOULATOU, P.F. SARRIS*, E.A. TRANTAS, D.S. STAVROU, K.B. SIMOGLOU, F.N. VERVERIDIS and D.E. GOUMAS Technological Educational Institute of Crete, School of Agricultural Technology, Department of Plant Sciences, P.O.Box 1939, GR-710 04 Heraklion, Greece. *Present Address: The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

During the years 2007 and 2011, signifi cant were biochemically identical to the refer- bacterial infections were observed in pota- ence strain Dickeya sp. IPO2222, while bio- to crops (Solanum tuberosum cv. Spunta) on chemically distinct from the related refer- the island of Crete in Greece. Similar infec- ence strain Dickeya dianthicola BPIC2098, a tions were observed at Nevrokopi, Drama, pathogen of potato. Molecular analysis with during the spring period of 2012 (cvs. Ban- a) repPCR (BOX and ERIC fi ngerprinting), b) ba and Safari). Aff ected plants had black- specifi c primers for the PCR amplifi cation of leg and rotting symptoms at the stem base, the pel (pectate lyase) gene, and c) sequenc- brown discolouration of vascular tissues, ing of the dnaX gene, revealed identical pro- wilting and soft rot in daughter tubers. Dis- fi les among the isolated strains and the ref- ease incidence varied from 5%-50%. The erence strain Dickeya sp. IPO2222; which aim of this study was to identify the patho- were clearly distinct from the those of relat- gen responsible for the blackleg disease iso- ed species D. dianthicola BPIC2098, P. caro- lated from both of the above mentioned tovorum TEIC3036, P. atrosepticum TEIC3211 regions. Isolations were made from symp- and other Erwinia spp. Koch’s postulates tomatic tissues onto CVP (crystal violet pec- were fulfi lled by inoculation onto pota- tate) and NAG (nutrient agar glucose) media. to plants and tubers and re-isolation from Single bacterial colonies having characteris- the sites of observed typical disease symp- tic pectinolytic activity (CVP) and/or “fried toms. This is the fi rst report of potato black- egg” colonies (NAG) were sub-cultured, pu- leg caused by Dickeya sp. biovar 3 (D. solani) rifi ed, and used for further characterisation. in a region outside of Crete (Nevrokopi, Dra- Twenty fi ve isolates were characterized as ma). As D. solani, species include very infec- Dickeya sp. biovar 3 (syn: Erwinia chrysan- tious strains, further spread of the pathogen themi, Pectobacterium chrysanthemi) based in potato production in Greece is expected on standard biochemical tests. These strains to have a high economic impact.

Molecular characterization of Pseudomonas viridifl ava strains from several hosts E.E. MPALANTINAKI, P.F. SARRIS*, E.A. TRANTAS, F.N. VERVERIDIS and D.E. GOUMAS Technological Educational Institute of Crete, School of Agricultural Technology, Department of Plant Sciences, P.O.Box 1939, GR-710 04 Heraklion, Greece. *Present Address: The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

The pectinolytic species Pseudomonas virid- and basal stem and root rots. In Greece this ifl ava is characterized as a weak opportunis- pathogen has been reported as a disease tic plant pathogen, with a wide host range, causal agent in a wide range of host plants causing foliar and stem necrotic lesions including celery, blitum, eggplant, acanthus,

© Benaki Phytopathological Institute 30 Hellenic Plant Protection Journal - Special Issue tomato, artichoke, rocket, among others. lation analyses, where the 18 isolates were This work reports the biochemical and mo- tested by rep-PCR fi ngerprinting (BOX- and lecular characterisation of bacterial isolates ERIC-PCR) as well as by Multilocus Sequence primarily identifi ed as Pseudomonas viridi- Typing (MLST), utilizing the partial sequenc- fl a v ,a that have been maintained for the last es of the genes gyrB, rpoB and rpoD. In con- twenty years in the collection of Bacteriolo- clusion, the biochemical tests and pathoge- gy lab, TEI of Crete. Amongst the 64 isolates, nicity profi ling did not reveal any variability 26 isolates obtained from several hosts were among the isolated strains studied. How- selected and their taxonomy was confi rmed ever, the molecular fi ngerprinting patterns based on their phenotypic, biochemical and and housekeeping gene sequences clear- physiological characteristics, as well as their ly demonstrated a signifi cant genomic vari- pathogenicity on diff erent hosts. These re- ability among the 18 isolates tested. sults were confi rmed by molecular popu-

Characterization of isolates of Pseudomonas corrugata and P. mediterranea, causal agents of «pith necrosis» disease of tomato on Crete M.G. PENTARI, E.E. MPALANTINAKI, P.F. SARRIS*, E.A. TRANTAS, F.N. VERVERIDIS and D.E. GOUMAS Technological Educational Institute of Crete, School of Agricultural Technology, Department of Plant Sciences, P.O.Box 1939, 71004 Heraklion, Greece. *Present Address: The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

Pseudomonas corrugate is a plant pathogen 36 isolates, which were analyzed by classi- that causes pith necrosis in tomato. Infect- cal bacteriological tests, 28 were classifi ed ed plants were identifi ed by a general chlo- as P. mediterranea and the remaining 8 as rotic appearance, a gradual withering and P. corrugata, including the reference strains pith necrosis. Recent fi ndings proposed that CFBP5447T and NCPPB2445 respectively. A Pseudomonas mediterranea, which is isolat- similar identifi cation was also observed used frequently from cultivated tomato fi elds ing the antiserum anti-PC14 for P. corrugata of countries in the Mediterranean basin, is in immunofl uorescence tests. These results a related bacterium acting as a new patho- were further confi rmed by molecular anal- genic agent causing pith necrosis. This ysis using a) the species specifi c PCR prim- work reports the characterization of bacte- er pairs PC1 and PC5, for the identifi cation rial isolates from pith necrosis of diseased of P. mediterranea and P. corrugata respec- tomato and pepper plants on the island tively, b) by rep-PCR (BOX and ERIC) fi nger- of Crete, obtained within the period 1991- printing, and c) by Multilocus Sequence Typ- 2009. Amongst the 72 isolates deposited in ing (MLST) utilizing the partial sequences the collection of the Bacteriology Lab, TEI of of the genes gyrB, rpoB and rpoD. Phyloge- Crete, 36 were chosen based on phenotyp- netic analysis of the isolated strains did not ic characters such as the absence of fl uores- reveal signifi cant genetic variability among cence and a positive oxidase reaction, the them. This is the fi rst report of Pseudomo- ability to elicit the typical hypersensitive re- nas mediterranea as a pathogen of pith ne- action on tobacco plants and their patho- crosis disease of tomato and pepper plants genicity on tomato plants. Among these in Greece.

Comparative genomics of multiple strains of Pseudomonas cannabina pv. alisalensis, a potential model pathogen of both monocots and dicots

P.F. S ARRIS1*, E.A. TRANTAS1, D.A. BALTRUS3, C.T. BULL4, W.P. WECHTER5, S. YAN6, C.D. JONES7, J.L. DANGL7,8, N.J. PANOPOULOS2,9, F.N. VERVERIDIS1, B.A. VINATZER6 and D.E. GOUMAS1 1Department of Plant Sciences, School of Agricultural Technology, Technological Educational Institute of Crete, Greece. 2Department of Biology, University of Crete, Heraklion, Greece. 3School of Plant Sciences, The University of Arizona, Tucson, Arizona, USA. 4United States Department of Agriculture, Agricultural Research Service, Salinas, CA, USA. 5United States Department of Agriculture– Agricultural Research Service, Charleston, South Carolina, USA. 6Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, Virginia, USA. 7Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 8Howard Hughes Medical Institute. 9Professor Emeritus, University Crete, Heraklion, Greece and University of California, Berkeley, CA, USA. *Present Address: The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

Comparative genomic study of closely re- nage pv. maculicola ES4326 as identical to Pcal lated pathogens with diff erent host range ES4326, the model pathogen P. syringae pv. provides insights into mechanisms of host- tomato DC3000 of Arabidopsis thaliana and pathogen interaction, diff erential virulence tomato, and P. syringae pv. syringae B728a factors, and pathogen evolution. Moreover, bean pathogen. All Pcal strains harbour two sequencing of various strains of the same genomic islands containing genetic elements pathogen can reveal additional information for type VI secretion systems (T6SSs). Surpris- concerning pathogen diversity and the mo- ingly, one of the Pcal T6SS is phylogenetical- lecular basis of virulence diff erences between ly closer to P. aeruginosa T6SS-I than the P. sy- strains. In the current study we present draft ringae T6SSs. All Pcal strains also harbour a genome sequences and a comparative ge- hrp/hrc gene cluster coding for a type III se- nomics analysis of four strains of Pseudomo- cretion apparatus (T3SS), which in regard to nas cannabina pv. alisalensis (Pcal), one of the structure and DNA sequence is most similar causative agents of bacterial blight of cruci- to Psy B728a, although Pst DC3000 is the clos- fers, isolated from geographically distant ar- er relative, suggesting horizontal gene trans- eas of Greece and the USA. Since Pcal causes fer of the hrp/hrc cluster between the strains’ disease in a wide range of plant species and ancestors. Although the overall genetic con- can infect both monocots and dicots, includ- tent of each of the four Pcal genomes appears ing the model plants Arabidopsis thaliana and to be highly similar, the repertoire of the type tomato, draft genome sequences of four Pcal III eff ector proteins (T3EPs) was found to be strains were obtained to develop hypothe- signifi cantly divergent between the exam- ses regarding the molecular basis of virulence ined strains, reinforcing previous molecular and host range determinants of this patho- data suggesting the existence of two distinct gen. Genomes were also compared to the ge- lineages within this pathovar of Pseudomonas nome of the recently reclassifi ed strain P. syri- cannabina.

Study of the resistance of tomato hybrid CLX3731 transgenic plants overexpressing the gmgstu4 gene after infection with the bacterium P. syringae pv. tomato Ν. ΤZIOUTZIOU1, P. MADESIS2 and I. ΝIANIOU-ΟBEIDAT1 1Laboratory of Genetics and Plant Breeding, School of Agriculture, A.U.TH. , GR-541 24 Thessaloniki, Greece. 2Institute of Agrobiotechnology, C.E.R.TH., 6th km Charilaou- Thermis Road, GR-570 01 Thermi, Thessaloniki, Greece

In tomato cultivation biotic stresses result in of the transgene in the lines (Β3 και Β7), and lower production and product quality. The to study transgene expression, respectively. GST isoenzymes participate in the antiox- To examine resistance, in vivo transgenic and idative defence mechanism of plants con- wild type plants of 4-6 weeks old were in- tributing to resistance against biotic stress fected with the bacterium P. syringae pv. to- factors. The basic aim of our study was the mato. The response of the transgenic plants formation of transgenic lines overexpressing to infection was evaluated 5 days later. The the gmgstu4 gene using the CLX3731 toma- transgenic lines showed 1.5 times fewer in- to hybrid and to study their response to bac- fected leaves and 6.4 times fewer necrotic terial speck disease. For the genetic transfor- lesions per plant, 1.7 times less electrolyte mation, cotyledons of 12-15 days old were leakage and 3.7 times more glutathione per- cocultivated with transformed Agrobacteri- oxidase enzymatic activity when compared um tumefaciens and subsequently grown on with the wild type plants. The study of the

MSR medium (0.1 mg/L ΙΑΑ, 0.5 mg/L Ζ, 250 gstu4 gene in genetically modifi ed tomato mg/L Cf και 100 mg/L Km). PCR and RT-PCR plants may suggest new ways to engineer were conducted to verify the the presence resistance against biotic stress factors.

First report of a “Candidatus phytoplasma solani” related strain infecting two accessions of jimsonweed (Datura stramonium) in Greece L. LOTOS1, J.T. TSIALTAS2, V.I. MALIOGKA1, N. KALOUMENOS2, I. G. ELEFTHEROHORINOS2 and N.I. KATIS1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece. 2Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, Faculty of Agriculture, Laboratory of Agronomy, GR-541 24 Thessaloniki, Greece

Datura stramonium is a common weed in (acc. no. HE598778 and HE598779 for D. stra- spring crops in Greece and worldwide, which monium f. stramonium and D. stramonium f. includes two accessions (D. stramonium f. tatula, respectively) exhibited 99% similarity stramonium, D. stramonium f. tatula). During a with the “Ca. Phytoplasma solani” reference fi eld trial conducted at the Aristotle Universi- strain (AF248959). Infected plants of D. stra- ty Farm, where the two accessions were eval- monium f. stramonium and D. stramonium f. uated for growth rate and alkaloid content, tatula showed 49-69% and 38% reduction of phytoplasma-like symptoms were observed. the above-ground fresh weight, respective- Plant samples were taken and tested with ly, as compared with the healthy ones. These a generic nested-PCR targeting the high- fi ndings suggest that the phytoplasma is an ly conserved 16S rRNA gene of the phyto- important pathogen of jimsonweed, which plasma genome. The ~1200 bp amplicon ob- reduces its vigour and makes this weed spe- tained only from the symptomatic plants of cies a good host-reservoir for this disease. both Datura accessions was sequenced and This is, to our knowledge, the fi rst report of compared with the NCBI database isolates, a “Ca. Phytoplasma solani” related disease in using the BLASTn algorithm. Both isolates jimsonweed found in Greece.

Pseudomonas cichorii is the causal agent of tomato pith necrosis in Crete E.A. TRANTAS, P.F. SARRIS*, M.G. PENTARI, E.E. MPALANTINAKI, F.N. VERVERIDIS and D.E. GOUMAS Technological Educational Institute of Crete, School of Agricultural Technology, Department of Plant Sciences, P.O.Box 1939, GR-710 04 Heraklion, Crete, Greece.

*Present Address: The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Recent taxonomic advances based on bio- was achieved using diff erent molecular typ- chemical as well as genotypic analysis have ing methods, including rep-PCR (BOX- and demonstrated that plant pathogen species ERIC), as well as Multilocus Sequence Typing Pseudomonas cichorii consists of a cluster (MLST), utilizing the partial sequences of the of closely related genomic groups collec- genes gyrB, rpoB and rpoD. Likewise, a num- tively defi ned as the P. cichorii complex. Pri- ber of biochemical tests were also used for or to this study, three pathovar groups had the biochemical identifi cation of the toma- been described, all sharing common pheno- to P. cichorii isolates. To our knowledge, this typic and biochemical characters but pre- is the fi rst complete biochemical, molecular senting variability regarding their DNA con- and phylogenetic analysis of P. cichorii spe- tent. All entities of the complex can cause cies as the causal agent of tomato pith ne- disease in a variety of hosts, including let- crosis. Our results clearly demonstrate the tuce, celery and chrysanthemum, among emergence of a new genomic group in the others. In this study, we present the isola- P. cichorii complex, consisting of strains that tion and biochemical and molecular charac- could consistently be separated from other terisation of P. cichorii isolates as the caus- members of the complex. Finally, this is the al agent of pith necrosis in tomato plants. A fi rst record of P. cichorii as a pathogen of to- detailed characterization of the genetic vari- mato pith necrosis in Greece. ability among strains belonging to P. cichorii

Infection of watermelon, melon and cucumber plants by a phytopathogenic bacterium of the genus Acidovorax in Greece M.C. HOLEVA, P.E. GLYNOS and C.D. KARAFLA Benaki Phytopathological Institute, Department of Phytopathology, Laboratory of Bacteriology, 8 St. Delta Str., GR-145 61 Kifi ssia, Athens, Greece

During the period of March-May 2012, spec- al streaming from lesion margins. Bacterial imens of young seedlings of watermelon, isolates obtained from the leaf lesions were melon and cucumber from a greenhouse consistently identifi ed on the basis of cul- crop in the area of Thiva (Boeotia), exhibit- tural, biochemical and serological assays, as ing necrotic, brown, irregular-shaped leaf well as a tobacco hypersensitivity test, as a lesions, of variable size and most often sur- phytopathogenic species of the genus Aci- rounded by a chlorotic halo, were examined dovorax. Sequencing of the 16S rDNA region in the Laboratory of Bacteriology of the Insti- showed signifi cant sequence identity of the tute. The lesions were observed on the coty- isolates to A. valerianellae, whereas PCR as- ledons of watermelon, melon and cucumber says showed them to diff er from reference plants, as well as on a small number of true strains of A. citrulli. Studies are ongoing to leaves of the melon plants. The outbreak was further characterize the isolates at the spe- reported by the agronomist who supervised cies level. This is a preliminary report on the the crops to have aff ected 10 to 100 % of the presence of a phytopathogenic species of plants. In all specimens, microscopic exam- Acidovorax causing economically important ination of aff ected leaves revealed bacteri- damage to crops of cucurbits in Greece.

First report of infection of sweet basil plantlets by the phytopathogenic bacterium Pseudomonas viridifl ava in Greece

M.C. HOLEVA, C.D. KARAFLA and P.E. GLYNOS Benaki Phytopathological Institute, Department of Phytopathology, Laboratory of Bacteriology, 8 St. Delta Str., GR-145 61 Kifi ssia, Athens, Greece

In November 2011, specimens of young ic examination of sections of aff ected leaves plantlets of sweet basil (Ocimum basilicum revealed bacterial streaming from lesion L.) cv. Genovese from a commercial hydro- margins. Bacterial isolates obtained from ponic culture in the area of Acharnes (Attiki), the leaf lesions were consistently identifi ed exhibiting irregular-shaped, necrotic, black on the basis of cultural, physiological and lesions of variable size at leaf margins were biochemical assays as well as a pathogenic- examined in the Laboratory of Bacteriolo- ity test, as Pseudomonas viridifl ava.To the gy of the Institute. This outbreak was report- best of our knowledge this is the fi rst report ed by the agronomist in charge to have af- of P. viridifl ava naturally infecting sweet ba- fected about 80 % of the plants grown in a sil in Greece. Further characterization of the 1000m2 cultivated area. Symptoms were ob- isolates and their comparison to P. viridifl ava served in a relatively small number of fully reference strains is in progress. expanded true leaves per plant. Microscop-

Abstracts - 16th Hellenic Phytopathological Congress 37

INVITED LECTURE

Deep sequencing and the identifi cation without prior knowledge of phytoviruses: applications to plant disease etiology and to metagenomics T. CANDRESSE1, A. MARAIS1, C. FAURE1, L. SVANELLA-DUMAS1, B. BERGEY1, Y. LAIZET1, M. CAMBRA2 and A. OLMOS2 1UMR 1332 BFP, INRA and Université de Bordeaux, BP81, 33883 Villenave d’Ornon cedex, France. 2Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, Spain

Similar to viruses infecting other organisms, an eff ort to identify viruses in Prunus stone plant viruses are collectively characterized fruit material, illustrating the power and the by an amazing diversity, encoding their ge- potential interest of these approaches. In nome on various kinds of nucleic acids and our hands, the best results so far were ob- sharing as a group no single common gene tained with dsRNA which can be analyzed in or determinant. The direct consequence of a multiplexed format in order to reduce in- this situation is that although the detection dexing costs. Although a detailed sensitivity of a known virus is now generally straight- comparison with biological indexing has yet forward, the detection, without prior knowl- to be performed, the cost of this new tech- edge, of all viruses present in a plant sample nique already compares favourably. remains a signifi cant challenge. The devel- The second area where these strategies are opment of novel sequencing technologies arousing wide interest is the global anal- (NGS: next generation sequencing) allows ysis of viral populations associated with unprecedented sequence data generation plants in an environment (metagenomics). at a fraction of the cost of previous technol- Such questions were formerly not accessi- ogies and has drastically altered this situa- ble to experimentation but are now feasible tion. Indeed, it is now possible to generate thanks to the democratization of NGS. We vast amounts of sequence information from are developing this approach in two con- a plant sample and to then use bioinformat- trasted environments, the simplifi ed eco- ics tools to sift through this data in order to system of the Kerguelen islands, the second identify any virus that might be present. Var- most isolated archipelago on earth, and in a ious templates have been sequenced in such less constrained but anthropized temperate approaches, including messenger RNAs (mR- horticultural setting. The fi rst results indi- NAs), small interfering RNAs (siRNAs), dou- cate a very high proportion in dsRNA viruses ble stranded RNAs (dsRNAs) or nucleic acids in the Kerguelen islands and, conversely, an extracted from semi-purifi ed viral particle enrichment in pathogenic ssRNA viruses in preparations. In the case of DNA plant virus- the agricultural context. They also provide es, which to date all have circular genomes, a fi rst glimpse at virus biodiversity in these the sequencing of RCA (Rolling Circle Ampli- two highly contrasting situations. fi cation) products has been used. Outside of these fi rst results demonstrating The fi rst area in which these approaches are the interest and the potential of these nov- likely to have a major impact concerns the el approaches, it is clear that new technical fi eld of etiology and the conceptually linked and conceptual developments are to be ex- certifi cation and quarantine fi elds. In each pected in the coming years. On the techno- case, the key problem is to be able to quickly logical side, NGS is progressing, allowing identify with great sensitivity any viral agent for ever increasing volumes of sequence at that may be present in a sample. We have re- lower cost. Challenges are therefore to be cently used siRNA and dsRNA sequencing in found in the processing of samples (autom-

© Benaki Phytopathological Institute 38 Hellenic Plant Protection Journal - Special Issue atization, multiplexing etc.) and in the pro- the conceptual fi eld, these developments cessing of the vast amount of sequence data are taking us to a new, more integrated vi- generated, were many questions remain. In sion of viral ecology.

