efe.aua.grefe.aua.gr/publications/11th_congress_english.pdf43 Phytopathol. Mediterr. (2006) 45,...

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Phytopathol. Mediterr. (2006) 45, 43–87

Summaries of invited lectures, oral and poster presentationsgiven at the Eleventh Hellenic Phytopathological Congress

Preveza, Greece, October 1–4, 2002

The 11th National Phytopathological Congress, organised every two years by the Hellenic Phytopatho-logical Society (HPS), was held in Preveza, Greece on October 1–4, 2002, celebrating the 25th anniversa-ry of the HPS. This meeting was attended by more than 400 participants, and 60 oral presentations, 51posters and 5 invited speeches were presented dealing with plant diseases caused by fungi, bacteria,viruses, and non-parasitic disorders and with disease control. In addition, one round-table discussionwas held on “Applied plant pathology: long-lasting problems and possible solutions”. Abstracts of the oralpresentations, the invited papers and the posters of the congress are presented in this issue.

BIOLOGICAL AND INTEGRATED CONTROL

Invited lectures

The methyl bromide crisis: lessons to be learnt andpotential solutions. J. KATAN. Department of Plant Pa-thology and Microbiology, The Hebrew University of Je-rusalem, Faculty of Agricultural, Food and Environmen-tal Quality Sciences, Rehovot 76100, Israel.

Soil pests such as bacteria, fungi, nematodes, parasiticplants, arthropods and other organisms frequently causeheavy losses to major crops, affecting both yield andquality. In severe cases, they may totally destroy thecrop, forcing the farmer either to abandon the land or toshift to less susceptible, but often less profitable crops.In intensive agriculture, especially, protected crops arefrequently planted and replanted in the same land,sometimes for years in succession, thus leading to a rapidbuildup of pest populations in the soil, especially those

causing root diseases. There is, therefore, a need to de-velop effective soil-pest control methods to ensure cropproductivity and yield stability. In addition to being ef-fective, these methods have to be economically, environ-mentally and technologically sound.Soil disinfestation is one way to control soilborne path-ogens causing root and other diseases, and is especiallycommon with high-value crops. It is a sophisticated andexpensive, but effective method, which has great advan-tages, but also some limitations. The basic principle isto eradicate a wide spectrum of harmful agents in thesoil before planting, usually by drastic chemical or phys-ical means, while minimizing the damage to beneficialmicroorganisms.Methyl bromide (MB) is the major soil fumigant in useworldwide and is highly effective in controlling a varie-ty of soilborne pests, especially in intensively growncrops. However, because of its role in the depletion ofthe ozone layer, international agreement has been

ABSTRACTS

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reached to reduce MB consumption and to phase it outcompletely by the year 2005 in developed countries.Hence there is a need to develop means to reduce itsuse now, and to find alternatives to it. MB has severaladvantages: it has a broad spectrum of control againsta wide variety of pests, it can be applied under a rangeof conditions and has behind it several decades of expe-rience during which methods of its application weredeveloped to a number of intensively grown crops. Be-cause of its high effectiveness, MB was widely adoptedand the search for alternatives was often, though fortu-nately not always, neglected. Many intensively growncrops thus became wholly dependent on MS. If by 2005a suitable alternative to MB is not found, a severe lossby soilborne pests can be expected. Strawberry grownin California and other places is such an example.One way of reducing MB dosages without loss of effec-tiveness is by using plastic sheets that are less imper-meable to MB, thus delaying its escape into the atmos-phere. Lower dosages can also be achieved by improv-ing the method of application and by combining MB withother methods of control.Many potential and existing alternatives to MB, bothnon-chemical and chemical, are available, but no singlealternative can totally replace MB in all circumstances.Since MB controls a wide range of pests, an integratedpest management (IPM) approach is called for. IPM aimsat integrating all the available, effective and environ-mentally acceptable methods of management, usingthem only when necessary with due regard to the envi-ronmental, social, economic and legal requirements ofeach. A combination of methods, which is the heart ofIMP, can give a wide spectrum of effective control. Asuitable combination can become an alternative to MB.However, it will be necessary to have different combi-nations for different situations, and that is less conven-ient than having only one method. Potential methods tobe used in such combinations are fumigants at low dos-ages combined with solarization or cultural or biologi-cal control methods, soil solarization alone, biologicalcontrol, the use of resistant plant varieties, grafting andparticular cultural practices. For the future, a depend-ence on any single method of control should be avoided,in favour of alternating and/or combined approaches,and non-chemical methods of control should also be in-cluded. The alternatives that will be developed have tobe made known and available, and education and ex-tension tools also need to be further developed, to ascer-tain that they are appropriately introduced for the farm-ers to use.The task of developing effective, economically feasible andsafe alternatives to MB for the various crops is difficultand complex, but seems realisable with correct planningand the necessary investment in research, developmentand manpower. It is not known, however, whether MBalternatives for all major crops will be ready by 2005.

Success in this task will serve as a model for solving sim-ilar problems with other pesticides in the future, whilefailure will result in severe economic losses. We shouldmake every effort to avoid replacing MB with chemicalsor other alternatives that may have negative or unknownconsequences for human health or the environment. It isessential to avoid replacement of MB with methods thatmay be more damaging than MB itself.