ORAL & POSTER PRESENTATIONS

Analysis of siRNAs using a new generation sequencing platform for the detection and characterization of viruses and viroids present in a citrus sample A. OLMOS1, C. VARVERI2, M.C. MARTÍNEZ1, E. BERTOLINI1, T. CANDRESSE3,4 and M. CAMBRA1 1Instituto Valenciano de Investigaciones Agrarias (IVIA). Plant Protection and Biotechnology Center, Moncada-Naquera km5, 46113 Moncada, Valencia, Spain. 2Benaki Phytopathological Institute, Laboratory of Virology, 8 St. Delta Str., GR-145 61 Kifi ssia, Greece. 3INRA, UMR1332 BFP, BP81, 33883 Villenave d’Ornon cedex, France. 4Université de Bordeaux, UMR 1332 BFP, BP81, 33883 Villenave d’Ornon cedex, France

The population of virus derived small inter- isolates, which allowed the determination fering (si) RNAs (21-24 nucleotides) induced of almost the full-length 19.251 nt genome by Dicer processing of dsRNAs formed dur- of this L192GR-CTV isolate. Only a small gap ing RNA virus replication can be sequenced of 18 nt was identifi ed and was later deter- by Ion Torrent next generation sequencing mined by RT-PCR and direct sequencing. technology. This strategy has been success- Phylogenetic analysis of the L192GR isolate fully used to analyze a sample derived from revealed high molecular homology with a lemon tree of the “Lemonodasos” area in the Israeli VT-CTV isolate (GenBank Acc. No Poros (Trizinia, Prefecture of Piraeus) where EU937519.1) with 98% sequence identity. In Citrus tristeza virus (CTV) had been previously addition, the de novo analysis of siRNA se- detected. Initially, lemon samples were graft- quences allowed the reconstruction of the ed on seedlings of sweet orange cv. Madam complete full-length genomes of Citrus exo- Vinous and then siRNAs were isolated. The cortis viroid and Hop stunt viroid and the re- Ion Torrent platform allowed the determina- trieval of partial sequences of Citrus viroid tion of 432.632 sequences. The de novo bio- III and Citrus viroid IV. These results demon- informatic analysis confi rmed the presence strate the great potential of this next-gen- of CTV by detecting several large contigs eration sequencing platform, opening new of this virus species. The isolate present in possibilities for the diagnosis and character- the sample was reconstructed by mapping ization of citrus viruses and viroids. the specifi c siRNAs against several reference

Development of a nested RT-PCR for the detection of Little cherry virus-1 and study of its presence in several host species Α.T. KATSIANI, Ε.V. DROUGKAS, V.Ι ΜALIOGKA and Ν.Ι.ΚATIS Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece

Little cherry virus-1 (LChV-1) and Little cherry ily Closteroviridae, are associated with lit- virus-2 (LChV-2), both members of the fam- tle cherry disease. In previous surveys, only

LChV-1 was detected in sweet cherry or- fying a 200 bps product within the gener- chards in Greece. Due to the high intraspe- ic nested RT-PCR amplicon, were designed. cies diversity of LChV-1, which can result in The new assay exhibited a broader detec- unreliable virus detection, we developed tion range when compared with other avail- a new molecular assay which detects all able methods and it was also able to detect LChV-1 isolates. For that purpose, several se- the virus in sweet cherry trees throughout quences of Greek LChV-1 isolates were ob- the year. Surveys conducted with this assay tained with a generic nested RT-PCR, which in stone fruit orchards has shown that LChV- amplifi es the 5΄part of the HSP70h gene 1 was present in sweet cherry (70/162), plum (500 bps) of closterovirids. These sequences (2/82), peach (1/54) and sour cherry (2/5) were aligned with others available in the da- while it was not detected in almond (0/126) tabase and new degenerate primers, ampli- and apricot trees (0/7).

Complete nucleotide sequencing and genome analysis of Eggplant mottled dwarf virus (EMDV) P.G . PAPPI 1, C.I. DOVAS 2, K.E. EFTHIMIOU 1, V.I. MALIOGKA 1, N.I. KATIS 1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece. 2Aristotle University of Thessaloniki, Faculty of Veterinary Medicine, Laboratory of Microbiology and Infectious Diseases, GR-541 24 Thessaloniki, Greece

Eggplant mottled dwarf virus (EMDV) is en- cleocapsid protein, X an unknown 10.8 kDa demic in the Mediterranean region since protein, P the 32.5 kDa phosphoprotein, Y a the end of 60’s. However, it remains one of putative 31.7 kDa movement protein, M the the less studied members of the genus Nu- 27.7 kDa matrix protein, G the 69 kDa glyco- cleorhabdovirus in the family Rhabdoviridae. protein and L the 221.7 kDa polymerase. Ad- The virus has a broad host range including ditionally, the genome also contains 198 and cultivated and ornamental plants. Until re- 90 nts long untranslated leader and trailer cently, only a small part of the glycoprotein sequences, respectively. The untraslated re- gene of EMDV was sequenced. Thus in this gions among the virus genes appear highly study, the full nucleotide genome sequence conserved and were used as a tool for the de- was determined, which is 13.093 nucleotides termination of the virus genome sequence. (nts) long. The negative sense, single-strand- Phylogenetic analysis confi rmed the classifi - ed RNA of the virus contains seven ORFs, cation of EMDV among nucleorhabdoviruses which are organized in the order 3’-5’ as N-X- and showed its close evolutionary relation- P-Y-M-G-L, where N encodes the 52.0 kDa nu- ship with Potato yellow dwarf virus (PYDV).

Study of the genetic variability of Eggplant mottled dwarf virus (EMDV) P.G . PAPPI1, C.I. DOVAS2, K.E. EFTHIMIOU1, V.I. MALIOGKA1 and N.I. KATIS1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece. 2Aristotle University of Thessaloniki, School of Veterinary Medicine, Laboratory of Microbiology and Infectious Diseases, GR-541 24, Thessaloniki, Greece

Eggplant mottled dwarf virus (EMDV) has a lanaceae, Cucurbitaceae) and ornamental wide host range including cultivated (So- plants. Although the entire genome of a vi-

© Benaki Phytopathological Institute 40 Hellenic Plant Protection Journal - Special Issue rus isolate from eggplant was recently se- netic variability in the gene encoding the X quenced, studies concerning the genet- protein which was up to 22% at nt level and ic variability among isolates from diff erent 23% at amino acid level. N, G and Y genes plant species are still limited. In the present were the most conserved displaying a max- study, a comparative analysis of gene se- imum variability of 14, 15 and 16% at nt lev- quences encoding structural and function- el (1 to 4% at amino acid level). The virus iso- al proteins between virus isolates from var- lates from eggplant, cucumber, tobacco and ious hosts such as eggplant, caper (Caparis hibiscus constitute a distinct phylogenet- spinosa), honeysuckle (Lonicera japonica), to- ic subgroup which diff ers from the respec- mato, tobacco, cucumber, rose mallow (Hi- tive consisting of EMDV isolates from toma- biscus syriacus) and Pittosporum tobira was to, caper, honeysuckle and P. tobira. Certain conducted. More specifi cally, the compari- isolates from caper plants are of particular son was held among sequences of the N, X, interest as they have a truncated G ORF, 92 Y and G ORFs (849, 294, 633 and 1257 nt, re- amino acids shorter compared to that of the spectively). The analysis revealed high ge- homologous ORF of the other isolates.

Detection and molecular characterization of viruses belonging to the family Betafl exiviridae (CNRMV, CGRMV and CVA) in sweet cherry orchards E.V. DROUGKAS, A.T. KATSIANI, E. DELIGIANNIS, C. KTORI, N.I. KATIS and V.I. MALIOGKA Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece

Cherry is susceptible to a number of virus Cherry green ring mottle virus (CGRMV) and species belonging to the family Betafl exiviri- Cherry virus A (CVA). In order to further study dae. However, their presence has not been their incidence, the generic nested RT-PCR studied extensively in Greece. For that rea- assay was modifi ed to include specifi c prim- son surveys were conducted in the spring of ers for the detection of CNRMV, CGRMV and 2006, 2007 and 2009 and 166 cherry samples CVA. The assays performed showed high were randomly collected from 12 diff erent incidence of CNRMV (64/166), followed by areas in Northern Greece. The samples were CVA (44/166) and CGRMV (21/166). Sequence tested for the presence of viruses of the fam- analysis of PCR products confi rmed the spec- ily Betafl exiviridae by using a generic nested ifi city of the methods used and showed high RT – PCR that amplifi es part of the viral poly- intraspecies variability of the viruses tested. merase of betafl exiviruses. Sequence analy- The detection methods developed are cur- sis of selected samples showed high similar- rently applied for studying the presence of ity with published homologous sequences CNRMV, CGRMV and CVA in other stone fruit of Cherry necrotic rusty mottle virus (CNRMV), trees.

Molecular Characterization and genetic diversity of Little Cherry Virus-1 (LChV-1) populations Α.T. KATSIANI, V.I. ΜALIOGKA, K.E. EFTHIMIOU and N.I. KATIS Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece

Little Cherry Virus-1 (LChV1), a member of the ruses associated with Little Cherry disease family Closteroviridae, is one of the two vi- (LChD) which causes severe losses in sus-

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 41 ceptible varieties by aff ecting fruit quality. (partial), RNA depended- RNA polymerase During a survey conducted in sweet cher- (RdRp), the small hydrophobic protein (p4), ry orchards for the presence of closterovi- the HSP70 homolog, the 61kDa protein and ruses, one sample in which LChV-1 and -2 the capsid protein (CP). High sequence di- could not be detected with specifi c prim- vergence was found compared to the other pairs, showed positive reaction in Closter- er known LChV-1 isolates which was ranging ovirus infection using a generic nested RT- from 7-10% in the RdRp and up to 29-35% in PCR. Sequencing and comparative analysis aminoacids in the p61 protein. Phylogenetic of the product amplifi ed with the generic analysis using part of the HSP70h, RdRp and assay revealed the presence of a virus dis- CP genes of several Greek isolates with oth- tantly related to the known LChV-1 isolates ers indicated the presence of diff erent evo- and therefore it was further characterized. lutionary clades. The eff ect of the genetic A 7.100 nucleotide part of the virus genome recombination in LChV1 evolution is under was determined including the Helicase investigation.

Epidemiology and genetic characterization of criniviruses associated with tomato yellows disease in Greece C.G. ORFANIDOU1, C. DIMITRIOU,1 L.C. PAPAYIANNIS,2, V.I. MALIOGKA1 and N.I. KATIS1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Plant Pathology Laboratory, GR-541 24, Thessaloniki, Greece. 2Agricultural Research Institute, P.O. Box 22016, Nicosia 1516, Cyprus

Tomato infectious chlorosis virus (TICV) and ilies) and 1041 adult whitefl ies were collect- Tomato chlorosis virus (ToCV) are transmit- ed. In tomato crops, results revealed the ted by whitefl ies in the semi-persistent prevalence of TICV (87%) over ToCV (13%), manner and are associated with yellows a fact that signifi es the direct correlation disease in tomato crops in Greece. In 2009- of their distribution with the whitefl y-vec- 2012, an extensive survey was conducted in tor species that prevails in each geograph- order to identify the presence of these vi- ic area. Weeds seem to play a signifi cant role ruses in open fi eld and greenhouse tomato in the epidemiology of both viruses as 26 crops. Moreover, the presence of TICV and species belonging to 15 diff erent families ToCV was also investigated in a number of were found to be infected with TICV or/and other vegetable crops that showed mild yel- ToCV. It is worth mentioning that for the fi rst lowing symptoms and in weed species (re- time in Greece lettuce plants showing mild gardless of symptoms); whitefl ies were also yellowing symptoms were found infected collected for typing from the aff ected crops. with ToCV. Finally, sequence analysis of the For the detection of TICV and ToCV a nest- CP and CPm genes from tomato and weed ed multiplex RT-PCR was used, while white- isolates of ToCV and TICV showed that their fl y identifi cation was done by real time RT- populations show very low intraspecies ge- PCR. In total, 1206 tomato, 4 lettuce, 1339 netic diversity. weed samples (42 diff erent species, 17 fam-

Tomato yellow leaf curl disease in the eastern Mediterranean basin: virus species, incidence, hosts and transmission properties L.C. PAPAYIANNIS1, A. PARASKEVOPOULOS2 and N.I. KATIS3 1Agricultural Research Institute, P.O.Box 22016, Nicosia 1516, Cyprus. 2Directorate of Agriculture, Plant Protection, GR-245 00 Kyparissia, Messinia, Greece. 3Aristotle

University of Thessaloniki, School of Agriculture, Plant Pathology laboratory, GR-541 24 Thessaloniki, Greece

During 2005-2012, an extensive survey was Molecular identifi cation of B. tabaci biotypes conducted in Cyprus, on Crete, the Dode- showed that Q was the only biotype found canese and the Ionian islands, as well as on in the mainland of Greece, Peloponnese and mainland Greece, in order to identify the vi- on the island of Crete. Both biotypes (B and rus species and Bemisia tabaci biotypes in- Q) are involved in TYLCD spread in Cyprus volved in Tomato yellow leaf curl disease and the Dodecanese islands. Forty nine dif- (TYLCD) epidemics. Approximately 8000 ferent weed species belonging to 15 botan- symptomatic tomato samples, 4500 weeds ical families tested positive to TYLCV under and 3000 whitefl y samples were collected fi eld conditions, suggesting that the host and analyzed. The host range of TYLCV and range of the virus is far more extensive that TYLCSV isolates was studied using whitefl y previously documented. Transmission stud- transmission tests in several plant species. ies showed that TYLCV isolates had a broad- Transmission effi ciency of TYLCV and TYL- er host range as well as a higher transmis- CSV was evaluated using diff erent B. tabaci sion effi ciency than TYLCSV. Finally, TYLCV biotypes colonies which harboured diff erent transmission was somehow correlated with bacterial endosymbionts. Results showed the presence of Hamiltonella sp. within the that in Greece, TYLCV was the most preva- body of the B. tabaci insect, as colonies that lent Begomovirus species (94.5%), whereas harboured this bacterium transmitted the TYLCSV was found in 4.5% of the total sam- virus more effi ciently both from and to to- ples tested. In Cyprus, TYLCV was the only mato plants. species found to be associated with TYLCD.

Citrus tristeza virus on the island of Crete: a survey and detection protocol applications M. SHEGANI1, D. TSIKOU1, A. VELIMIROVIC1, H. AFIFI1, A. KARAYANNI1, A. GAZIVODA1, K. MANEVSKI2, I. MANAKOS2 and I.C. LIVIERATOS1 1Plant Virology Laboratory, Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepion, GR-731 00, Chania, Crete, Greece. 2Department of Geoinformation in Environmental Management, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepion, GR-731 00, Chania, Crete. Greece

Over a period of two years, more than in all tissues tested. A PCR-amplifi ed dig-la- 5,000 citrus trees were tested for the pres- belled CTV p23 DNA probe was applied to ence of Citrus tristeza virus (CTV) on the is- stem and leaf prints, and to crude and to- land of Crete, resulting in thirty eight posi- tal RNA leaf extracts, using non isotopic hy- tives. Comparisons of the relative transcript bridization. This technique, when applied to levels of CTV p23, coat protein (CP), poly- stem or leaf prints, and particularly to total merase (POL) and an intergenic (POL/p33) RNA, unequivocally provided strong signals region using quantitative RT-PCR, revealed with minimal backgrounds. Moreover, an consistent diff erences in abundance for antiserum with high sensitivity and specifi c- each of these RNAs among fl owers, stems, ity of CTV detection as shown by DAS and young fruits and leaves of infected orange immunoprint ELISA was produced against trees. CTV p23 RNAs accumulated at highest bacterially-expressed CTV CP. By the former levels, reaching a maximum in the fl owers, method, stems and fl owers contained high- with lower levels in the leaves, while POL er levels of CTV CP when compared to leaf RNAs consistently accumulated at low levels extracts. Taking into account Cretan geog-

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 43 raphy and the importance of citrus to the is- en. The observations, materials and meth- land, systematic surveys for CTV eradication, ods presented here may assist all three tasks sustainable control measurements and epi- at a local and at a national level. demiological studies need to be undertak-

RNA silencing pathways may have a positive eff ect on Potato spindle tuber viroid infectivity in Nicotiana benthamiana E. DADAMI1,2*, K. KATSAROU1, A. BOUTLA1*, N. VRETTOS1, 2**, S. TZORTZAKAKI1, I. KARAKASILIOTI1, 2 and K. KALANTIDIS1,2 1Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, P.O. Box 1527, GR-711 10 Heraklion, Crete, Greece. 2Department of Biology, University of Crete, Heraklion, Crete, Greece. Current address: *B.A., BSRC “Alexander Fleming” P.O. Box 74145 GR-166 02, Varkiza, Greece; **V.N., Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

The role of RNA silencing during viral infec- stranded RNA (dsRNA) molecules produced tion in plants is well studied. However, the during its replication, may serve as substrate role of RNA silencing during viroid infec- for Dicer-like (DCL) enzymes. In order to ex- tion still remains elusive. Potato spindle tu- amine the role of RNA silencing upon PST- ber viroid (PSTVd) is a naked single strand- Vd infection, we produced Nicotiana bentha- ed RNA (ssRNA) plant pathogen, which does miana knock down DCL lines (DCLi lines) by not encode for any protein. PSTVd infection RNA silencing and then tested PSTVd infec- leads to the accumulation of abundant vi- tivity in the DCLi background. Our analyses roid small interfering RNAs (vd-siRNAs), the indicate that PSTVd infectivity is facilitated latter being able to trigger the degradation by DCL1, DCL3 and DCL4, but seems to be of homologous RNA sequences. Yet, PST- unaff ected by DCL2. In addition, viroid repli- Vd genomic RNA itself seems to be resistant cation seem also to be infl uenced in double to siRNA-directed degradation. Sequenc- DCLs knock down plants leading to diff ering of PSTVd siRNAs during infection has re- ent theories about their role in its infectivity. vealed a hotspot-confi ned origin, indicating Collectively, our results indicate that specif- that the rod shape-like structure of the ma- ic RNA silencing pathways may be advanta- ture PSTVd ssRNA, rather than the double geous for viroid infectivity.

Heterologous RNA silencing suppressor proteins support Plum pox virus infection V.I. MALIOGKA*, M. CALVO, A. CARBONELL, J.A. GARCIA and A. VALLI1 Centro Nacional de Biotecnologia-CSIC, Campus Universidad Autonoma de Madrid, 28049 Madrid, Spain. *Current address: Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece

HC-Pro is a multifunctional protein encod- by the P1b protein of Cucumber vein yellow- ed by the genome of potyviruses within the ing ipomovirus, an RSS coming from a virus family Potyviridae, which acts as a suppres- of the same family which however doesn’t sor of RNA silencing (RSS) in plants. Recent show any sequence similarity with HC-Pro. studies have shown that the HCPro of Plum In this study in order to clarify whether the pox virus (PPV) can successfully be replaced presence of a specifi c RSS is necessary for

© Benaki Phytopathological Institute 44 Hellenic Plant Protection Journal - Special Issue the establishment of a successful potyvirus including some of animal virus origin. Inter- infection, we tested the capacity of heterol- estingly, the capacity of a protein to replace ogous RSSs with a diff erent mode of action HCPro was not strongly associated with its coming from animal and plant viruses to re- ability to suppress silencing. Overall, the replace the HC-Pro. For this purpose infectivity sults of this study show that not all RNA si- studies took place using recombinant infec- lencing suppression strategies are equally tious clones of PPV which showed that the suitable for the eff ective escape of PPV and HC-Pro of the virus can be functionally re- the establishment of a successful infection. placed by some but not all the RSSs studied,

Studies on the adaptation of Potato virus Y to pepper plants N. VASSILAKOS1, V. SIMON2, A. TZIMA1, O. KEKTSIDOU1 and B. MOURY2 1Laboratory of Virology, Benaki Phytopathological Institute, St. Delta Str. 8, 14561 Kifi ssia, Greece. 2INRA, UR407 Pathologie Végétale, F-84140 Montfavet, France

Potato virus Y (PVY) isolates (genus Potyvirus) binations the entire virus genome. The in- are classifi ed into four major phylogenet- fectivity experiments on pepper plants us- ic groups, O, N, C1 and C2. Only isolates being these hybrid viruses have revealed that longing to group C1 are able to systemical- the major determinants of PVY infectivity on ly infect pepper plants. In order to identify pepper were located within the P3/PIPO and the regions of PVY genome that determine the CI coding regions. In addition, fl uores- the ability of C1 isolates to infect pepper cent microscopy of a PVY-N isolate incorpo- plants, a series of full length infectious virus rating the GFP reporter gene showed restric- clones have been constructed comprising of tion of the virus to a small number of cells in hybrids between a PVY-C1 and a PVY-N or the inoculated leaves, providing a fi rst in- a PVY-C2 isolate, covering in diff erent com- sight into the mechanism of resistance.

Further identifi cation of virus-host protein interactions (Pepino mosaic virus-tomato) and plant responses to viral infection M.M. MATHIOUDAKIS1,2 and I. LIVIERATOS1 1Plant Virology Laboratory, Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepion, GR-731 00, Chania, Crete, Greece. 2Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, P.O. Box 269, GR-541 24 Thessaloniki, Greece

Pepino mosaic virus (PepMV), belonging to nicity determinants is the result of virus-host the genus Potexvirus, was fi rst reported in protein interactions. We have recently iden- Peru (1980) in Solanum muricatum (pepi- tifi ed two tomato-PepMV protein interac- no). PepMV has spread over the last decade tions; Hsc70-PepMV CP, and catalase-PepMV in tomato crops throughout Europe, due TGBp1 (p25) by yeast two–hybrid interac- to its ready mechanical transfer and seed tion, electron microscopy and fl uorescence transmission, and has been included on the microscopy assays. In the present study, fur- alert list of pathogens necessitating con- ther in vivo and in vitro confi rmation of these trol strategies. Four PepMV genotypes exist interactions is presented. Tomato responses with sequence divergence that aff ects host to PepMV infection, as revealed using mo- range/symptomatology and most likely also lecular and biochemical methods are also pathogenicity as a result of specifi c interac- presented. tions with the hosts. One group of pathoge-

In vitro template-dependent synthesis of Pepino mosaic virus positive- and negative-strand RNA by its RNA-dependent RNA polymerase T.A.M. OSMAN1,2, R.C.L. OLSTHOORN3 AND I.C. LIVIERATOS1 1Plant Virology Laboratory, Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepion, GR-731 00, Chania, Crete, Greece. 2Department of Agricultural Botany, Faculty of Agriculture, Fayoum University, Fayoum 63514, Egypt. 3Department of Molecular Genetics, Leiden Institute of Chemistry, PO Box 9502, 2300 RA Leiden, The Netherlands

Pepino mosaic virus (PepMV)-infected toma- plus- and minus-strand RNAs. Rabbit poly- to plants were used to develop an in vitro clonal antibodies against an immunogenic template-dependent system for the study epitope of the PepMV RdRp (anti-RdRp) de- of viral RNA synthesis. Diff erential sedimen- tected a protein of approximately 164 kDa tation and sucrose-gradient purifi cation of in the membrane-bound and template-de- PepMV-infected tomato extracts resulted in pendent RdRp preparations and specifi cally fractions containing a transcriptionally ac- inhibited PepMV RNA synthesis when add- tive membrane-bound RNA-dependent RNA ed to the template-dependent in vitro tran- polymerase (RdRp). In the presence of Mg2+ scription system. The 300 nucleotide long ions, 32P-labelled UTP and unlabelled nucle- 3’-terminal region of the PepMV genome otides, the PepMV RdRp catalysed the con- containing a stretch of at least 20 terminal version of endogenous RNA templates into adenosine (A) residues, was an adequate ex- single- and double-stranded (ds) genomic ogenous RNA template for RdRp initiation RNAs and three 3’-co-terminal subgenomic of the minus-strand synthesis but higher dsRNAs. Hybridisation experiments showed transcription effi ciency was observed as the that the genomic ssRNA was labelled only in number of A residues increased. This obser- the plus strand, the genomic dsRNA mainly vation might indicate a role for the poly(A)- in the plus strand and the three subgenom- tail in the formation and stabilisation of ic dsRNAs equally in both strands. Following secondary structure(s) essential for the initi- removal of the endogenous templates from ation of transcription. The template-depen- the membrane-bound complex, the purifi ed dent specifi c RdRp system described in this template-dependent RdRp could specifi cal- article will facilitate identifi cation of RNA el- ly catalyse transcription of PepMV virion ements and host components required for RNA, in vitro-synthesized full-length plus- PepMV RNA synthesis. strand RNA and the 3’-termini of both the

Incidence of graft-transmissible pathogens in newly established peach orchards K. EFTHIMIOU, P.G. PAPPI, L. LOTOS, A.T. KATSIANI, S. GAVALEKA and N.I. KATIS Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece

Production of certifi ed plant propagation sence of Plum pox virus (PPV), Apple chlorot- material is regulated by the Hellenic Minis- ic leaf spot virus (ACLSV), Prunus necrotic ring- try of Rural Development and Food which spot virus (PNRSV), Prune dwarf virus (PDV), defi nes the requirements concerning its im- Peach latent mosaic viroid (PLMVd) and the port and export. Concerning the stone fruits, European stone fruit yellows phytoplasma certifi cation includes, among others, the ab- (ESFY). In order to determine the presence

© Benaki Phytopathological Institute 46 Hellenic Plant Protection Journal - Special Issue of these pathogens in Greece, fi eld samples gens PLMVd (66.66%) and PNRSV (16.1%), from new peach orchards of the prefecture while PPV (0.83%), ACLSV (1.11%), PDV (1.38 of Imathia were collected during the years %) and ESFY (0.55%) were detected very 2010-2011. Overall, 360 samples of diff erent rarely. According to these results, it is obvi- varieties of nectarine and peach were col- ous that the implementation of the recent lected and tested for the presence of these regulation for the production and distribu- viruses by Polymerase Chain Reaction (PCR) tion of certifi ed propagative material should protocols developed for their detection. The be mandatory. results revealed high incidence of the patho-

First report of Plum pox potyvirus in almond in Greece in the context of phytosanitary control M. KAPONI1, I.A. AXARLI2, P. KOUTRETSIS2, N. NIKOLOUDAKIS2, P. DROGOUDI3 and M.G. BERBATI4 15 Myrtiotissas Str., GR-151 26 Maroussi, Athens, Greece. 2Ministry of Rural Development and Food, Control Station for Vegetative Propagation Material, 2 Antheon Str., GR-151 23 Maroussi, Athens, Greece. 3Institute of Deciduous Trees, General Administration of Agricultural Research, ELGO ‘DEMETRA’, GR-590 35 Naoussa, Greece. 426 Doxis Str., GR-181 22, Korydallos, Piraeus, Greece

Phytosanitary control is critical for crop oto’ and ‘Retsou’, collected at Hemathia and health and for transport and certifi cation Attica, respectively. The leaves tested were of plant propagation material, especially in collected from all directions of the canopy case of quarantine pathogens, such as Plum and showed no or mild symptoms (mottle, pox potyvirus (PPV). PPV has been naturally distortion, chlorosis and vein clearing). In found in almond (Prunus dulcis, syn. Prunus order to detect the virus in almond, immu- amygdalus Batsch) in Hungary, Romania, Bul- nocapture RT-PCR (IC-RT-PCR) was tried in garia, Russia, Turkey and India. In addition, DAS-ELISA- and PPV-positive almond sam- almond resistance to PPV has been well es- ples, in the presence of proper positive con- tablished. In the Control Station for Vegeta- trols. This method is economically effi cient tive Propagation Material of Greek Ministry for conducting tests, as no separate nucle- of Rural Development and Food, phytosani- otide extraction is needed. Application of tary control is always practiced by applying this method will allow for the rapid detec- DAS-ELISA, an immunochemical method tion of the virus in extensive phytosanitary implementing polyclonal antibodies. Dur- controls in almond, a concealed host of this ing the years 2010-2012 PPV was detected in important pathogen. leaf samples from almond trees cvs ‘Drepan-

Phenotypic and functional analysis of SERRATE in Nicotiana tabacum plants N. KRIOVRISANAKI1,2, T. ALEXIADIS1,2 and K. KALANTIDIS1,2 1Department of Biology, University of Crete, Vassilika Vouton, GR-714 09 Heraklion, Crete, Greece . 2Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Greece, GR-711 10 Heraklion, Crete, Greece

RNA silencing is an evolutionarily conserved (sRNAs, 21-24nt-long), including microRNAs mechanism present in most eukaryotes. It is (miRNAs) and small–interfering RNAs (siR- implicated in sequence-specifi c regulation NAs), are key components of the RNA silenc- of gene expression, genome stabilization, ing mechanism. miRNAs are involved in the as well as in antiviral defense. Small RNAs regulation of developmental processes and

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 47 are also central to plant immunity against the response of these plants to viral and vi- bacterial, fungal and viral pathogens. In roid infection. SERRATE knockdown tobac- plants, SERRATE (SE) encodes a C2H2 zinc-fi n- co plants show abnormal leaf development ger protein and is considered a post-tran- and phase transition delay from the vege- scriptional regulator of miRNAs levels, pro- tative to reproductive phase, compared to cessing pri-miRNA precursors through its wild type plants. Our experimental data also direct interaction with HYL and DCL1. SER- suggest that SERRATE, and as a consequence RATE mutants in Arabidopsis thaliana aff ect miRNAs, participate in plant biotic defence. leaf polarity, phase transition, meristem ac- Further studies are required in order to un- tivity, and infl orescense architecture. Here, derstand the pathway through which SER- we present the phenotypic analysis of N. RATE aff ects plant defence mechanisms. tabacum SERRATE-knockdown plants and

First report of Malva vein clearing virus (MVCV) and Bean yellow mosaic virus (BYMV) in Greece L. LOTOS, C.G. ORFANIDOU and N.I. KATIS Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece

Genus Potyvirus is one of the largest plant Malva sylvestris exhibiting symptoms of vein virus genera consisting of 146 members, clearing and intraveinal chlorotic patterns many of which have been found to infect or- and plants from the genus Cassia with mosa- namental plants. During the spring-winter ic infl icted leaves were found to be infected. of 2011, ornamentals and weeds exhibiting The generic PCR amplicon was sequenced viral-like symptoms were observed in the and the nucleotide sequence was compared central area of Thessaloniki city. Sampling of with the ones deposited in the NCBI data- the aforementioned plants was performed base using the BLASTn algorithm. The Malva which were in turn subjected to a two step isolate was found to be 97 % similar with the generic RT-PCR capable of detecting mem- Malva vein clearing virus and the Cassia sp. bers of the Potyvirus genus. The presence of one was 91 % identical to Bean yellow mosa- potyviruses was detected in two plant spe- ic virus. This is, to our knowledge, the fi rst re- cies. More specifi cally, plants of the species port of these two viruses in Greece.

First report of a BLRV-like virus in three Fabaceae species L. LOTOS1, J.T. TSIALTAS2 and N.I. KATIS1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece. 2Aristotle University of Thessaloniki, Faculty of Agriculture, Laboratory of Agronomy, GR-541 24 Thessaloniki, Greece

Bean leafroll virus (BLRV) belongs to genus sp. sativa), bitter vetch (Vicia ervilia (L.) Willd.) Luteovirus and is one of the most impor- and alfalfa (Medicago sativa L. subsp. sativa). tant viruses of Fabaceae, as an early infec- Leaf samples were collected from the infect- tion causes severe yield losses of the aff ected plants, as well as from healthy ones of the ed plants. During the spring of 2011 typical same species, which were later on checked symptoms of a luteovirus infection were ob- for the presence of BLRV with a specifi c RT- served in common vetch (Vicia sativa L. sub- PCR. This method amplifi es a 391bp portion

© Benaki Phytopathological Institute 48 Hellenic Plant Protection Journal - Special Issue of the viral coat protein (CP), which was ob- sults with the use of a luteovirus generic de- tained from all of the symptomatic samples tection method did not yield the expected but not from the healthy controls. Sequenc- amplicons. The inability to obtain the ampl- ing of the amplicon and comparison via the icons from both ORF regions could possibly BLASTn algorithm showed 98-99% similari- indicate the existence of variability in the ty of the common vetch (HE601635), bitter new isolates. This is, to our knowledge, the vetch (HE601636) and alfalfa (HE601637) iso- fi rst report of a BLRV-like virus in these Fa- lates with BLRV. An attempt to verify the re- baceae species.

Molecular detection of Grapevine virus A (GVA) and Grapevine rupestris stem pitting associated virus (GRSPaV) in grapevine cultivars and rootstocks K. MORAKI1, C.G. ORFANIDOU1, V.I. MALIOGKA1, G. GRAMMATIKAKI2, A. AVGELIS3 and N.I. KATIS1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece. 2Σχολή Τεχνολογίας Γεωπονίας, Τεχνολογικό Εκπαιδευτικό Ίδρυμα Κρήτης. 3Laboratory of Plant Virology, Institute of Grapevine, Greek Agricultural Organization “Demeter”, Heraklion, Crete

Grapevine virus A (GVA) and Grapevine rup- 20 grafted cultivars. Detection of GVA and estris stem pitting associated virus, GRSPaV) GRSPaV was done by RT-PCR. In the case of are members of the genera Vitivirus and Fo- GVA an RT-PCR was developed and applied veavirus (family Betafl exiviridae), respective- which uses degenerate primers binding to ly. They are both widely distributed world- the capsid protein and shows wide detec- wide and they are associated with diff erent tion range whereas detection of GRSPaV diseases of the Rugose wood complex. The was based on a published method. The re- purpose of this study was to evaluate the sults showed that only GRSPaV (7/26) was presence of GVA and GRSPaV in self-rooted, detected in the rootstocks, whereas only grafted grapevine cultivars as well as in root- GVA (12/60) was detected in self-rooted cul- stocks which are cultivated in Greece. In to- tivars. In grafted cultivars, both viruses were tal, we tested 26 samples originating from detected with GVA (44/136) being dominant 20 diff erent rootstocks, 60 samples from 17 followed by GRSPaV (26/136). self-rooted cultivars and 136 samples from

Etiology of yellows disease in cucurbit crops in Greece C.G. ORFANIDOU1, L.C. PAPAYIANNIS2, L. LOTOS1, C. DIMITRIOU1, E. DIOGENOUS1, V.I. MALIOGKA1 and N.I. KATIS1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece. 2Agricultural Research Institute, P.O. Box 22016, Nicosia 1516, Cyprus

Cucurbit yellows disease (CYD) is a wide- DV) and Cucurbit aphid-borne yellows virus spread disease in cucurbit crops in Greece. (CABYV) are usually implicated. BPYV, CYS- However, its etiology has not been studied DV and CABYV are endemic in Greece. BPYV suffi ciently, despite the fact that it is well and CYSDV are transmitted with the whitefl y known that Beet pseudo-yellows virus (BPYV), species Trialeurodes vaporariorum, and Bemi- Cucurbit yellow stunting disorder virus (CYS- sia tabaci, respectively, while CABYV is trans-

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 49 mitted persistently by the aphid species ed with the presence of the whitely-vector Aphis gossypii and Myzus persicae. During ex- species that exists in the geographic area. tensive surveys conducted in 2011-2012, leaf Moreover, CYSDV is the predominant virus samples were collected from cucurbits ex- (35.64%) in greenhouse crops, followed by hibiting yellowing symptoms in diff erent ar- CABYV (23.76%) and BPYV (23.76%). In con- eas of Greece. From the aff ected crops, we trast, CABYV prevails (74.1%) in open-fi eld also collected arable weeds and adult white- crops, followed by CYSDV (66.6%), where- fl ies. RT-PCR was used for the determina- as BPYV was not detected in open-fi eld cu- tion and discrimination of viruses and Taq- curbit crops. Infected weeds seem to play an Man PCR was used for typing of whitefl y important role in the epidemiology of these species. In total, 128 samples from 4 cucur- viruses, as neither of CYSDV, BPYV or CABYV bit crops (cucumber, zucchini, melon, water- is seed-transmitted. It is worth mentioning melon), 34 samples from 6 weed species and that for the fi rst time, CYSDV and CABYV were 241 adult whitefl ies were collected. Results detected in watermelon plants that showed showed that the etiology of CYD is correlat- mild yellowing symptoms in Greece.

Detection and characterization of Citrus tristeza virus (CTV) in Cyprus using molecular techniques L.C. PAPAYIANNIS, A. KYRIAKOU and T. KAPARI-ISAIA Agricultural Research Institute, 1516, Nicosia, Cyprus

Citrus tristeza virus (CTV) was fi rst reported virus strains identifi ed in Cyprus, six prim- in Cyprus in 1968 and until recently virus de- er pairs were designed suitable for applica- tection has been mainly based on Mexican tion in conventional or real-time PCR assays. lime (Citrus aurantifolia) indexing and ELI- Primer specifi city was based on short prim- SA tests. In view of a national project for dis- er lengths (12-15 nucleotides), and on the ease management and characterization, a incorporation of modifi ed bases known as new diagnostic protocol based on real-time locked nucleic acids (LNAs), which increase reverse transcription and the polymerase hybridization range and melting tempera- chain reaction (Real-Time TaqMan RT-PCR) ture. Evaluation of these primers in isolates was developed and optimized. The proto- from Cyprus, Greece, and from other geo- col is suitable for the generic detection of graphical regions showed that they were all virus isolates associated with severe or able to discriminate diff erent CTV strains ef- mild symptoms in the Mediterranean ba- fi ciently and rapidly. sin. In addition, to discriminate between

Development of a quantitative one-tube Real Time Reverse Transcription PCR (qRT-PCR) for the detection and quantifi cation of EMDV isolates in diff erent species P.G . PAPPI1, C.I. DOVAS2, K.E. EFTHIMIOU1 and N.I. KATIS1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece. 2Aristotle University of Thessaloniki, School of Veterinary Medicine, Laboratory of Microbiology and Infectious Diseases, GR-541 24 Thessaloniki, Greece

Eggplant mottled dwarf virus (EMDV) is en- terranean basin since 1969. Recently, the endemic in countries surrounding the Medi- tire genome of a Greek isolate originating

© Benaki Phytopathological Institute 50 Hellenic Plant Protection Journal - Special Issue from eggplant was sequenced but epidemi- conditions of qRT-PCR were optimized. The ological data concerning the presence and amplifi cation effi ciency was 96.9% and the dispersal of the virus is still limited. The pur- linear range of quantifi cation was from 20 to pose of the present study was the develop- 2×108 RNA transcripts. The above assay was ment of a rapid and sensitive method of re- applied to total RNA extracts from tissues of verse transcription PCR (Real Time qRT-PCR) eggplant, honeysuckle (Lonicera japonica), to detect and quantify EMDV in plant tis- tomato, tobacco, caper (Caparis spinosa), cu- sues and insect vectors. A 210 nucleotides cumber, Pittosporum tobira and hibiscus (Hi- long conserved region of the polymerase biscus syriacus) in which the virus was suc- (L) gene was used as target of the assay. In cessfully detected. The developed method vitro synthesized EMDV-RNA transcripts of proved to be a simple and reliable tool for known concentration were used for the eval- the detection and quantifi cation of the virus uation of the system. Moreover, the extrac- in host plants as well as in insect vectors. tion method of the viral RNA as well as the

Development of a semi-nested RT-PCR for the detection of ApLV and study of its presence in Greece I. FOTIOU, P.G. PAPPI, V.I. MALIOGKA and N.I. KATIS Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24 Thessaloniki, Greece bind to conserved sequences of the 5’ un- Apricot latent virus (ApLV) is a member of translated region of the viral genome and the genus Foveavirus, family Betafl exiviridae, the 5’-end of the RNA dependent RNA poly- and infects a wide range of plant species of merase (RdRp) gene. The method was suc- the family Prunoideae. It is spread in diff er- cessfully evaluated by using four charac- ent countries. Although it is usually latent, terized virus isolates (Casserta 12, LA2, SB, in some host plants it causes symptoms but A18) and proved to have a wider detection without severe yield losses. The available range compared to the available methods detection methods of ApLV are not reliable as it detects all virus isolates tested during as they do not detect all virus isolates. In this this study. Finally, the developed method study, we developed an improved detection was used to test 546 stone fruit samples (al- method of ApLV which was also used for mond, apricot, cherry, peach, ornamental testing diff erent plant species of the fam- plum) from diff erent regions of Greece for ily Prunoideae for the virus presence. For the presence of ApLV. The virus was not de- this purpose, a semi-nested RT-PCR was detected in any of them. veloped using degenerate primers which

Abstracts - 16th Hellenic Phytopathological Congress 53

ORAL & POSTER PRESENTATIONS

Nematodes as soil health bioindicators in an experimental sunfl ower fi eld E. KARANASTASI1, V. TRIANTAFYLLIDIS2, L. ECONOMOU3, N. MANTZOS4, G. MANOS5 and I. KONSTANTINOU4 1TEI of Messolongi, Faculty of Agricultural Technology, Department of Greenhouse Crops and Floriculture, GR-302 00 Messolongi, Greece. 2University of Western Greece, Department of Business Administration of Food and Agricultural Enterprises, GR-301 00 Agrinio, Greece. 3Benaki Phytopathological Institute, Department of Pesticides Control & Phytopharmacy, Laboratory of Biological Control of Pesticides, 8 St. Delta Str., GR-145 61 Kifi ssia, Greece. 4University of Western Greece, Department of Environmental and Natural Resources Management, GR-301 00 Agrinio, Greece. 5TEI of Epirus, Faculty of Agricultural Technology, Department of Crop Production, GR- 471 00 Arta, Greece

A 1000m2 experimental sunfl ower fi eld, lo- case, 2 with 5% inclination and 2 with 1%. cated in Kostakioi, Arta, was divided into 16 No herbicides were applied in the other 8 plots 40m2 each, 8 of which had a 5% incli- plots, 4 cultivated, 2 with 5% and 2 with 1% nation and 8 of 1%. The soil was tilled, the inclination, respectively, and 4 uncultivat- pilot blocks delineated, the water collection ed, 2 with 5% inclination and 2 with 1%. Soil system constructed and sunfl ower seeds sampling was performed at 0-10cm depth. were seeded in 8 of the plots, 4 with 5% in- Nematodes were isolated, the total popula- clination and 4 with 1%, in grooves 4-5cm tion per cm3 estimated, species identifi ed deep, at 70cm distance between the lines based on food preference and / or at fami- and 20cm on the track. The remaining 8 ly or genus level, where possible, and statis- plots were left uncultivated. There were two tical analysis of data using the program STA- applications of herbicides, one of oxyfl u- TISTICA v. 7.0. The experimental fi eld was orfen against broadleaf weeds, and one of installed following the principles of a split- quizalofop-p-ethyl against grasses. The her- split-plot experimental design and statisti- bicides were applied in 8 plots, 4 with sun- cally signifi cant interaction was observed fl ower and 4 without cultivation, and in each for each sample (p<0.05).