Chemical and non-chemical alternatives toMethyl Bromide in Italy. G. CARTIA. Dipartimentodi Agrochimica e Agrobiologia, Università degli Stu-di Mediterranea di Reggio Calabria, Piazza S. Franc-esco 2, 89061 Gallina (Reggio Calabria), Italy.

In intensive agriculture, from the point of view of cropprotection, vegetables are continuously planted and re-planted in the same field, leading to a rapid buildup ofsoilborne pests (SPs) attacking the plant roots. Becauseof our climatic conditions, many pathogens induce dis-eases in more than one major crops, causing damageand economic losses. Heavy damage is also caused bynematodes in the genera Meloidogyne, Dytilenchus andPratylenchus, and by weeds.To ensure stable crop production and yield it is neces-sary to treat soil with steam or fumigants so as to re-duce disease damage to below tolerance limits. The highcost of soil disinfestation by steam restricts its use tohigh-value crops, therefore soil fumigation has becomethe most common approach for SP control. Chemicalsallowed for soil-disinfestation prior to planting are veryfew (methyl bromide, metham sodium, 1,3 dichloropro-pene and dazomet) and of these methyl bromide (MB)was the most widely used, due to its relatively shorttreatment periods and high effectiveness against pests.Unfortunately MB has been found to contribute to strat-ospheric ozone depletion, so it will gradually be phasedout (except for critical use) from now until 2005. Re-searchers have to play an important role in ensuringthe continued safe use of MB at progressively lower dos-ages until its complete ban. It has been found that insoil naturally infested with pathogens and nematodes,MB is still active at dosages of 40 and 20 g m-2, whenthe soil is covered with films virtually impermeable(VIFs) to gas (Cartia and Minuto, 1998).In Italy, studies on controlling diseases of tomatoes andvegetables with methods alternative to MB started in1980, with soil solarization (SS) (Garibaldi and Cartia,1991). Studies carried out in our climatic conditions overa twenty-year period, showed that this method was ef-fective in reducing the population density of many path-ogens and nematodes.To optimize alternatives to MB and improve knowledgeand techniques of soil solarization, a project (POM, A12)was carried out. The project was implemented underthe supervision of the Department of Agrochemistry and

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Eleventh Hellenic Phytopathological Congress

Agrobiology and financed by the European Community,and was designed to be carried out in various regions,and to involve five research institutes and researchersfrom many different fields (pathologists, nemathologists,physicists, engineers) as well as the extension servicesfrom Apulia, Calabria and Sicily (Di Primo et al., 1999).Nurserymen, fumigation and pest control companies,mulching film manufacturers, producers of machineryapplying films to the field, film recycling industries andfarmers were also involved. Central to the project wasSS applied alone or as part of an integrated system (IPM)including: low dosages of chemicals; manure (biofumi-gation); biological antagonists; resistant/tolerant varie-ties and rootstocks. The project aims to reduce the costand increase the quality and environmental safety ofagricultural products.During the three-year period 1998–2001 studies werecarried out on:• thermal regimes in soils subject to solarization

(Gutkowski and Terranova, 1991);• the effectiveness of innovative mulching films in-

tended to enhance SS effects (Di Primo and Cartia,1998);

• the effectiveness of biological agents against rootrot (Cartia and Causarano, 1996);

• the efficacy of SS and IPM in controlling soilbornepathogens and nematodes (Cartia et al., 1997).

Biofumigation (BF), combining SS with organic amend-ments, offers additional options for SP management inits chemical, physical and biological aspects. Additivessuch as chicken manure releasing volatile ammonia andincreasing soil temperature, controlled overwinteringforms of SPs more effectively, than soil solarization alone(Di Primo and Cartia, 1998).Mulching film is an important factor in creating favour-able conditions for SS and in reducing soil reinfesta-tion. Recent studies in this area indicate the importantrole of coextruded plastic film with different photomet-ric properties and color, in comparison with traditionalPE mulching film. Green coextruded film (GCF), whichalso inhibits weeds, can be left in the field after solari-zation to reduce reinfestation by pathogens in the so-larized soil (Cascone et al., 2000).Ethylene tetrafluoroethylene (ETFE) is a greenhousecover film that improves soil thermal regimes and SSeffectiveness (Cascone et al., 2001).Space structural solarization (SSS) is a promising dis-ease management technique that is a complementarysoil disinfestation to control inoculum surviving ongreenhouse structures. Control was achieved in closedtunnel or greenhouses, during July– August, when airtemperatures reached as high as 60–65°C.To verify the effectiveness of alternative methods to MB,various experimental trials to control SPs of tomato,melon, onion, and carrot were carried out. The mainresults are given below.