Distinction of live/dead eggs of potato cyst nematodes using propidium monoazide (PMA) M. CHRISTOFOROU, I.S. PANTELIDES, L. KANETIS, D. TSALTAS and N. IOANNOU Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus

Potato cyst nematodes (PCN), Globode- and labour consuming and frequently leads ra pallida and G. rostochiensis cause severe to overestimation of viable PCN inocula. losses in potato crop. Crop losses are relat- Furthermore, molecular assays such as Real- ed to nematode population densities in soil, Time PCR cannot directly assess the viability which are therefore used to support deci- of PCN inocula, since DNA of both live and sion making for the management of PCN. dead cells can be amplifi ed, thus quantify- The standard method to determine the via- ing the total amount of DNA. Recent studies bility of PCN is the microscopic visualization report the use of a DNA-intercalating dye, of nematodes stained with Meldola’s Blue Propidium Monoazide (PMA), in combina- dye. Meldola’s Blue assay, however, is time tion with Real-Time PCR assays for the dis-

© Benaki Phytopathological Institute 54 Hellenic Plant Protection Journal - Special Issue tinction of live/dead cells in several microor- viable cells is amplifi ed. In this study, we re- ganisms. The novelty of the method lies in port a novel method for the enumeration of the fact that cell membranes are imperme- viable PCN eggs in soil, based on Real-Time able to PMA. Thus PMA can be selectively PCR in combination with PMA. Quantitative used to intercalate the exposed DNA from analysis of DNA from viable eggs was per- dead cells, rendering it unavailable for PCR formed by using species specifi c Taqman amplification, and therefore only DNA from probes and primers.

“Seed Blight”, a non parasitic disease of sunfl ower A.I. ANASTASIADIS Hellenic Agricultural Organization “Demeter”, Center “Demeter” Drama, 5th km. Drama-Thessaloniki, GR-661 00 Drama, Greece

High temperatures, the sharp rise in tem- es R6-R7. Optically heads looks healthy, but perature and bright sunshine in the process when we rub the hand over the fl owers of of fi lling the seed, could aff ect the seeds of the head to reveal the seeds, then there is a sunfl ower, causing tanning (burning) accom- spot of seeds that shows in “silver-gray” col- panied by a complete absence of the en- or. Over the time they dry out and the same dosperm or have rudimentary endosperm. happens to the fl owers of the aff ected area This phenomenon is created when the seed of the head as a result making them seem fi lling stage, in the fl owers of the head are literally “burnt”. Aff ected seeds beneath the directly exposed to solar radiation (at the dried fl owers have a brown tint and are eas- top of the head). “Burning” of seeds was ob- ily rubbed from the head and they are slight served during the 2011 growing season, es- because most are hollow or have rudimen- pecially in a particular variety, which lacks a tary endosperm. In some cases the “burn- strong inclination of the head. The phenom- ing” of the seeds, proceeds to the top half enon was noticed in late June between stag- of the head.

Measurement of ozone concentrations using passive sampling in 13 fruit tree growing areas of the Imathia, Pella and Magnisia prefectures in Greece, during three years P. D ROGOUDI1, G. PANTELIDIS1 and G.D. NANOS2 1Pomology Institute, National Agricultural Research Foundation, Hellenic Agricultural Organization “Demeter”, 38 R.R. Station, GR-590 35 Naoussa, Greece. 2Laboratory of Pomology, School of Agricultural Science, University of Thessaly, Fitoko Str., GR-384 46 Volos, Greece

Ambient ozone (O3) concentrations were Imathias and Zagora and Chania Magnisias), measured using passive samplers and UV in comparison with those at lower altitude or HMOS monitors, in fruit tree growing ar- (Kampohori and Ammos Imathias and Veles- eas of Imathia (9 sites), Pella (1 site) and Mag- tino Magnisias). In 2007 the highest O3 con- nisia (3 sites) in Greece, during the sum- centrations were recorded, compared with mer months of three consecutive years. The 2005 and 2006. Accumulative AOT(40) val-

O3 concentrations were also monitored at ues during the day in Naoussa were greater diff erent positions in an orchard. Results by 1.5 and 3.5 times at 6.000 ppb.h. Diurnal showed that ten-day O3 concentrations re- O3 concentrations in Naoussa and Rodo- corded using passive samplers were great- chori followed a campanoid curve with lower in areas with higher altitude (Rodochori est values occuring during the early hours of

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 55 the morning, and maximum values during opy and between rows. Ozone phytotox- the mid- to late afternoon. In each orchard, ic symptoms were observed in the leaves of the measured O3 concentrations were great- sweet cherry trees, raspberry, sorbus, clover er above canopy compared to within can- and rose plants in Imathia.

BIOLOGICAL & INTEGRATED CONTROL

Abstracts - 16th Hellenic Phytopathological Congress 59

ORAL & POSTER PRESENTATIONS

Study the eff ectiveness of diff erent natural compounds for the protection of pomegranate trees from spring frost P. D ROGOUDI, A. DELLA and G. PANTELIDIS Pomology Institute, National Agricultural Research Foundation, Hellenic Agricultural Organization “Demeter”, 38 R.R. Station, GR-590 35 Naoussa, Greece

Spring frosts can cause serious damage to before assessments were made of their frost pomegranate orchards, and this was the case sensitivity using controlled freezing tests. in many areas of Northern Greece during Frost damage was assessed using the elec- 2011. During spring the sensitivity of trees trolyte leakage test, tetrazolium stain tests in to frost increases and this has been found to pith and cortex, and visual assessment of inju- be proportional to decreased levels of sug- ry. The results showed that molasses spray ap- ars and the osmotic potential of cell sap. The plication provided protection as indicated by aim of the present study was to assess the all test methods used to reveal frost damage. eff ectiveness of the external application of Glucose application protected from low tem- molasses, glycine-betaine, ethylene glycol, peratures in all methods apart from the visu- glycerine, sucrose and glucose on conferring al rating assessment. Finally, glycine-betaine, frost resistance in pomegranate. Pomegran- ethylene glycol and sucrose application con- ate trees were sprayed with solutions of the ferred a lesser degree of protection that was above compounds in the fi eld 8 and 3 days only detectable by tetrazolium tests.

Phytochemicals with nematicidal activity in IPM programs for the control of rootknot nematodes Meloidogyne spp. N.G. NTALLI1 and U. MENKISSOGLU-SPIROUDI2 Pesticide Science Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece. E-mail: [email protected]; 2rmenkis@ auth.gr

Amongst all agricultural pests aff ecting tally safer than the synthetic derivatives cur- crops, root-knot nematodes (RKN; Meloido- rently available. An interesting way to search gyne spp.) represent possibly the most dam- for biorational nematicides is to screen nat- aging worldwide, with a wide host range, a urally occurring compounds in plants. Plant short generation period, a high reproduc- derived pesticides may fi nd favour in organic tive rate, and with the ability to form disease food production because many are environ- complexes with other soil borne pathogens mentally friendly, pose less risk to humans like fungi. In the past phyto-nematode con- and animals, have a selective mode of ac- trol has been mainly based on chemical tion, avoid the emergence of resistant races nematicides, but recently many synthetic of pest species, and as a result can be safely nematicides have been banned or are un- used in Integrated Pest Management (IPM) der evaluation (Regulation 2009/1107/EU programmes. However, botanical nemati- & 2009/128/EU), due to environmental and cides have yet to form the focus of research, health concerns. Additionally, the economic unlike insecticides and pesticides. This is a cost of research and registration to develop review of our recent studies on Meloidogy- new nematicides is not easily sustained by ne spp. control using natural substances of the industry. There is therefore, a great need plant origin. We report on the most potent for novel nematicides, that are environmen- nematicidal botanical extracts, as well as on

© Benaki Phytopathological Institute 60 Hellenic Plant Protection Journal - Special Issue the chemical groups of substances exhibit- paramount importance in nematode con- ing substantial nematicidal activity. The ac- trol as it enhances nematicidal activity in ad- tivity of the extracts was verifi ed against jacent, untreated soil layers. various growth stages of the parasite in vivo; Insights into the development of natural ne- while enzyme (AchE) inhibition activities maticidals for use in ecofriendly agriculture were determined in vitro. Fumigant prop- are presented. erties were also evident, an eff ect that is of

Eff ect of several plant extracts and chitosan on Chrysanthenmum stunt viroid (CSVd) infections of chrysanthemum plants I.N. BOUBOURAKAS and M. HOSOKAWA Laboratory of Vegetable and Ornamental Horticulture, Department of Agronomy and Horticultural Sciences, Graduate School of Agriculture, Kyoto University

Chrysanthemum stunt viroid (CSVd) consti- Time RT-PCR. C. chinense, Houttuynia, Bou- tutes a serious problem for chrysanthemum gainvillea and chitosan were the treatments cultivation worldwide and especially in Ja- that stood out, resulting in statistically sig- pan. Infection is extremely diffi cult to avoid, nifi cant (t-test) reductions in CSVd titres, as chrysanthemum is propagated vegeta- ranging from 35 to 50%. The application of tively, CSVd is readily transmissible, and the chitosan to ex vitro plants prevented CSVd disease has a long latent period. The pres- titre increase (50% to 70%) compared to ent study investigated whether extracts of control and seemed to have a positive eff ect Mirabillis japala, Pelargonium sp., Phytolac- on plant height. Experiments using CSVd ca americana, Capsicum chinense, Oleae eu- RNA treated with plant extracts and chito- ropaea, Brassica sp., Bougainvillea sp., Hout- san were also performed in order to quan- tuynia sp., Dendranthema grandifl orum tify viroid degradation. Results showed that Kitam cv. Sei-no-issei, Allium cepa, Allium sa- pepper and chitosan extracts were as eff ec- tivum plants and Chitosan-100 could reduce tive as sodium hypochlorite treatment. In CSVd titre in highly infected plants in vitro, conclusion, chitosan and extracts of Capsi- and/or prevent CSVd titre increase in new- cum sp., Houttuynia sp. and Bougainvillea sp. ly-infected plants ex vitro. Ten to 15 plants were found to be promising for use in chry- were used per treatment and the viroid load santhemum stunt viroid disease manage- was estimated weekly using one step Real ment.

Impact of Cucumber mosaic virus (CMV) infection and benzothiadiazole (BTH) application on the quantitative and qualitative traits of marketable tomato fruits M. KALOGIROU1,2, C. VARVERI1, N. VASSILAKOS1 AND L.A. TERRY2 1Laboratory of Virology, Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia, Athens. Greece. 2Plant Science Laboratory, Cranfi eld University, College Road, Bedfordshire, MK43 0AL, United Kingdom

This study evaluated the eff ect of Cucum- grown F1 Delos plants during two successive ber mosaic virus (CMV) infection and benzo- seasons. CMV caused the most severe stunt- thiadiazole (BTH) application (BION 50 WG, ing of tomato plants compared to the oth- 50 mg/L), individually or in combination, on er treatments (CMV+BTH, BTH, healthy con- several quantitative and qualitative traits of trol plants) and resulted in signifi cant loss tomato fruits produced from greenhouse- of marketable fruits, although the total fruit

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 61 number was higher compared to the other the BTH treatment, except for the lycopene treatments. Fruits derived from CMV-infect- and β-carotene contents, which were signif- ed plants ripened later than those from all icantly higher than in control fruits. BTH, ap- other treatments and showed signifi cantly plied before and after CMV inoculation, in- enhanced antioxidant capacity, ascorbate, duced systemic acquired resistance (SAR) lycopene and β-carotene contents. Week- and clearly suppressed CMV infection, not ly BTH application to healthy plants nega- only reducing the virus infection rate but im- tively aff ected plant growth, fruit size and proving plant growth and fruit size of plants marketable yield. The nutritional status of subsequently infected with CMV. Repeated tomatoes, as defi ned by nonstructural car- foliar applications of BTH could be used to bohydrates, organic acids and antioxidants reduce the damage caused by CMV in seri- content, was not signifi cantly aff ected by ous viral epidemics.

Evaluation of the eff ectiveness of soil solarization with impermeable plastic fi lms in combination with half the recommended dose of soil fumigants for the control of soil borne pathogens P.P. A NTONIOU1 J.O. GIANNAKOU2 and E.C. TJAMOS1 1Department of Plant Pathology, 2Laboratory of Agricultural Zoology and Entomology, Department of crop Science, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

The aim of this work was to evaluate the rum, on the severity of the disease caused eff ectiveness of soil solarization with im- by Fusarium oxysporum f.sp. radicis lycoper- permeable plastic fi lms (ORGASUN), for 4 sici and the percentage of diseased plants and 6 weeks under greenhouse conditions with symptoms of vessel discoloration and on Crete, in combination with half of the stem rot, was also examined. Moreover, the recommended dose of soil disinfectants, eff ect of treatment on the percentage of dis- against soil borne pathogens. The imperme- eased tomato plants caused by Pyrenochae- able plastic fi lm was compared with com- ta lycopersici was also studied. The following mon polyethylene fi lm. The disinfectants conclusions were drawn: the soil tempera- and doses that were used in the experimen- tures that were recorded during soil solar- tation were CONDOR: 20 l/1000m2. (100%) ization in the Tympaki and Kalessa green- greenhouse, 10 l/1000m2. (50%) greenhouse. houses were initially suitable to achieve VAPAM: 55l/1000m2. (100%) greenhouse, for eff ective soil solarization. However lat- 27 l/1000m2. (50%) greenhouse. CHLO- er, due to the low air temperatures the mar- ROPICRINE: Pic (67:33 v/v) l/1000m2 (100%) ginal levels of eff ectiveness of this meth- greenhouse, l/1000m2 (50%) greenhouse. od were reduced. The fungal populations The experiments were carried out in toma- were very high in the control greenhouses to greenhouses in the Tympaki region and (disease index 8.46-9.33). Soil solarization in at the experimental station in Kalessa, Her- combination with fumigants resulted in a aklion. The experiment began in July 2011, very low disease index. Also an exception- and the evaluation took place between Oc- al reduction the in natural soil populations tober 2011 and April 2012 at the end of the of Fusarium oxysporum was noted, with ef- cultivation period. The following were stud- fective control of Fusarium wilt and crown ied: the eff ect of treatments in soil temper- rot caused by Fusarium oxysporum f.sp. radi- ature, on the population and in the disease cis lycopersici and corky root rot caused by index of knot nematodes (Meloidogyne sp). Pyrenochaeta lycopersici was achieved with Additionally, the eff ect of treatments on the the combined use of half doses of fumigants population dynamics of Fusarium oxyspo- and the impermeable plastic fi lm Orgasun.

It should be noted that from the control observed of up to 16% (January 2012) were greenhouses, high rates of diseased toma- removed to avoid mass aerial contamination to plants infected with Fusarium oxysporum by this pathogen. f.sp. radicis lycopersici with infection rates

Eff ect of essential oils of fi ve medicinal plants on the mycelial growth and conidia germination of the fungi Penicillium expansum and Aspergillus niger V. KOUTSIOUKI, N. BROCHARIDIS, G. VLACHOS, A. FILOTHEOU, TH. THOMIDIS and E. PANOU- FILOTHEOU Alexander Technological Education Institute of Thessaloniki, Department of Crop Production, GR-574 00 Sindos, Thessaloniki, Greece

Fruit rots are some of the most important trose agar supplemented with the essen- plant diseases worldwide. Because of the tial oils at diff erent concentrations. Results policy of the European Union for the pro- were collected by recording the diameter of duction of fruits without pesticide residues, the colony 4 days later. In addition, a conidi- new biological methods to control fruit rot al suspension (2x106 / ml) of the above fungi must be investigated. The main aim of this was added to petri dishes containing potato study was to investigate the eff ects of essen- dextrose agar supplemented with essential tial oils extracted from origanum, (Origanum oils at diff erent concentrations. The results vulgare subsp. hirtum), rosemary (Rosmari- were collected by recording the percentage nus offi cinalis), creeping rosemary (Rosmari- of germinated conidia or germ tube elonga- nus offi cinalis Prostratus), basil, (Ocimum ba- tion 24 hrs later. The results showed that all silicum) and sage (Salvia offi cinalis) on the the essential oils used in this study inhibited mycelial growth and conidia germination of mycelial growth and conidia germination of the fungi Penicillium expansum and Asper- both fungi. However, the essential oils from gillus niger. Mycelial discs of each fungus, origanum and basil were eff ective at much 6 mm in diameter, were placed in the mid- lower concentrations. dle of petri dishes containing potato dex-

Establishment of the pathogenic fungi Puccinia punctiformis and Colletotrichum gloeosporioides in the fi eld for the biological control of the weeds Cirsium arvense and Salsola kali L.A. LAGOPODI1, J. KASHEFI2 and D.K. BERNER 3 1Plant Pathology Laboratory, School of Agriculture, Aristotle University of Thessaloniki. 2USDA ARS European Biological Control Laboratories, Thessaloniki, Greece. 3USDA-ARS-Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD

Cirsium arvense and Salsola kali are indige- genic fungi as potential biocontrol agents to nous weeds in Greek agriculture, whereas control the weed populations, and to study in the USA they are considered as invasive aspects of the epidemiology of the two dis- species. In the present study, a methodolo- eases. C. gloeosporioides was isolated from gy for the establishment in the fi eld of their naturally infected S. kali plants in a fi eld in host-specifi c, native Greek, pathogens Puc- Chalkidiki. The fungus was grown in a large cinia punctiformis and Colletotrichum gloeo- volume substrate containing rice, in order to sporioides was developed. The purpose of produce a great number of acervulli. A sin- the research was to study the two patho- gle treatment with the inoculum, in disease-

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 63 free experimental fi elds in Kozani, resulted vense rosettes, in a disease-free experimen- in a signifi cant reduction in the S. kali weed tal fi eld in Kozani, has shown that successful population, and its almost complete eradi- inoculations take place only during autumn. cation over the following 2-3 years. The dis- The percentage of successful inoculations tribution of the inoculum was performed in was low, and multiple inoculum applica- a natural manner, and commenced with the tions were necessary during this period. fi rst rains at the end of August. Naturally in- However, the establishment of the patho- fected C. arvense leaves, bearing the telio- gen in the fi eld was successful. The progress sori of P. punctiformis were collected from of the disease over the following years is be- a fi eld in Kozani. Inoculation of young C. ar- ing studied.

Screening of Greek Trichoderma isolates as potential biocontrol agents of soilborne fungal pathogens G.A. BARDAS, P. AGGELOPOULOU, H. EFSTRATIOU, G. TZINOGLOU, G. MOUTOULI, A. TSANAKTSIDOU G. PALATOS and S. STEFANOU Alexander Technological Educational Institution of Thessaloniki, GR-574 00 Sindos, Thessaloniki, Greece

The general inadequacy of chemical fun- ic activity on pathogen colonies. Regarding gicides for the control of Verticillium dahli- antibiotic activity, two Trichoderma viride ae, Rhizoctonia solani, Pythium ultimum and isolates exhibited increased pathogen sup- Sclerotinia sclerotiorum on several plant pression due to the production of volatile hosts has led to a survey for biocontrol so- and non volatile metabolites. The chitino- lutions. Thirty two Trichoderma spp. iso- lytic (chitinase and NAGase) activity of the lates were isolated and identifi ed, on the Trichoderma species tested varied, as to the basis of their morphological characteris- carbon source (colloidal chitin, R. solani cell tics and by molecular analysis of the inter- wall material) targeted and the location of nal transcribed spacer 1 and 2 regions (ITS1 these enzymes (mycelium, liquid). In detail, and ITS2). These isolates were evaluated for Trichoderma harzianum B2 and Trichoderma their antagonistic activity against the soil viride B4 isolates were characterized as be- borne plant pathogens in vitro. Diff erent iso- ing highly productive of endochitinase, ex- lates showed varying degrees of biocontrol ochitinase and β-Ν-acetylhexosaminidase. activity. Specifi cally, regarding mycoparasit- In conclusion, the promising results of this ism tested using a dual culture technique, research could form the basis of an extend- seven Trichoderma sp. isolates, identifi ed ed study, in order to evaluate the effi cacy of as Trichoderma asperellum and Trichoderma these biocontrol agents in planta. harzianum, showed enhanced mycoparasit-

Evaluation of the biocontrol effi cacy of Paenibacilus alvei strain K165 against Fusarium oxysporum f.sp. melonis S.E. TJAMOS, A. CHARALAMPOUS and E.J. PAPLOMATAS Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

The plant pathogen Fusarium oxysporum with diff ering host specifi cities. In the pres- constitutes one of the most signifi cant ent research, the effi cacy of the biocontrol threats to farmers’ revenue worldwide, hav- agent Paenibacilus alvei K165 to control Fu- ing more than a hundred formae specialis sarium oxysporum f.sp. melonis was studied.

For this purpose, a talc preparation of Κ165 eff ective treatment of 10% but at the same was either incorporated into the soil (in a time the severity of the disease in this group proportion of 1%, 5%, 10% and 20%) that was not statistically diff erent to that in the was used to grow melon plants, or was used non-treated controls. It is tempting to spec- as a seed coating. At the stage of the 2nd leaf ulate that this diff erence may be attributa- the plants were transplanted to soil infest- ble to the phenomenon of quorum sensing ed with 103 Fusarium oxysporum chlamy- as the size of the rhizosphere population of dospores per gram of soil. It was observed the 20% treatment group was 2-4 fold high- that the 10% treatment alone resulted in er than that of the 10% treatment. On the a statistically signifi cant reduction in dis- other hand, the size of the K165 rhizosphere ease severity. It is noteworthy that applica- population of the other ineff ective treat- tion of the biocontrol agent at 20% result- ments (1%, 5% and seed coating) was sub- ed in partial reduction in disease severity stantially lower than that seen with the ef- that was not statistically diff erent from the fective treatment of 10%.

Isolation of new biocontrol agents against crown and root rot disease of tomato caused by Fusarium oxysporum f. sp. radicis – lycopersici N.N. KAMOU1, L. LOTOS, G. MENEXES2 and L.A. LAGOPODI1 1Plant Pathology Laboratory, School of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece. 2Agronomy Laboratory, School of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece

The need to limit the irrational use of chem- of tomato plants with the fungus was signif- ical fungicides in plant protection, has led to icantly reduced by the selected rhizobacte- a tendency to use biological control. In the ria. Moreover, the ability of these rhizobac- present research, 383 rhizobacteria, isolat- teria to promote the growth of tomato was ed from cultivated plants from diff erent re- studied in pot experiments. Finally, the gions of Greece, were studied. Fusarium ox- presence of antifugal metabolites in the liq- usporum f.sp. radicis – lycopersici (Forl), which uid culture supernatants of the biocontrol causes crown and root rot disease in tomato agents and their organic extracts was inves- was chosen as a target pathogen. The inhib- tigated. The eff ect of the metabolites on the itory activity of the bacteria on the fungus, growth of the fungus was studied by fi lter was tested in vitro and in planta. For the in paper, TLC, and microtitre bioassays. The re- vitro experiments, a lysis bioassay was per- sults demonstrated that the strains Bacillus formed and the presence of a potential inhi- cereus S76 and S79, Serratia rubidaea S55 and bition zone of fungal growth was examined, S49, Serratia marcescens PiHa5II, S52 and S47 in multiple and dual cultures of the fungus and Pseudomonas chlororaphis ΤοZa7 are ef- and the bacteria in Petri dishes. The most ef- fective biocontrol agents that successfully fective bacteria in the two previous bioas- inhibit the growth of Forl, and the disease says were identifi ed by sequencing the 16S intensity caused by this fungus in tomato. In rRNA gene, or the intergenic spacer region addition, the strains S. marcescens S47, B. ce- between 16S and 23S rRNA. In the in planta reus S76 and S79 and P. chlororaphis ToZa7, tests that followed in a gnotobiotic system in showed a statistically signifi cant promotion pots, and in one indicative fi eld experiment, of tomato plant growth in pots and in the the disease caused by artifi cial inoculation fi eld.

Biological control of the toxigenic fungus Aspergillus fl avus and afl atoxins produced in shelled pistachio nuts cv. “Eginis”

D.F. ANTONOPOULOS1,2, M. GEORGIADOU3 , S.P. AGORITSIS1, S. GIANNIOTIS3 and D.I. TSITSIGIANNIS1 1Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55, Athens, Greece. 2Laboratory of Crop Protection, School of Agricultural Technology, ΤΕΙ of Kalamata, Antikalamos, GR-241 00 Kalamata, Greece. 3Laboratory of Food Process Engineering, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55, Athens, Greece

One of the most signifi cant threats for food quality and safety are the mycotoxins, particularly toxic and carcinogenic low molecu- These 2 yeast strains were further tested on lar weight metabolites produced by certain shelled pistachio nuts cultivar “Eginis” for fungal species. Afl atoxins produced by the their role in afl atoxin production (assessed fungi Aspergillus fl avusand A. parasiticus are by High Performance Liquid Chromatogra- highly carcinogenic mycotoxinsthat several phy-HPLC) and led to a signifi cant decrease times have been detected at high concentra- by 89% (FR6) and 85% (MR7), respectively, tions levels in pistachio nuts in Greece. The in comparison to the control. Evaluation of aim of this study was to evaluate a collec- the non-toxigenic A. fl avus strains as biolog- tion of 20 yeast isolates and 4 non-toxigenic ical control agents of the disease and myco- strains of A. fl avus, isolated from experimen- toxin in pistachio orchards, AF38, AF51 and tal pistachio orchards located at Fthiotida AF57 reduced signifi cantly the production County for the management of A. fl avus and of the afl atoxins (assessed by HPLC) AFΒ1, Β2 afl atoxins. The yeast isolates MR7 (Candida and G1 of the Δ1.3 AF2 strain by 41%, 48% sp.) and FR6 (Aureobasidium pullulans) were and 69%, respectively, whereas AF45 strain selected as the most eff ective against the did not show any eff ect in comparison to afl atoxigenic pathogen A. fl avus, strain Δ1.3 the control. The results of this study contrib- ΑF2 because they led to a 40-50% reduction ute to the development of environmentally of Aspergillus growth and to a signifi cant re- friendly methods of biological management duction in conidiogenesis by approximate- of mycotoxins in pistachio nuts. ly 1000 times, in comparison to the control.

Τhe swarming motility of biocontrol antagonistic Pseudomonas strains possibly inhibits the growth of phytopathogenic fungi A. VENIERAKI1, K. PAPAMELETIOU1, A. PAPADOPOULOU1, M. DIMOU1, P. ANTONIOU2 and P. KATINAKIS1 1Laboratory of General and Agricultural Microbiology, Department of Agricultural Biotechnology, 2Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

More than 30 antagonistic culturable bacte- 16S rRNA analysis. Most were found to be- rial strains were isolated from solarized, to- long to the Pseudomonas group (P. fl uores- mato rhizosphere soil in greenhouses and cens, P. poae, P. lurida, P. synxantha. P. moorei characterized for their potential to inhib- etc). Strains were tested for their swarming it the growth of plant pathogenic fungi. In motility capacity under diff erent environ- in vitro experiments they showed remark- mental conditions. Two strains, designat- ably antagonistic activity against Fusarium ed as P3 and P23, Pseudomonas lurida and oxysporum, Phytophthora sp. and Rhizoc- Pseudomonas azotoformans, respectively, tonia solani. The antagonistic isolates were showed hyper-swarming motility and inhib- fi rst characterized using classical microbio- ited the growth of phytopathogenic fungi. logical methods and further assessed using The results suggest that swarming activity

© Benaki Phytopathological Institute 66 Hellenic Plant Protection Journal - Special Issue may contribute to fungal growth inhibition teria under greenhouse conditions and to in soil. In this respect it will be of great inter- characterize the bacterial genes involved in est to investigate the behavior of these bac- fungal growth inhibition.

Selection of non-toxigenic strains of Aspergillus fl avus from pistachio and cottonseed as biological control agents for afl atoxin management K. VICHOU, G. VARDOUNIOTIS and D.I. TSITSIGIANNIS Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

The presence of mycotoxins in agricultural gillus section Flavi was performed by plating products is considered as one of the most soil-water suspensions on plates of selec- serious food safety concerns worldwide. tive medium. Furthermore, several strains of One of the most carcinogenic mycotoxins is Aspergillus section Flavi were isolated from afl atoxin produced by the fungi Aspergillus pistachio during harvest from experimental fl avus and A. parasiticus. Afl atoxin has been fi elds of Aegina, as well as from pistachio ker- detected at high concentrations in Greece, nels from several other sources in the mar- including in pistachio. A promising and ef- ket. Using thin layer chromatography (TLC) fective strategy to reduce afl atoxin levels is the collection of isolates was evaluated for the application in the fi eld of endemic non- their ability to produce afl atoxins in order toxigenic Aspergillus spp. strains. This tech- to discover non-afl atoxigenic strains that nique is based on the gradual substitution could be used in biological control experi- and exclusion of A. fl avus and A. parasiti- ments. Finally, in vitro competition assays cus toxigenic strains by the non-toxigenic were carried out using two wild toxigen- strains due to competition. The goal of this ic and 12 non-toxigenic strains, in order to study was to create a collection of greek en- study the potential for the reduction of the demic non-toxigenic Aspergillus spp. strains. afl atoxigenic capacity of toxigenic strains by Initially, soil samples were collected from non-toxigenic strains. Most non-toxigenic diff erent experimental fi elds in the Fthioti- strains reduced the afl atoxigenic activity of da region and transferred to the laborato- wild type strains, and some strains prevent- ry. The isolation of several strains of Asper- ed afl atoxin production completely.

Evaluation of rhizobacterial activity against soil-borne plant pathogenic fungi in vitro A. MALAFOURIS AND L.A. LAGOPODI Plant Pathology Laboratory, School of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece

Eight (8) rhizobacterial strains, known for Pseudomonas chlororaphis ToZa7, and the their in vitro and in planta activity against new strains ΤοΖa9-12, ΤοΖa1-5-10, ΤοΖa4-8 of the plant pathogenic fungus Fusarium ox- yet unknown genetic identity, were tested ysporum f. sp. radicis-lycopersici (Forl), and 3 against Verticillium dahliae, Sclerotium rolfsii, new strains, were tested in vitro for the for- and Rhizoctonia solani, with the latter three mation of an inhibition zone in the myceli- also being tested against Forl. In addition, al growth of 4 soil-borne plant pathogenic S76, PiHa5-II, ToZa7, ToZa9-12 and ToZa4-8 fungi. Specifi cally, the strains Bacillus cere- were tested in vitro against Forl and S. rolf- us S76 and S79, Serratia marcescens PiHa5- sii for the presence of volatile compounds II, S47 and S52, Serratia rubidea S55 and S49, that inhibit mycelial growth. The most im-

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 67 pressive results were obtained by combin- of volatiles was also apparent, as the strains ing all of the (11) rhizobacteria with S. rolfsii, PiHa5-II, ToZa7, ΤοΖa4-8 and ΤοΖα9-12 pre- or the combination of the 5 strains tested for vented mycelial growth and germination the eff ect of volatiles on the same fungus. In of sclerotia of S. rolfsii at a distance, where- almost all treatments mycelial growth was as S76 showed diff erent results in 3 replica- very limited or non-existent, and an inabil- tions. Finally, combination of V. dahliae and ity of sclerotia germination to hyphae was S52 gave interesting results with fungal observed. Treatments of S. rolfsii with PiHa5- growth being limited, and the formation of II and S47 were the only exceptions in which microsclerotia inhibited. the inhibition zone was limited. The eff ect

Evaluation of Greek Trichoderma isolates eff ect on plant growth characteristics of aronia (Aronia melanocarpa) plants G.A. BARDAS, P. PENTSOU, M. DOTSIOU, H. MANDRAS, G. MOUTOULI, A. TSANAKTSIDOU G. PALATOS and S. STEFANOU Alexander Technological Educational Institution of Thessaloniki, GR-574 00 Sindos, Thessaloniki, Greece

Aronia melanocarpa fruit production is a pared with the single treatments, with 80, novel crop in Greek agriculture and research 87, 94 and 95% root colonization observed is needed in order to defi ne and optimize at, 2, 7, 15 and 30 d.a.t., respectively. In ad- the cultivation techniques in biological pro- dition, the combined treatment was superi- duction systems. The present research was or with regard to all the growth characteris- focused on the evaluation of three Tricho- tics tested. Data from the single treatments derma sp. isolates, belonging to Trichoderma showed that plants treated with Trichoderma harzianum and Trichoderma asperellum spe- harzianum isolates B2 or B3 had higher dry cies, regarding their eff ect on plant growth root weights and numbers of leaves com- characteristics of aronia plants. All three pared to the untreated rootstocks, while T. Trichoderma sp. isolates, when applied sep- asperellum B1 treatment was better than the arately, colonized aronia roots to the same control with regard to dry root and shoot extent, with colonization levels ranging on weight. In conclusion, specifi c biocontrol average from 58% at 2 days after treatment isolates can form a complementary treat- to 94% at 30 d.a.t. However, the combined ment to assist Aronia melanocarpa commer- treatment resulted in asignifi cant increase cial fruit production in Greece. in the total number of colonized roots com-

Molecular identifi cation of epiphytic yeasts isolated from grapevines and their assessment as biological agents to control Aspergillus spp. Ι.S. PANTELIDES, O. CHRISTOU, D. TSALTAS and Ν. IOANNOU Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus

The aim of this study was the isolation and ‘Maratheftiko’ and ‘Cabernet Sauvignon’ molecular identifi cation of epiphytic yeasts originating from four areas of the Limassol from grapevines and their assessment as bio- district during 2010. Genomic DNA was iso- logical agents against black aspergilli (Asper- lated from each yeast and was used for the gillus section Nigri). Initially, epiphytic yeasts amplifi cation and sequencing of the D2 re- were isolated from grapes of the varieties gion of the nuclear large-subunit (LSU) ribo-

© Benaki Phytopathological Institute 68 Hellenic Plant Protection Journal - Special Issue somal RNA gene. The sequences were com- The isolates showing antagonism (the pres- pared to a library (MicroSeq® Fungal Gene ence of an inhibition zone) were also test- library) containing D2 sequence entries from ed in a detached berry test. Berries were im- more than 1000 validated species, using Mi- mersed in a suspension of the selected yeast croSeq® ID Analysis software. The isolates isolates and a conidial suspension of A. tub- identifi ed included 39 Aureobasidium pullu- ingensis was spot inoculated on a wound lans, 9 Cryptococcus magnus, 1 Pseudozyma made on each berry. The inhibition of fun- onfarclica aphidis, 1 Hanseniaspora opuntiae gal growth was determined 7 days after A. and 1 Αcremonium glaucum. After identifi ca- tubingensis inoculation. The isolates with tion, the isolates were screened for in vitro the highest inhibition percentage (93.78% antagonism against Aspergillus tubingensis and 90.08%) belonged to the species Aure- by the dual culture technique on PDA plates. obasidium pullulans.

Biological control of Aspergillus carbonarius in pomegranates P. S KIADAS, M. DIMAKOPOULOU, S.E. TJAMOS and E.J. PAPLOMATAS Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

The plant pathogen Aspergillus carbonar- granates were immersed in a suspension of ius causes one of the most signifi cant post the isolated microorganisms and after one harvest diseases in stored fruits leading to day they were infected with the pathogen rot and contamination with the mycotoxin by making an incision on the surface of the ochrotoxin A (OTA). The aim of the study was fruits and applying 10 μl of A. carbonari- the isolation of epiphytic microorganisms us spores at a concentration of 105 spores/ from pomegranates and the in vitro test- ml. Two out of the 30 isolated microorgan- ing of these microorganisms for their effi ca- isms signifi cantly reduced A. carbonarius rot cy against A. carbonarius. A number of mor- on the surface of the tested pomegranates; phologically distinct microorganisms were however, only one of these was capable of isolated and tested against A. carbonarius by reducing signifi cantly the total number of using a pomegranate bioassay. The pome- pathogen spores produced.

Screening of the biocontrol agents Paenibacilus alvei Κ165 and Fusarium oxysporum F2 talc preparations against the plant pathogen fungus Verticillium dahliae S.E. TJAMOS, D. AGGELOPOULOU, E. NASKA and E.J. PAPLOMATAS Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece

Verticillium dahliae is among the most patho- talc preparations of 2 BCAs, either Paeni- genic plant microorganisms, mainly due to bacilus alvei Κ165 or Fusarium oxysporum F2, the lack of eff ective chemical control strat- were mixed with potting soil at a ratio of 1%, egies. Therefore, it is evident that the devel- 5%, 10% and 20% and used as substrates for opment of biocontrol strategies against this eggplant cultivation, or were used for seed pathogen is of vital importance for agricul- coating. At the stage of the 3rd-4th leaf, the ture. The aim of this study was to develop plants were transplanted into soil infested and evaluate the effi cacy of easy to apply with 20 V. dahliae microsclerotia per gram formulations of already known biocontrol of soil. It was observed that the ratios 1%, agents (BCAs) of V. dahliae. For this purpose, 5%, 10% and 20% of both BCAs in soil were

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 69 equally eff ective in controlling Verticillium biocontrol preparations to control Verticil- wilt. However seed coating treatment was lium wilt refl ected the observed diff erenc- ineff ective in the case of K165, but F2 treat- es in the size of the BCA populations be- ment partially reduced disease severity. The tween the treatments of 1%, 5%, 10%, 20% diff erences in the effi cacy of the diff erent and seed coating.

Spread of hypovirulent strains of the fungus Cryphonectria parasitica after biological control of chestnut blight in the Region of Epirus G.T. TZIROS, C. PERLEROU, E. TOPALIDOU, V. CHRISTOPOULOS and S. DIAMANDIS Hellenic Agricultural Organization ‘’Demeter’’ (Ex NAGREF), Forest Research Institute, GR-570 06 Vassilika, Thessaloniki, Greece

Chestnut blight, a bark disease caused by (orchards) in Arta and from six plots in cop- the fungus Cryphonectria parasitica, was fi rst pice forest in Ioannina. The growth and the reported in Greece, Mount Pelio, in 1963. Un- characterization of the strains yielded into til 2002 it was spread all over the mainland virulent and hypovirulent were carried out country where chestnut is cultivated, while on PDA medium according to morphologi- in 2006 it was recorded in the islands of Les- cal features. In Arta 34.9 - 53.1% of the total vos and Crete. It is known that the above as- cankers yielded hv strains, while in Ioanni- comycete may be infected by dsRNA virus- na the percentage fl uctuated between 39.2 es called Cryphonectria hypoviruses (CHVs), and 68.5%. Furthermore, 35.9 - 61.85% of the a fact that reduces fungal virulence to levels non inoculated cankers sampled yielded hv ranging from avirulence to near-virulence. strains in the Prefecture of Ioannina, while Such infected strains with proved reduction the percentage was 32.3- 48.8% for Arta. The in their virulence termed as hypovirulent results show that, two years after the end of (hv) were used for the biological control of artifi cial inoculations, introduced hypovi- the pathogen in the period 2007-2009 in the lence was successful in all treated areas as Region of Epirus (Prefectures of Arta and Io- hypovirulent strains of the fungus have set- annina). Two years after the end of artifi cial tled in orchards as well as in coppice stands inoculations, samples from cankers were and began as well spreading to non inocu- randomly selected from three round plots lated cankers.

Determination of the optimal retention time for sclerotia in soil paste for the isolation of Sclerotinia sclerotiorum mycoparasites. A proposed method for the eradication of nematodes and mites. F.A. TSAPIKOUNIS* Department of Biology, University of Patras, GR-265 00 Rio, Patra, Greece. *Present address: Agiou Nikolaou 48, GR-270 52 Varda, Ilia, Greece

In order to determine the optimal time of For all treatments soil from the same fi eld sclerotia retention in soil paste, seven dif- was used. Subsequently the sclerotia were ferent retention times, each using four rep- disinfected and placed for 24 or 48 hours in licate s and 40 sclerotia in total were exam- a environment with 100% relative humidity. ined. The sclerotia were placed in the soil After 35, 30, 25, 20, 15, 10 and 5 days in soil, paste and incubated at 250C in the dark with the proportion of sclerotia in a good condi- collection and disinfection taking place eve- tion (not dissolved during the process of dery fi ve days, beginning at 5 days for the fi rst contamination) was 0, 10%, 17%, 20%, 80%, treatment and ending at 35 for the seventh. 97.5% and 100%. The optimal retention

© Benaki Phytopathological Institute 70 Hellenic Plant Protection Journal - Special Issue time for sclerotia within the soil paste was tia and any mycoparasites present were not found to be fi fteen days. If, by stereoscop- adversely aff ected. Subsequently, the plates ic examination nematodes or mites were de- were incubated for a further 24-48 hours at tected on the sclerotia, the aff ected plates 100% humidity until the appearance of ei- were then placed in an oven at 80° for four ther phytopathogenic microbes or myco- hours. Under these conditions both nema- parasites was detected. todes and mites were killed while the sclero-

The presence of mycoparasites of sclerotia of the phytopathogenic ascomycete Sclerotinia sclerotiorum in cultivated lands and a possible relationship with organic matter F.A. TSAPIKOUNIS* Department of Biology, University of Patras, GR-265 00 Rio, Patra, Greece. *Present address: Agiou Nikolaou 48, GR-270 52 Varda, Ilia, Greece

More than 80 soil samples from all over From each soil sample 5 candidate myco- Greece, from tree crops, open fi eld vege- parasites were isolated on average. Most tables, greenhouse vegetable crops, vine of them belonged to the genera of Gliocla- crops and raisins, and gardens, were collect- dium, Fusarium, Trichoderma, Coniothyrium ed in order to identify sclerotia and to iso- and the class of Phycomycetes. Mycopara- late mycoparasites. Cultivation was either site presence was directly proportional to by conventional or organic methods. For the the concentration of organic matter. Also, in detection of sclerotia two metal sieves with organically-cultivated vegetable crops, the holes of 4 and 7 mm, respectively were used. presence of parasitized sclerotia was great- For the isolation of sclerotial mycoparasites er. It seems that the parasitism of sclerotia using as traps sclerotia and pH, the texture and the overall presence of mycoparasites and organic matter were determined in soil is higher in soils where the concentration of samples. Sclerotia were detected in 22.4% of organic matter is increased. Mycoparasites the samples, mainly on vegetables crops, re- were also isolated from soil samples where gardless of whether cultivation system was only traces of organic matter were present. organic or conventional. Hundreds of candi- The treatment of organic matter should be date mycoparasites were isolated and pre- a top priority for all crops and basic fertiliza- liminary evaluation showed that the major- tion should be rational and based on analy- ity had mycoparasitic qualities or abilities. sis of the soil.

Evaluation of surfactants in activation of the innate immune system of the plant Arabidopsis thaliana F. AGGELOPOULOU, P.P. A NTONIOU, E.C. TJAMOS, J. KUC* and D.I. TSITSIGIANNIS Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece. *ex. Emeritus Professor, 5502 Lorna St., Torrance, CA 90503, USA

Verticillium wilt caused by the soilborne covery of alternative means for their control pathogen Verticillium dahlia and bacteri- is essential. Induction of systemic acquired al speck caused by Pseudomonas syringae resistance (SAR) could be an alternative pv. tomato are two very serious diseases of strategy of these diseases since there are crop plants. Management of these diseases data of successful control of various plant is mainly based on prevention, thus the dis- diseases with this method. The aim of this

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 71 study was the evaluation of fi ve diff erent ty by approx. 20%. In the case of P. syringae stimulants that belong to ammonium bro- pv. tomato, no signifi cant diff erences were mide surfactants (A, B, C, D, E), for their abil- observed in the percentage of the disease ity to induce the plant defense mechanisms. for all the treatments with the fi ve factors. Pathogenicity experiments were performed Finally, in preliminary experiments investi- in Arabidopsis thaliana plants infected with gating the mode of action of the fi ve factors, V. dahliae or P. syringae pv. Tomato and to- it was found that factors C and D (at the con- mato plants infected with P. syringae pv. to- centration of 10 mM) had the ability to form mato. The fi ve surfactants were applied as a zone of inhibition against the aforemen- droplets in various concentrations on leaf tioned two pathogens. Gene expression surfaces in order to determine the optimum studies demonstrated that ammonium bro- concentration of each agent that can limit mide surfactants A and B have the potential the disease spread. It was found that in case to induce genes that play a key role in plant of V. dahliae, factor D at the concentration of defense in A. thaliana such as the PR-1 and 10 mM, was the most eff ective compared to PR-5 Pathogen Related proteins. other factors decreasing the disease severi-

Risk management measures to eradicate and prevent the spread of the quarantine potato fungus Synchytrium endobioticum (Schilbersky) Percival in Greece Η. ELEFTHERIADIS1, Ι. SARIGKOLI1, Κ.Β. SIMOGLOU1, I. VLOUTOGLOU2 and D. GILPATHI3 1Region of Eastern Macedonia & Thrace, Regional Unit of Drama, Rural Economy & Veterinary Directorate, Department of Quality and Phytosanitary Control, Dioikitirion, GR-661 00 Drama, Greece. 2Benaki Phytopathological Institute, Department of Plant Pathology, Laboratory of Mycology, 8 St. Delta Str., GR-145 61 Kifi ssia, Athens, Greece. 3Ministry of Rural Development and Food, Directorate General of Plant Produce, Directorate of Plant Produce Protection, 150 Sygrou Avenue, GR-176 71 Athens, Greece In August 2011, during offi cial plant health Ministerial Directives 259959/1984 (B’ 260) surveys, the quarantine organism Synchytri- and 456/5861/18-01-2012 (B’ 159). The two um endobioticum (Council Directive 2000/29/ fi elds were offi cially designated as infest- EC) was detected for the fi rst time in Greece, ed, placed under phytosanitary supervi- in two potato (Solanum tuberosum L.) fi elds sion, and the produce was destroyed. A buf- in Perithori, Regional Unit of Drama. Galls of fer zone was defi ned around the infested irregular shape and various sizes were ob- fi elds, based on the pathogen biology, the served on the stolons and tubers of infected disease epidemiology and the area topog- plants. Pathotype identifi cation (as patho- raphy. In the present work, the phytosani- type 18) was made by the National Refer- tary measures implemented in the area with ence Centre (The Netherlands) on gall sam- the aim to manage the risk of disease spread ples originating from the infested fi elds. The (e.g. a ban on grazing animals, prevention traceability of the propagating material was of the movement of infested soil, disinfec- investigated and a large number of potato tion of agricultural machinery and packing fi elds in the area were inspected and test- equipment, etc.) and to eradicate the path- ed, with negative results. The local potato ogen (e.g. a ban on potato cultivation in the producers were informed about the disease, infested fi elds, cultivation of resistant pota- its impacts and the phytosanitary measures to cultivars in the buff er zones, etc.) is pre- to be implemented, in compliance with the sented.

Evaluation of zeolite and Agri-fos 600® in wilt disease management and the bacterium Pseudomonas syringae pv. tomato C. LAGOGIANNI1, F. AGGELOPOULOU1, G. ZAKYNTHINOS2 and D.I. TSITSIGIANNIS1 1Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece. 2Technological Educational Institute (A.T.E.I.) of Kalamata, Department of Food Technology, Kalamata, Greece

Plants, during their long evolutionary path sorbent and catalyst. Zeolite is common- have developed several complex mecha- ly used as a soil improvement substance, nisms for resistance to a plethora of invad- but its role in disease management has not ing pathogens (fungi, bacteria and viruses). been studied. Agri-fos 600® is a special for- The phenomenon of induced and system- mulation consisting of potassium phospho- ic acquired resistance is part of the innate nate anions that, apart from their role as immune system of plants and may be po- nutrients, have the ability to induce the de- tentiated by biochemical or chemical stim- fence mechanism of plants. Pathogenicity ulation of latent resistance mechanisms, experiments were performed in Arabidop- through the the use of nonpathogenic mi- sis thaliana infected with Verticillium dahli- croorganisms, or chemical compounds. The ae or Pseudomonas syringae pv. tomato and objective of this study was the evaluation in aubergine infected with Verticillium dahli- of zeolite and Agri-fos 600® in the control of ae, where zeolite and Agri-fos 600® were ap- Verticillium wilt disease and bacterial speck plied in the form of root drench at various caused by Pseudomonas syringae pv. toma- doses. Virulence assays showed that zeolite to. Zeolite is a microporous, aluminosilicate and Agri-fos 600®have the ability to reduce mineral with specifi c physicochemical prop- the rate of Verticillium wilt in eggplant and erties commonly used as a commercial ad- in A. thaliana.

Evaluation of Renovation Sekamosa to control Verticillium wilt of eggplant E.K. LIGOXIGAKIS1, E.A. MARKAKIS2 and E. VLACHOGIANNAKIS3 1Laboratory of Plant Pathology, Plant Protection Institute of Iraklio, Hellenic Agricultural Organization “Demeter”, Mesa Katsampas, GR-710 03 Iraklio, Crete, Greece. 2Laboratory of Plant Pathology, School of Agricultural Technology, Technological Educational Institute of Crete, Stavromenos, GR-710 04 Iraklio, Crete, Greece. 3Biogenus E.P.E., 5 Anapafseos Str., GR-716 01 Nea Alikarnassos, Iraklio, Crete, Greece

The ability of the plant extract “Renovation greenhouse experiments. The lower disease Sekamosa” to control the soilborne patho- symptoms associated with the use of “Reno- gen V. dahliae in vitro and in planta was eval- vation Sekamosa” were associated with de- uated. “Renovation Sekamosa” successful- creased fungal biomass in the xylem ves- ly inhibited the growth of V. dahliae in vitro sels of the eggplants, whereas plant growth with the EC50 determined between 0,025- promotion, as indicated by the higher to- 0,05%. “Renovation Sekamosa” suppressed tal number of leaves and the fi nal height of signifi cantly Verticillium wilt symptoms in plants, was also observed.

Suppressive eff ects of compost against Verticillium wilt of eggplant on the basis of phenolic composition E.A. MARKAKIS1, M.N. ANDROULIDAKI1, M. FOUNDOULAKIS2, G. DASKALAKIS2, E.K. LIGOXIGAKIS3

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 73 and D. GOUTOS1 1Laboratory of Plant Pathology, 2Laboratory of solid waste and wastewater management School of Agricultural Technology, Technological Educational Institute of Crete, Stavromenos, GR-710 04 Iraklio, Crete, Greece. 3Laboratory of Plant Pathology, Plant Protection Institute of Iraklio, Hellenic Agricultural Organization “Demetra”, Mesa Katsampas, GR-710 03 Iraklio, Crete, Greece

Evaluation of the suppressive eff ect of six composts (A, B, C, D, E and Z) against verticillium wilt of eggplant was carried out. The sup- er total phenol content in plant stems, in- pressive action of the compost, on the ba- dicating lower levels of pathogen infection. sis of its phenolic composition was further The total phenol content of the pure com- investigated. Results showed that composts posts as well as of mixtures (consisting of C, D, E and Z reduced verticillium wilt symp- 20% compost - 80% substrate) where the toms, as well as the fungal biomass in the plants grew with diff ering disease severity, xylem vessels, despite the high inoculum was also assessed. Results showed that the density (45 microsclerotia g-1 soil) which was total phenol content of composts with sig- applied. Composts E and Z (originating from nifi cant suppressive eff ect against V. dahliae plant residues and olive leaves, olive mill ex- (C, D, E and Ζ) was signifi cantly higher that tracted press cake, and olive mill waste wa- that of the non suppressive composts. In ad- ter, respectively) showed the highest sup- dition, the total phenol content of mixtures pressive eff ects, reducing signifi cantly the (20% compost - 80% substrate) with a sig- disease severity, disease incidence and rel- nifi cant suppressive eff ect was about 400% ative area under disease progress curve (rel- higher than those of the pure substrate and ative AUDPC). The observed decrease in the non suppressive compost A, indicating symptom severity was associated with sig- the importance of these compounds for dis- nifi cant reduction of V. dahliae biomass in ease suppression mechanisms. the vascular tissues as well as with a low-

Comparative study of phytopathogenic fungi growth in two greenhouse tomato farming systems M. PAPADOPOULOU Technological Educational Institution (TEI - K) Antikalamos, GR-241 00, Kalamata, Greece

Soil microorganisms signifi cantly aff ect Chloroform Fumigation Extraction Meth- plant growth, but in agricultural ecosystems od (CFE), the respiratory activity of micro- their growth, diversity and activity depend organisms (Rbasal) was examined, and the to a large degree on the management of the total number of soil fungi (phytopathogen- soil. The purpose of this study was to inves- ic or not) was determined in vitro by the tigate the eff ect of diff erent cultivation tech- method of successive dilution and colony- niques on the development of soil microor- forming units (CFU) assay. The nutrient me- ganisms and phytopathogenic fungi. Soil dia used for the isolation of fungi were PDA, samples were collected (2009-2010), from oatmeal agar, corn meal agar, strep RBA, three greenhouses (Triphylia area, Kyparis- Botrytis sporulation agar, Czapek (Dox) agar, sia) sown with diff erent tomato varieties, and Fusarium medium. A comparative eval- and exposed to diff erent regimes of appli- uation of the samples showed that the var- cation of fertilizer and pesticides. Soil mi- iation in soil species biodiversity depends crobial biomass was determined using the on the addition of organic matter and im-

© Benaki Phytopathological Institute 74 Hellenic Plant Protection Journal - Special Issue port of nutrients by fertilization. By increas- frequently the groups of fungi appeared in ing the content of organic matter and of the cultivated soils in comparison to non-culti- macro elements in soil samples, microbial vated soils. Sclerotia of Botrytis sp, Sclerotium biomass and the total number of diff erent sp were isolated from the soil of the green- groups of microorganisms was increased. house where solarization had taken place. There was also a change with regard to how

Sustainable use of chemical fumigants for the control of soil-borne pathogens in the horticultural sector (LIFE 2008 -SustUse Fumigants) D.Ι. TSITSIGIANNIS, P.P. A NTONIOU, S.E. TJAMOS, S.D.KOUNTOURI, E.J. PAPLOMATAS and Ε.C. TJAMOS Agricultural University of Athens, Department of Crop Science, Laboratory of Phytopathology, 75 IeraOdos Str., GR-118 55 Athens, Greece

The project “SustUse Fumigants” is funded following: by the European Union under the “LIFE + • To promote the wider adoption of more Environment Policy and Governance 2008” sustainable crop protection strategies for and is intended to promote the sustaina- soil-borne diseases in horticulture ble use of chemical fumigants and the pro- • To promote the sustainable use of chem- motion of non-chemical practices (e.g. soil ical fumigants in horticultural cropping solarization, resistant rootstocks and bio- systems logical control agents) for the control of soil- • To maintain competitiveness of European borne pathogens in the horticultural sector, horticulture in a globalized market, and in in two agro-ecosystems of Mediterranean particular Italian, Greek and Polish mar- agriculture (Greece and Italy) and in a sys- kets. tem typical for Central Europe (Poland). The • To increase eff ectiveness of research on project encourages the judicious use of pes- sustainable use of pesticides ticides by applying principles of Integrated • To promote the awareness of growers, Pest Management (IPM) in order to support fumigators, advisors, policy makers and the EU policy for successful and sustainable general public on sustainable crop pro- use of pesticides. tection strategies at National and Europe- The specifi c objectives of SustUse are the an Level.

Suppression ability of composts prepared using municipal solid wastes and olive oil byproducts against the tomato pathogen Fusarium oxysporum f.sp. radicis-lycopersici A. CHOURDAKI1,2, E. BARBOPOULOU1 and N. KAVROULAKIS1 1Institute of Olive tree & Subtropical plants, NAGREF, Agrokipio, GR-731 00 Chania, Crete, Greece. 2Department of Crop Science, Technological Educational Institute of Crete, Iraklio, Crete, Greece

This trial examined the suppressive ability of nicipal solid waste. Both composts were two diff erent composts on the tomato soil- used at a low ratio mixture (5-10% w/w) with borne pathogen Fusarium oxysporum f.sp. sphagnum peat in soilless plant growth me- radicis-lycopersici. The fi rst compost was pre- dia, due to their high electrical conductivity. pared using a mixture of agricultural wastes The results showed great potential for the (olive press cake and olive leaves) while the suppression of the pathogen by both com- second utilized the organic fraction of mu- posts. Disease reduction ranged between

10 and 50% compared to the controls, and miclofl ora rather than the direct eff ect of was dependent on the ratio of the compost the compost on the pathogen. The micro- to the soilless growth media. The eff ective organisms most likely eff ect pathogen sup- control of Fusarium oxysporum f.sp. radicis- pression through various means, including lycopersici could be explained by the sup- competition for nutrients, antibiosis and/or pressive activity of the indigenous compost the induction of host resistance.

CHEMICAL CONTROL & DEVELOPMENT OF RESISTANCE

Abstracts - 16th Hellenic Phytopathological Congress 79

INVITED LECTURES

Biological properties of Xemium, the new SDHI from BASF G. STAMMLER and K. KLAPPACH BASF SE, Agricultural Center, Limburgerhof, Germany

Despite of their long history of use, succi- such as cereals, oilseed rape, potatoes and nate dehydrogenase inhibitors (SDHI) form others. After foliar application, the molecule one of the most interesting classes of agri- is systemically (acropetally) distributed with- cultural fungicides with a fascinating line in the crop and continuously released from of innovations. Being introduced in the late depots on the leaf. Xemium provides excel- 60s of the last century, SDH inhibitors are lent preventive, curative and long lasting ef- among the longest studied group of agricul- fi cacy and contributes strongly to green leaf tural fungicides. While the fi rst generation tissue and high yield. With the investigation of SDHI fungicides have a narrow spectrum of SDHI resistance, a complex picture is form- of disease control against basidiomycetes, ing. Several mutations in the target protein and are mainly used in seed treatment ap- at diff erent positions, conferring resistance plications (e.g. carboxin), the disease and to SDH inhibitors were detected. Diff erenc- crop spectrum has broadened and the in- es in the impact of such mutations on the trinsic effi cacy levels have increased signif- effi cacy level of single SDHI molecules has icantly with the new generations of SDHIs lead to sophisticated scientifi c and techni- (e.g. boscalid). Xemium, the latest SDHI mol- cal discussions. The high value of this group ecule from BASF is a broad-spectrum fungi- of fungicides for farmers worldwide and the cide that controls a wide range of economi- complexity of SDHI resistance in agricultur- cally important diseases from the classes of al pathogens call for eff orts to maintain effi - basidiomycetes, ascomycetes and deutero- cacy of SDHIs. Monitoring assays based on mycetes in specialty crops including vegeta- genetic analysis have been developed to bles, stone fruits, and pome and other fruits, improve the sensitivity of monitoring of a berries and grapes and also in arable crops number of pathogens targeted by SDHIs.

Dynali 60/30 DC, the combination of difenoconazole with cyfl ufenamid creates a new benchmarking product for the management of powdery mildew on vineyards F. M EIER-RUNGE1, S. PARAGIOU2, N. MYTILEKAS3, and E. LOLOU4 1Development Lead Specialty Crops & Potato Fungicides, Development Centre North-East, Syngenta Agro GmbH. 2Technical & Development Support Manager, Syngenta Hellas AEBE. 3Campaign Manager Specialty Crops, Syngenta Hellas AEBE. 4Registration Supervisor, Regulatory Aff airs, Stewardship & Technical support, Syngenta Hellas AEBE

Dynali 60/30 DC is a dispersible concen- dery mildews, rusts and scab of annual and trate (DC) containing 60 grams per litre (g/L) perennial crops. It is active against plant difenoconazole and 30 grams per litre (g/L) pathogens belonging to the Deuteromyco- cyfl ufenamid for use on grapes. Difenocon- ta, Basidiomycota and Ascomycota. Cyfl ufe- azole is a translaminar (weakly xylem-mo- namid belongs to a novel and a chemically bile) triazole fungicide with long-lasting unique class of fungicides, the amidoximes). preventative and curative broad-spectrum- Although the biological mode of action of control, including leaf spot diseases, pow- cyfl ufenamid against pathogens is still un-

© Benaki Phytopathological Institute 80 Hellenic Plant Protection Journal - Special Issue known, it has been demonstrated to diff er activity is extremely useful in practice, even from those of commercial fungicides such in the broadly recommended preventative as benzimidazole, demethylation inhibitor spray programmes. Under fi eld conditions, (DMI) and strobilurin. Cyfl ufenamid is high- especially under very high disease pressure, ly active on a wide range of powdery mil- purely preventative action is frequently not dews, with preventative, residual and cu- enough to provide reliable control because rative activity. On the plant, it has contact, several stages of the pathogen are simulta- translaminar and vapour properties. The neously present. For powdery mildew man- combination of difenoconazole and cyfl ufe- agement it is strongly recommended that a namid in Dynali 60/30 DC provides a broad mixture of products is used as well as alter- spectrum foliar and bunch fungicide to con- nating products with diff erent modes of ac- trol Erysiphe necator (powdery mildew), tion, in order to avoid the selection of strains Guignardia bidwellii (black rot) and Pseudo- that are resistant to single site active sub- pezicula tracheiphila (syn. Pseudopeziza tra- stances. For this reason the availability of cheiphila) (rotbrenner), with excellent crop new products is essential in order to design safety. The curative properties of the triaz- effi cient and long-lasting eff ective crop pro- ole difenoconazole are enhanced by the cu- tection programmes. rative activity of cyfl ufenamid. The curative

ORAL & POSTER PRESENTATIONS

Study of the inherent risk of resistance to dicarboximides and phenylpyrroles by Alternaria solani Ε.L. LAFKA, Α.Α. MALANDRAKIS and Α.Ν. MARKOGLOU Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos Str., GR- 118 55 Athens, Greece

Mutants of Alternaria solani moderately dioxonil-resistant strains tested. Interesting-

(RF: 15, based on EC50s) and highly (RF: 150 ly, a signifi cant reduction in the sensitivity to >1000) resistant to phenylpyrroles were of a mutant strain to pyraclostrobin was ob- isolated at high mutation frequency af- served. Study of fi tness determining param- ter UV-mutagenesis and selection on me- eters showed that the mutation(s) confer- dia containing fl udioxonil. Cross resistance ring resistance to phenylpyrroles did not studies with other fungicides showed that aff ect, or only slightly aff ected, the myceli- the mutation(s) that conferred resistance al growth rate, osmosensitivity, pathogenic- to fl udioxonil also reduced the sensitivity ity and conidial germination in most mutant of mutant strains to the aromatic hydrocar- strains tested. On the contrary, most of the bon (quintozene) and dicarboximide (iprod- fl udioxonil-resistant strains produced few- ione and vinclozolin) fungicides. No eff ect er conidia than the wild type strain. Almost of phenylpyrrole resistance mutation(s) was all fl udioxonil-resistant strains retained their observed on the fungitoxicity of the triaz- resistance levels even after 9 subcultures on ole fl usilazole, the imidazole imazalil, the fungicide-free medium. The above data in- carboxamide boscalid and the QoIs pyra- dicate that there is a considerable inherent clostrobin and azoxystrobin. Furthermore, risk of Alternaria solani resistance to fl udiox- an increased sensitivity (RF: 0.5-0.07) of the onil and that appropriate anti-resistance mutant strains to the anilinopyrimidine fun- strategies should be implemented to avoid gicide pyrimethanil was observed in all fl u- future control failures.

Characterization and distribution of fungicide resistant phenotypes in Botrytis cinerea originating from lettuce crops in Greece M. CHATZIDIMOPOULOS and A. C. PAPPAS University of Thessaly, Department of Agriculture Crop Production and Rural Environment, GR-384 46 N. Ionia Volos, Greece

The minimum inhibitory concentration (MIC) imidazoles counted 11% in total, and mainly for spore germination and mycelial growth of originated from open fi eld lettuce grown in 447 B. cinerea isolates to nine fungicides from the Macedonia or Peloponnese regions. Phe- diff erent chemical groups was determined in notypes with various resistance combina- vitro, by the point inoculation method. Three tions to 1-5 chemical groups were found less phenotypes with multiple resistance to sto- frequently (<5%). No resistant phenotypes to bilurins (QoI), carboximides (Bos), hydroxya- fl uazinam (dinitroanilides) and chlorothalonil nilides (Hyd), anilinopyrimidines (Ani), phe- (phthalonitriles) were detected. B. cinerea nylpyrroles (Phen), dicarboximides (Dic) and phenotypes exhibiting multiple resistance benzimidazoles (Βen), [QoIΗRBosΗRHydHRAniΗRP to 7 diff erent groups of fungicides, high re- henMRDicMRBenHR, QoIΗRBosΗRAniΗRDicMRBenHR, sistance to fenhexamid (Hyd) and moderate QoIΗRBosΗRDicMRBenHR] were detected at fre- resistance to fl udioxonil (Phen), were record- quency 7%, 12% and 6%, respectively. These ed for the fi rst time in Greece. To reduce grey phenotypes were mainly isolated from a mould losses the application of anti-resis- glasshouse lettuce crop located in Thessaly. tance strategies is considered necessary. Isolates with single high resistance to benz-

Molecular characterization, methodology of sdhB mutation identifi cation, and cross-resistance in SDHI-resistant Botrytis cinerea isolates T. VELOUKAS1, M. LEROCH2, M. HAHN2 and G.S. KARAOGLANIDIS1 Plant Pathology Laboratory, Faculty of Agriculture, Aristotles University of Thessaloniki, GR-541 24 Thessaloniki, Greece

Succinate dehydrogenase inhibitors (SDHIs) to boscalid, a primer introduced restriction have been used against grey mould since analysis PCR (PIRA PCR) was developed. The the end of the previous decade. Howev- method was successfully applied to the en- er, shortly after their introduction into the tire resistant subpopulation. To study the spray programmes resistance emerged cross-resistance relationships, 30 isolates (5 both in Greece and worldwide. The current per genotype) were characterized for their study was conducted to: i) investigate the sensitivities to 8 SDHI fungicides. The results molecular mechanism of resistance in 25 B. showed diff erent sensitivities and cross re- cinerea isolates showing diff erent levels of sistance patterns between structurally dif- resistance to the SDHI fungicide boscalid, ferent SDHIs. P225F mutants were resistant ii) develop molecular methods for the rap- in vitro to all SDHIs tested. Similarly, isolates id identifi cation of resistance mutations possessing the H272L mutation were high- and iii) investigate the cross-resistance re- ly resistant to boscalid, but showed low to lationships among members of the SDHI moderate levels of resistance to other SDHIs. group. Sequence analysis of the sdhB, sdhC The N230I mutants were moderately resis- and sdhD subunits of the gene revealed fi ve tant to boscalid, fl uopyram and fl uxapyrox- mutations leading to amino acid substitu- ad and showed low levels of resistance to tion in the SdhB subunit (P225F, N230I και isopyrazam, bixafen, fenfuram, benodanil H272L/R/Y). To facilitate rapid detection of and carboxin. The H272R mutants showed these mutations associated with resistance moderate levels of resistance to boscalid

© Benaki Phytopathological Institute 82 Hellenic Plant Protection Journal - Special Issue and low resistance levels to isopyrazam, but showed an increased sensitivity to ben- fenfuram and carboxin, but retained the odanil and fl uopyram (negative cross-resis- wild-type sensitivity to fl uopyram, bixafen, tance). The fi ndings of the current research fl uxapyroxad and benodanil. Similarly, the can be useful both for future sensitivity H272Y showed moderate levels of resistance monitoring programmes and in the imple- to boscalid, and very levels of resistance to mentation of anti-resistance strategies. isopyrazam, bixafen, fenfuram and carboxin,

Resistance of Botrytis cinerea populations from strawberry and greenhouse-grown tomato to botryticides P. K ALOGEROPOULOU, T. VELOUKAS and G.S. KARAOGLANIDIS Plant Pathology Laboratory, Faculty of Agriculture, Aristotles University of Thessaloniki, GR-541 24 Thessaloniki, Greece

Botrytis cinerea is a high risk pathogen for the population), in the region of Preveza the fungicide resistance development and dur- predominant phenotype was that of single ing the past has presented the problem of resistance to benzimidazoles (57.4% of the fungicide resistance development world- population), while in the Kyparissia region wide. The present study was conducted to the sensitive isolates dominated the pop- investigate the fungicide sensitivity pro- ulation (57.4%). In contrast, within the pop- fi le of pathogen isolates obtained from ulations obtained from strawberry fruits, a strawberry (from the Pieria and Ilia regions) high proportion of isolates (57%) exhibited and greenhouse-grown tomato (regions multiple resistance to anilinopyrimidines, of Preveza, Kyparissia, and on Crete). In to- benzimidazoles, QoIs and SDHIs. None of tal 1,160 single spore isolates of the patho- the isolates tested was found to be resis- gen were tested for sensitivity to the SDHI tant to hydoxyanilide or phenylpyrrole fun- fungicide boscalid, the QoI pyraclostrob- gicides. The results revealed a widespread in, the anilinopyrimidine cyprodinil, the hy- prevalence of boscalid resistant pheno- droxyanilide fenhexamid, the phenylpyrrole types in the strawberry population rang- fl udioxonil, and the benzimidazole car- ing from 46.7% to 76.8% in the Pieria and bendazim. The isolates were characterized Ilia regions, respectively. The resistance fre- as either sensitive or resistant to each of the quency in the tomato population was signif- fungicides using a bioassay technique with icantly lower, with boscalid-resistance fre- distinct discriminatory fungicide doses. The quency values of 0.8, 9.8 and 13.2% in the isolates were grouped based on their resis- Crete, Messinia and Preveza populations, re- tance phenotype profi le. Results showed spectively. In addition, in SDHI-resistant iso- that in fungal populations obtained from lates the sdhB mutations associated with re- green-house grown tomatoes on the island sistance were identifi ed using a PIRA-PCR of Crete, the predominant phenotype was technique, which showed that H272R was that of double resistance to benzimidazole the predominant mutation in all of the sam- and anilinopyrimidine fungicides (48.9% of pled populations.

Fitness and competitive ability of Botrytis cinerea strains possessing diff erent mutations in the sdhB gene T. VELOUKAS1, P. KALOGEROPOULOU1, Α. ΜARKOGLOU2 and G.S. KARAOGLANIDIS1 1Aristotle University of Thessaloniki, Faculty of Agriculture, Plant Pathology Laboratory, POB 269, GR-541 24 Thessaloniki, Greece. 2Agricultural University of Athens, Laboratory of Pesticide Science, 75 Iera Odos Str., GR-118 55 Athens, Greece

Succinate dehydrogenase inhibitors (SDHIs) ters measured. In terms of fi tness, the H272R are a fungicide class with increasing rele- isolates most closely resembled the sensi- vance in grey mould control. However, they tive isolates. In contrast, the H272Y/L, N230I are also a fungicide class at high risk for re- and P225F isolates showed reduced fi tness sistance development. Recent studies have values when compared to the sensitive iso- shown that resistance of B. cinerea to SDHIs lates. In the competition experiments it was is associated with mutations in the sdh gene. found that, in the absence of fungicide se- The objective of the current study was to in- lection pressure, after 4 disease cycles the vestigate the eff ect of the H272Y/R/L, N230I sensitive isolates dominated the population and P225F mutations in the respiratory ac- in all the mixtures tested. In contrast, when tivity, the fi tness, and the competitive- the competition experiment was conduct- ness of B. cinerea isolates. Fitness parame- ed under the selective pressure of boscalid, ters measured were: i) mycelial growth and a gradual decrease in the frequency of sen- conidial germination in vitro, ii) aggressive- sitive isolates was observed while the fre- ness and sporulation capacity in vivo and iii) quency of H272L and P225F isolates in- sclerotia production in vitro and sclerotia vi- creased. When the competition experiment ability under diff erent storage conditions. was conducted in the presence of fl uopy- The competitive ability of the resistant iso- ram the sensitive isolates were eliminated, lates was measured both in the absence and in some cases after a single disease cycle, in the presence of boscalid and fl uopyram. and the P225F mutants dominated the pop- Measurements of oxygen uptake showed ulation. These results indicate that the sdhB that N230I mutants had the lowest respi- mutations adversely aff ect the mutants. The ratory activity, followed by the H272Y mu- observed dominance of sensitive isolates in tants. In contrast no diff erences in respi- the competition experiments conducted in ratory activity were observed among the the absence of fungicides suggest that the H272R/L, P225F mutants and the wild-type application of SDHIs in alternation schemes isolates. The measurements revealed sig- may delay the selection or reduce the fre- nifi cant diff erences in most of the parame- quency of SDHI-resistant mutants.

Phytopathological and molecular characterization of Penicillium expansum mutant strains resistant to new succinate dyhydrogenase inhibitors K.N. VATTIS1, G.S. KARAOGLANIDIS2 and A.N. MARKOGLOU1 1Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece. 2Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, POB 269, GR-541 24 Thessaloniki, Greece

Mutants of Penicillium expansum highly re- es, such as the triazole fl usilazole, the phe- sistant (RF: >300, based on EC50s) to the new nylpyrrole fl udixonil, the anilinopyrimidine succinate dehydrogenase inhibitors (SDHIs) cyprodinil and the benzimidazole benomyl. were isolated at a mutation frequency of An increased sensitivity of most boscalid-re- 2.3×10-6 after UV-mutagenesis and selec- sistant mutant strains to fl uopyram and pyr- tion on media containing boscalid. Cross aclostrobin was observed. Studies of fi tness- resistance studies with other fungicides determining parameters showed that the showed that the mutation(s) for resistance to mutation(s) for resistance to boscalid may boscalid also reduced the sensitivity of mu- or may not aff ect the mycelial growth rate, tant strains to isopyrazam, fl uopyram, thifl u- but had no adverse eff ect on sporulation, zamide and carboxin, but not to fungicides conidial germination or pathogenicity. Anal- aff ecting other cellular pathways or process- ysis of mycelial extracts from the wild types

© Benaki Phytopathological Institute 84 Hellenic Plant Protection Journal - Special Issue and mutant strains, using thin layer chroma- sitivity to fl uopyram, while the H272Y mu- tography (TLC) and high performance liquid tation resulted in increased sensitivity to chromatography (HPLC), showed that most fl uopyram. However, in one strain no muta- P. expansum mutant strains produced patu- tions were found in the Ip gene, though the lin and citrinin in vitro at signifi cantly high- strain showed high resistance to boscalid er concentrations than the wild-type parent (RF: 300) and medium-high resistance to fl u- strains. Similar results were found in tests opyram (RF: 10). This is the fi rst study report- on artifi cially inoculated apples. Gene se- ing P. expansum strains resistant to SDHIs quence analysis of the SdhB subunit of com- due to the biochemical mechanism of tar- plex II, revealed that single point mutations get-site modifi cation, resulting from ami- within a highly conserved region of the iron- no acid substitutions in the SdhB subunit of sulphur protein (Ip) conferred resistance in complex II. Moreover, the data of the pres- most cases. Mutations resulted in replace- ent study indicate for the fi rst time that P. ment of histidine with arginine (H272R) or expansum have the genetic and biochemi- tyrosine (H272Y) at position 272. The H272R cal potential for the appearance of SDHI-re- mutation had no eff ect on the strains’ sen- sistant isolates in the fi eld.

Molecular characterization and PCR-RFLP detection of the E198A benzimidazole resistance mutation in fi eld isolates of Monilinia laxa from Greece A. MALANDRAKIS, A.N. MARKOGLOU and B.N. ZIOGAS Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos Str., GR- 118 55 Athens, Greece

Sensitivity to benzimidazoles in isolates of cal classes revealed no cross-resistance rela- the brown rot pathogen Monilinia laxa col- tionships between benzimidazoles and the lected from stone fruit in central and north- dicarboximide iprodione, the phenylpyrrole ern Greece was evaluated and the molecular fl udioxonil, the hydroxyanilide fenhexamid, basis for resistance was investigated. M. laxa the carboxamide boscalid, the triazole tebu- isolates were classifi ed as benzimidazole conazole and the strobilurin-type fungicide sensitive (S) or highly resistant (HR) based pyraclostrobin, indicating that a target site on their sensitivity profi les to carbendaz- modifi cation is probably responsible for the im. Thirty seven percent of the isolates be- resistant phenotypes observed. Compari- longed to the HR phenotype, carried no son of the b-tubulin gene DNA sequences apparent fi tness penalties and exhibited re- between resistant and sensitive isolates resistance factor values (based on EC50 values) vealed a point mutation resulting from the greater than 500. Highly resistant isolates E198A substitution of the corresponding were also less sensitive to the benzimida- protein in all HR isolates tested. An Eco31I zoles benomyl and thiophanate-methyl but restriction site in the b-tubulin gene, which were more sensitive to the N-phenylcarbam- was destroyed in HR M. laxa isolates, al- ate diethofencarb and the benzamide zox- lowed the development of a PCR-RFLP diag- amide, when compared to isolates belong- nostic for the detection of the E198A resis- ing to the S phenotype. Fungitoxicity tests tance mutation. with fungicides belonging to other chemi-

Penthiopyrad (Fontelis®20SC) a new fungicide for the control of a large number of diseases of fruits and vegetable crops in protected and open fi eld environments D. PANORIOS, Y. STAMATAS, J.L. GENET and R. MATYSIAK

DuPont Hellas S.A.,2 Hydras str. & 280 Kifi ssias ave., GR-152 32 Chalandri, Athens, Greece

Penthiopyrad (Fontelis® 20SC) is a novel fun- is also under registration for the control of gicide belonging to the chemical family of grey mould on fi eld and greenhouse vege- carboxamides (FRAC Group 7 - SDHI, Suc- tables (tomato, eggplant, pepper, cucumber cinate DeHydrogenase Inhibitors). Penthi- and zucchini). High effi cacy and its unique opyrad controls a broad spectrum of crop characteristics, (good translaminar pene- diseases caused by many Ascomycetes and tration and local systemic movement, very Basidiomycetes. In Greece, it is currently un- good rainfastness, a favorable toxicological der registration for the protection of ap- and eco-toxicological profi le) make it high- ple and pear against apple and pear scab, ly compatible with Integrated Pest Manage- powdery mildew and brown spot of pear. It ment programmes.

Biological evaluation of INITIUM® against downy mildew diseases. Field trial data on grape, potato and tomato crops K. BOZOGLOU, K. TSAKIRI, T. KLITSINARIS, S. BITIVANOS and D. SERVIS BASF Hellas SA, 449 Mesogeion Av., GR-153 43 Agia Paraskevi, Athens, Greece

Initium is a new fungicidal active ingredient diseases, on a preventive scheme, or when developed by BASF. The innovative com- ambient conditions are conducive for the pound belongs to a new chemical class, the disease and the crop is at a sensitive stage. triazolo-pyrimidylamines. Initium is a mito- Initium controls all major oomycete diseas- chondrial respiration inhibitor interfering es, e.g. downy mildew caused by Plasmo- with the complex III (complex bc1) in the para viticola in grapes, late blight caused by electron transport chain of the pathogen, Phytophthora infestans in potatoes and to- thus ATP synthesis in the fungal cells is in- matoes, and a broad range of downy mil- hibited. Research has demonstrated that dews and late blights in vegetables (e.g. cu- Initium does not show cross-resistance with curbits, onions, and lettuce). fungicide classes such as Qo inhibitors, phe- In Greece, Initium was tested in replicated nylamides and carboxylic acid amides. Ini- fi eld trials in ready mix products with either tium is highly eff ective in the inhibition of dimethomorph or metiram during the 2006- zoospore formation and release, zoosporan- 2012 growing seasons and showed high ef- gia release, motility and germination. In nu- fectiveness compared to standard fungi- merous fi eld trials carried out worldwide, cides. Initium has been shown to be highly selec- The performance features of Initium in tive in a wide range of specialty crops and terms of biological effi cacy, selectivity and providest the best performance when ap- environmental safety, will contribute signif- plied as a protectant spray before disease is icantly to crop protection, and support in- established in the crop. Initium applications tegrated production of high quality agricul- should begin before infection of the target tural products.

Investigation of the eff ect of pyraclostrobin on grapevine physiology and disease control S. KOUNDOURAS1, G.S. KARAOGLANIDIS 2, N. THEODOROU1, A. PAPAVASILIOU 2, G. KOTSIOU1, E. CHATZIDIMITRIOU 3, A. KLITSINARIS 4, and C. BOZOGLOU 4 1Aristotle University of Thessaloniki, Faculty of Agriculture, Laboratory of Viticulture, GR-541 24, Thessaloniki, Greece. 2Aristotle University of Thessaloniki, Faculty

© Benaki Phytopathological Institute 86 Hellenic Plant Protection Journal - Special Issue of Agriculture, Laboratory of Plant Pathology, GR-541 24, Thessaloniki, Greece. 3Aristotle University of Thessaloniki, Department of Chemistry, Laboratory of Food Technology, GR-541 24, Thessaloniki, Greece. 4BASF Hellas, 48 Egialias Str., GR-151 25 Paradisos Marousi, Athens, Greece

The aim of this research was to investi- and grey mould infections were completely gate the eff ect of pyraclostrobin on physi- absent during the summer period. A high in- ological parameters, grape attributes and cidence of Aspergillus spp. presence was ob- disease control in grapevine. The experi- served on grape berries, possibly related to ment was conducted in a commercial vine- the warm and dry conditions that prevailed yard in the Amyntaio area (Greece), planted from the veraison until the harvest period. with cv. Sauvignon blanc onto 1103P. Ex- The lowest incidence of Aspergillus spp. was perimental design included fungicide spray observed in bunches obtained from 3S treat- programmes with one (pre-bloom: 1S), two ment suggesting that the application of pyr- (pre-bloom and berry set: 2S) and three aclostrobin at veraison contributed to better (pre-bloom, berry set and veraison: 3S) ap- fungal control. Pyraclostrobin applications plications of pyraclostrobin. Control plants signifi cantly increased the chlorophyll con- (C) received a standard fungicide spray pro- tent in Sauvignon blanc leaves, with highest gramme according to the local agricultural levels in 1S, 2S and 3S compared to C plants. practice. During the vegetative period, mea- Concerning vine physiological response to surements of downy and powdery mildew pyraclostrobin, results showed lower values severity on grape leaves and bunches were of stomatal conductance and assimilation rate conducted. The incidence of grey mould in all pyraclostrobin treatments compared to and black rot infections was measured af- C plants but increased transpiration levels, ter harvest. Black Aspergilli were identifi ed leading to lower instantaneous water use molecularly at a species level as either Asper- effi ciency in the pyraclostrobin treatments, gillus carbonarious or A. niger. Estimation throughout the study period. However, pyr- of physiological parameters included leaf aclostrobin treatments manifested high- chlorophyll, midday gas exchange parame- er photosynthetic nitrogen use effi ciency ters and leaf carbon isotope discrimination. than C plants which could allow for maxi- Grapes were picked at commercial harvest mum carbon gain at a lower nitrogen lev- and grape yield and composition attributes els. No diff erences were observed among were measured. Vinifi cation of each vine- treatments for yield and berry chemical yard block was carried out and experimental characteristics (with the exception of must wines were analyzed for standard chemical ammonia nitrogen, which was higher in the parameters and the precursor of Sauvignon pyraclostrobin treatments). No diff erenc- blanc varietal aroma. Measurements of pow- es were observed among treatments in the dery mildew severity showed that disease chemical composition of the experimental severity was very low without statistical dif- wines or in the concentration of the precur- ferences among treatments. Downy mildew sor of Sauvignon blanc varietal aroma.

Effi cacy evaluation of pyraclostrobin against bacterial and viral diseases of tomato and investigation of its mode of action N. SKANDALIS1, S. STAVROULAKIS1, A. TZIMA1, Ι. MALANDRAKI1, S. BITIVANOS2, A. KLITSINARIS2, C. VARVERI1 and N. VASSILAKOS1 1Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia, Athens, Greece. 2BASF Hellas S.A., 449 Mesogion Av., GR-153 43 Agia Paraskevi, Athens, Greece

Strobilurins have rapidly become an impor- in tomato plants treated with pyraclostrobin tant class of agricultural fungicides. Their was assessed in three independent experi- activity is due to their ability to inhibit mi- ments. Moreover, the eff ect of pyraclostrob- tochondrial respiration, thus disrupting the in against Cucumber mosaic virus (CMV) and energy cycle within the fungus. In addition Potato virus Y (PVY) was assessed in an en- to serving as potent fungicides, they have vironmentally controlled greenhouse, as been reported to off er protection to plants well as in fi eld trials in commercial (toll type) by increasing their capacity to activate cel- greenhouses. Disease incidence and devel- lular defence responses and promote plant opment was aff ected by pyraclostrobin ap- growth (greening eff ect). The strobilurin plication in all experiments conducted so far class fungicide pyraclostrobin is registered and further verifi cation of results and analy- for use in several crops across Europe. In this sis is in progress. In a second set of experi- work, we present the effi cacy evaluation ments, the elicitation of defence responses of pyraclostrobin against bacterial and vi- by pyraclostrobin, as indicated by a poten- ral diseases. Specifi cally, the incidence and tial increase in the transcription of systemic development of bacterial speck disease, acquired resistance (SAR)-linked genes, was caused by Pseudomonas syringae pv. tomato, assessed.

Chemical control of grey mould on lettuce grown in hydroponic systems and fungicide residues at harvest M. CHATZIDIMOPOULOS1, I. LIGAS2 and A.C. PAPPAS1 1University of Thessaly, Department of Agriculture Crop Production and Rural Environment GR-384 46 N. Ionia Volos, Greece. 2Cadmion Analytical Laboratory, E. Antistaseos 153, GR-202 00 Kiato Korinthias, Greece

For two consecutive years (2010, 2011) the nifi cantly the disease incidence and severity, eff ectiveness of various fungicide spray ap- compared to untreated controls. The fungi- plications against grey mould on lettuce cides Switch WG and Signum WG provided grown in hydroponics, was evaluated. Two the best control, whereas Teldor WG and Da- standard applications were made at seed- conil SC were the least eff ective. The level of ling stage followed up by one or two more the disease was further reduced with one or sprays after transplanting, at 15-day inter- two more fungicide applications after trans- vals. The last application was made at least planting. To avoid the development of re- 4 weeks before harvest. Applications at sistance, alternative fungicide applications the seedling stage with each of Daconil SC were found necessary. With the exception (chlorothalonil 50%) 3ml/lt, Teldor WG (fen- of applications of Daconil SC (chlorothalonil hexamid 50%) 1.5g/lt, Signum WG (boscalid 50%) after transplanting, in all other treat- 26.7% + pyraclostrobin 6.7%) 1.5 g/lt, Switch ments either no fungicide residues or resi- WG (fl udioxonil 25% + cyprodinil 37.5%) due levels below acceptable European MRLs 0.5g/lt and Ortiva Οpti SC (azoxystrobin 8% were detected at harvest. + chlorothalonil 40%) 2.5ml/lt, reduced sig-

Biological activity of the succinate dehydrogenase inhibitor fl uopyram against Botrytis cinerea and fungal baseline sensitivity T. VELOUKAS and G.S. KARAOGLANIDIS Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University, Thessaloniki, Greece

Succinate dehydrogenase inhibitors (SDHIs) the strawberry fruit. Similarly, fl uopyram constitute a fungicide class of increasing rel- showed a high curative activity when it was evance in crop protection. These fungicides applied at 100 μg ml-1 24h post-inocula- could play a crucial role in the successful tion, but when application was made 48 or management of grey mould disease. In the 96h post-inoculation disease control effi ca- current study the eff ect of fl uopyram, a nov- cy was modest or low. The measurement of el SDHI fungicide, on several developmental baseline sensitivity showed it to be unimod- stages of Botrytis cinerea was determined in al in all populations tested. The individual vitro, and its protective and curative activi- EC50 values for fl uopyram ranged from 0.03 ty against the pathogen was determined in to 0.29 μg ml-1. In addition, no correlation strawberry fruit. Furthermore, fungal base- was found between sensitivity to fl uopy- line sensitivity was determined in a set of ram and sensitivity to other fungicides in- 192 pathogen isolates. Germ tube elon- cluding cyprodinil, fenhexamid, fl udioxonil, gation was found to be the most sensitive iprodione, boscalid and pyraclostrobin. The growth stage aff ected by fl uopyram, while obtained biological activity, baseline sen- mycelial growth was found to be the least sitivity and cross-resistance relationships sensitive growth stage. Fluopyram provid- data suggest that fl uopyram could play a ed excellent protective activity against B. key-role in grey mould management in the cinerea when applied at 100 μg ml-1 96, 48 near future and encourage its introduction or 24h before the artifi cial inoculation of into spraying programmes.

Sensitivity of isolates of the fungi Aspergillus niger and Aspergillus carbonarius in registered fungicides in viticulture M. BERTOLI, M. DIMAKOPOULOU and D.I. TSITSIGIANNIS Agricultural University of Athens, Department of Crop Science, Laboratory of Plant Pathology, Athens, Greece

The fi lamentous fungi Aspergillus niger and for SHAM. First, cyprodinil showed higher A. carbonarius cause signifi cant rot prob- inhibition rates in all of the tested isolates lems in grapes at pre-harvest and post-har- compared to fl udioxonil at all concentra- vest level and produce various carcinogenic tion rates. After six days of incubation of the mycotoxins (i.e. ochratoxin A) in grape ber- plates with the fungal isolates, the two fun- ries and wine. The goal of this study was to gicides showed a statistical diff erence be- evaluate 14 A. niger and A. carbonarius iso- tween them only in the two lower concentra- lates from the fungal collection of the Labo- tions. The tested A. niger and A. carbonarius ratory of Plant Pathology in Agricultural Uni- isolates did not demonstrate any statistical- versity of Athens (isolated from grapes in ly signifi cant diff erence between them re- diff erent regions of Greece) for sensitivity in garding their sensitivity to the two active various fungicides that are included in viti- ingredients of fl udioxonil and cyprodinil. culture plant protection programs. The fi rst In azoxystrobin, all isolates showed moder- experiments were performed with the ac- ate sensitivity which was increased signifi - tive ingredients fl udioxonil, cyprodinil and cantly when SHAM was added in the medi- azoxystrobin used alone and/or in combi- um. The exception was the isolation 315 (an nation with salicylhydroxamic acid (SHAM- A. carbonarius strain isolated from the grape inhibitor of alternative cellular respiration). variety Corinthiaki Stafi da from Trikala in The applied concentrations of the active in- Corinthia) that showed a small inhibition in gredients fl udioxonil and cyprodinil were all four treatments of fl udioxonil, cyprodinil, 0.01, 0.05, 0.1 and 0.5 ppm for azoxystrob- azoxystrobin and azoxystrobin+SHAM. This in 0.01, 0.1, 1.0 and 10.0 ppm and 100.0 ppm particular isolate has preserved this resis-

© Benaki Phytopathological Institute Abstracts - 16th Hellenic Phytopathological Congress 89 tance even in the treatment of azoxystrob- sistance levels to any of the active ingredi- in 10.0 ppm+SHAM 100.0 ppm. The results ents tested that are registered for use in vi- of this study indicate that the isolates used ticulture. (except for 315) do not demonstrate high re-

Impact of fungicides contained in wastewaters from the fruit packaging industry on soil microbes E.S. PAPADOPOULOU1, D.G. KARPOUZAS2 and U. MENKISSOGLU - SPIROUDI1 1Pesticide Science Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece. 2Department of Biochemistry and Biotechnology, University of Thessaly, Ploutonos 26, GR-412 21 Larisa, Greece

Thiabendazole (TBZ), imazalil (IMZ), and or- decomposers, were determined by denatur- tho-phenyl phenol (OPP) are fungicides ating gradient gel electrophoresis (DGGE). used in fruit packaging plants for the control A general inhibitory eff ect was observed of Penicilium sp. and Geotrichum sp. infesta- for potential nitrifi cation in the presence tions during storage. Despite their indoor, of OPP, in contrast to TBZ which promoted post-harvest use high loads of these fungi- potential nitrifi cation. Enzymatic activities cides are annualy released in the soil envi- were signifi cantly aff ected by fungicides, ronment through direct land spreading of however no clear temporal patterns of fun- wastewaters from the fruit packaging indus- gicide eff ects were observed. DGGE analysis try. Taking into consideration the major role indicated a rather rich and complex soil ba- of microbes in soil nutritient cycling, a labo- sidiomycetes community. Multivariate anal- ratory study was conducted to monitor the ysis of the binary data matrix revealed that eff ects of these fungicides on soil microbial fungicides signifi cantly altered the structure diversity and function. Fungicides were ap- of basidiomycetes community, however as plied at 50 mg/kg assuming a direct soil dis- with enzymatic activities no clear temporal posal scenario and potential eff ects were re- patterns of fungicide eff ects were evident. corded on a temporal basis post application. To conclude, potential risk may exist to soil Potential nitrifi cation, β-glucosidase, acid microorganisms and their activities in soils and alkaline phosphatases, and fl uorescence treated routinely by wastewaters from the diacetate hydrolyzing activity were deter- fruit packaging industry, therefore manage- mined to assess impacts on soil functions. ment measures are necessary to control the Furthermore, structural changes on basid- waste disposal of spent application solution iomycetes community, being main carbon and prevent soil contamination.

In situ eff ectiveness of diff erent fungicides against selected resistant phenotypes of Botrytis cinerea originating from lettuce I. PAPASTERGIOU, M. CHATZIDIMOPOULOS and A.C. PAPPAS University of Thessaly, Department of Agriculture Crop Production and Rural Environment, GR-384 46 N. Ionia Volos, Greece

The effi cacy of seven fungicides with diff er- ilinopyrimidine), fl udioxonil (phenylpyrrole), ent modes of action against nine multiple fl uazinam (dinitroaniline) and chlorothalonil resistant isolates of B. cinerea, was tested in (phthalonitrile). Young lettuce leaves (c.v. Pe- situ. The fungicides used were: pyraclostrob- nelope RZ) were immersed in aqueous fun- in (strobilurin), boscalid (carboxamide), fen- gicide suspensions at the recommended hexamid (hydroxyanilide), cyprodinil (an- spraying concentrations (a.i.%). When the

© Benaki Phytopathological Institute 90 Hellenic Plant Protection Journal - Special Issue leaves dried they were placed in Petri dishes development of Botrytis lesions by the resis- containing sterilized water agar. Then, inoc- tant isolates, in all tests. Similar results were ulation was eff ected place by transferring ei- also obtained for boscalid (0.04%), cyprodinil ther an inverted 5 mm mycelial disc, or a drop (0.018% and 0.038%) and fl udioxonil (0.012% of a spore suspension (5x104/ml) onto the and 0.025%) when mycelial discs were used adaxial leaf surface (wounded or not). Fun- as the inoculum. In contrast, the fungicides gicide effi cacy was evaluated by measuring fl uazinam (0.02%) and chlorothalonil (0.15%) the size of the infection lesion after a 3 (my- were in all cases eff ective against all isolates celium) or 6 (spores) day incubation, respec- examined. These results show the inability tively, in a growth chamber at 18οC and 12 h of recently introduced fungicides to inhibit light. The fungicides pyraclostrobin (0.01%) infection by B. cinerea resistant phenotypes and fenhexamid (0.075%) failed to inhibit the obtained from lettuce crops.

PROLECTUSTM (Fenpyrazamine): A new fungicide for the control of Botrytis cinerea on grape, vegetables, strawberry and Monilia spp. on stone fruits S. TANAKA1, N. KIMURA1, Y. SENECHAL2 1Sumitomo Chemical Company, ltd., Tokyo, Japan. 2Sumitomo Chemical Agro Europe, Lyon, France

PROLECTUSTM is the trade name of a fenpyra- health of operators, workers and bystanders zamine (S-2188) based product developed by has lead to a non-classifi cation from a toxi- Sumitomo Chemical Co., Ltd., as a fungicide cological point of view. Regarding physico- for foliar applications. Fenpyrazamine be- chemical properties, the results of residue longs to the chemical family of Pyrazolino- trials support a 14 to 7 days PHI for grapes ne and is characterized by persistent action (wine or table) and 1 day PHI for vegetables. and high effi cacy against a range of fungi in- PROLECTUSTM is also safe for benefi cials. cluding Botrytis sp., Sclerotinia sclerotiorum PROLECTUSTM at a dose between 0.8 and 1.2 and Monilia sp. The product has translami- kg per hectare demonstrates a high effi cacy nar motion and is active against the agent of against Botrytis cinerea on grapevine, vege- grey mould by inhibiting germ tube elonga- table crops and strawberry. Its spectrum of tion, mycelium growth, and spore formation activity also extends to fungi of the genus in the lesions, as well as preventing lesion Monilinia and Sclerotionia. As the product development, by acting on ergosterol bio- exhibits excellent selectivity and rainfast- synthesis. No cross resistance was observed ness, a long-lasting action, favourable tox- with the dicarboximide, benzimidazole, stro- icity classifi cation and a short PHI, PROLEC- bilurin, triazole and pridinamine fungicides. TUSTM is therefore an ideal partner for IPM PROLECTUSTM will be released as water dis- programs, in line with the needs of a mod- persible granules containing 50% fenpyra- ern control strategy. zamine. The very good profi le regarding the

Εff ect of pyraclostrobin on the physiology of durum wheat and disease control I.T. TSIALTAS1, G.S. KARAOGLANIDIS2, A. PAPAVASILIOU2, A. KLITSINARIS3, C. BOZOGLOU3 and A. KONTSAS3 1Aristotle University of Thessaloniki, Faculty of Agriculture, Laboratory of Agronomy, GR-541 24 Thessaloniki, Greece. 2Aristotle University of Thessaloniki, Faculty of Agriculture, Plant Pathology Laboratory, 541 24 Thessaloniki, Greece. 3BASF Hellas, Egialias 48, GR-151 25 Paradisos Marousi, Athens, Greece

During the growing season 2010-11, a fi eld cation of pyraclostrobin at the stage BBCH experiment was established with two du- 39, exceeding by 8.69% the respective val- rum wheat cultivars (Agapi and Elpida) aim- ue of the controls. Treatments did not show ing to investigate the eff ect of pyraclostrob- any diff erentiation with regard to SLA. Com- in on both physiology and disease control pared to controls, all treatments had high- in wheat. Experimental design included sin- er ΔΤ (ΔΤ: Tcanopy-Tair) values. All treatments gle applications of pyraclostrobin at two dif- outyielded controls (2180 kg/ha) by 8% to ferent vegetative stages, double application 56.2%. Protein content and vitreousness of pyraclostrobin at both stages, and a tri- were higher in the treatments with single ple application, to investigate the eff ect on pyraclostrobin applications either at BBCH Fusarium head blight. Disease severity mea- 30 or BBCH 39. In conclusion, the present surements showed that pyraclostrobin ap- preliminary work indicates possible phys- plication signifi cantly reduced powdery mil- iological eff ects of pyraclostrobin on du- dew and Septoria tritici blotch severity in rum wheat, which also refl ected on yield both cultivars. The frequency of Fusarium- and quality. Most eff ective was the BC treat- infected kernels was relatively high, without ment (double spraying at BBCH 30 and 39) signifi cant diff erences being detected be- but even C treatment (spraying at BBCH 39) tween treatments. Chlorophyll content was could provide an acceptable eff ectiveness highest in the treatment of a single appli- regarding yield and quality traits.

Δεκέμβριος 2015 ISSN 1791-3691

Περιεχόμενα

Μυκητολογία 7-26

Βακτηριολογία 29-34

Ιολογία 37-50

Νηματωδολογία & Μη Παρασιτικές Ασθένειες 53-55

Βιολογική & Ολοκληρωμένη Αντιμετώπιση 59-75

Χημική Αντιμετώπιση & Ανάπτυξη Ανθεκτικόητας 79-91

Hellenic Plant Protection Journal www.bpi.gr © Benaki Phytopathological Institute December 2015 ISSN 1791-3691 H Hellenic Plant Protection Journal Contents e l l e

Mycology 7-26 n i

Bacteriology 29-34 c

P Virology 37-50 l a

Nematology & Non Parasitic Diseases 53-55 n t

Biological & Integrated Control 59-75 P

Chemical Control & Development of Resistance 79-91 r o t e c t i o n

J o u r n a l