Tomato

Fresh greenhouse tomatoes are a major vegetable cropin Italy. Among SPs of tomato, the most harmful are,Fusarium oxysporum f. sp. radicis-lycopersici (FORL),Pyrenochaeta lycopersici and nematodes (Meloidogynespp.), causing Fusarium crown and root rot (FCRR),corky root, and root-knot respectively.Preliminary studies in the open field showed that 12days of SS significantly reduced the survival of FORLpropagules. Control effectiveness was improved bycombining SS with manure, or by extending SS treat-ment to 27 days. In closed greenhouses SS and BFwith bovine manure effectively reduced the viabilityof FORL chlamydospores, lowered disease incidenceand increased commercial yield. Susceptible tomatoplants grafted on FCRR-tolerant hybrid rootstock (He-man), even when cropped in a severely FORL-infest-ed soil, remained healthy during the growing seasonand gave a profitable yield (Di Primo and Cartia,2001a).Trials to test the effectiveness of SS as compared withMB to control P. lycopersici and nematodes, were per-formed in greenhouses where the soil was covered withdifferent films. Mulching soil with EVA, GCF, and blackcoextruded films (BCF) for 373, 265 and 68 hours re-spectively resulted in soil temperatures over 42.5°C.Corky root and root-knot symptoms appeared with verylow frequency in MB and in the solarized plot mulchedwith EVA and GCF, and differred statistically from BCFand from the untreated untreated control (Cascone etal., 2000).Five biological agents were evaluated for control of FCRRon 1700 seedlings grown in nine greenhouses. Becauseof the high incidence of FCRR, only Fus Più controlledthis disease, and no yield increase was recorded (Polizziand Catara, 2001).

Melon

Fusarium oxysporum f. sp. melonis (FOM), the causalagent of muskmelon wilt, causes serious damage tocrops grown in unheated tunnels. The effectivenessof SS and BF in the control of FOM propagules placedin the soil at depths of 15 and 30 cm was tested infield and in tunnel trials. In the field short solariza-tion (12 days), caused a mortality of 52 and 65% topropagules placed at 30 and 15 cm depth respectively;a combination of SS with chicken manure (1 kg m-2), orextending SS treatment to 27 days, improved patho-gen control. In the tunnel trials the chlamydosporeswere set at the same depths and the soil was treatedwith SS, BF and MB. SS for 35 days killed all prop-agules at 15-cm depth and induced 83% mortality at30-cm depth. BF and MB caused 100% mortality atboth soil depths. All treatments reduced disease inci-dence and increased yield in melon plants (Di Primoand Cartia, 2001b).


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Onion

White rot caused by the fungus Sclerotium cepivorumis most common on onion crops; the sclerotia remainviable for many years and become a negative factor ininfested soils. In preliminary field trials carried out inCalabria, SS significantly reduced white rot by 89%, andincreased marketable yield by 64% (Polizzi et al., 1995).Soil solarization for 27 days completely destroyed scle-rotia buried at depths of 15 and 30 cm. BF adding chick-en manure to the soil significantly enhanced pathogenimncidence as early as 12 days after treatment. Sclero-tium mortality at the two tested depths reached 100%.SS of a field amended with organic substances gave bet-ter and faster control of sclerotia of S. cepivorum, thaneither treatment alone (Di Primo and Cartia, 1998).Good results were also obtained by mulching soil withF-clean and GCF. Viability of sclerotia, placed in the soilat 20 cm depth, decreased by 26 % with F-clean, and by21% with GCF, compared with 10 days for SS alone, andby 100 % after 20 days (Agosteo and Cartia, 2001).

Carrot

The effectiveness of two fenamiphos formulations in com-bination with SS (August 13–September 25) to controlDitylenchus dipsaci of carrots was studied in trials car-ried out in Sicily. When carrots were sown in October,all control plots and only one solarized plot showed manydead or stunted and yellowing plants in early March.Because of the several generations produced by the nem-atode, no significant differences were observed in thenumbers of nematodes in the soil and in the carrot taproots at the end of March. However, yield parametersshowed highly significant differences. At the end ofMarch the average carrot tap root weight was 58.5 g inplots that were only solarized, 62.5–77.2 g in plots so-larized and treated with fenamiphos and only 30.7 g inthe control plots. At harvest (17 April) the carrot taproot yield/0.8 m2 was 3.6 kg in the controls and as muchas 8–9.7 kg in the treated plots, while the average taproot weight was 38.7 g and 92–97g, respectively (Grecoand Cartia, 2000).Trials carried out in Apulia, Calabria and Sicily, duringa three-year period showed that in specific crops andsituations, various chemical and non-chemical methodsare able to replace MB to control SP activity. In our cli-matic conditions, SS treatment is effective in summer,particularly against pathogens sensitive to heat, and SSin combination with other control methods (IPM) is moreeffective, and can also be extended to regions located innorthern Italy. Researches are now evaluating problemsrelated to MB use, and at the same time assessing ifchemical alternatives to MB will increase together withenvironmental problems.The project “Toxicological impact of agricultural and en-ergetic activities” is carried out in Sicily to determine

which chemicals alternative to MB are being used inthe Ragusa area to control SPs. Preliminary investiga-tions found that 1,3 dichloropropene is extensively em-ployed to control soilborne nematodes. Its use appearspromising; so far no chemical residues have been foundin treated soil or in the cropped vegetables.

Literature citedAgosteo G.E. and G. Cartia, 2001. Valutazione dell’efficacia

della solarizzazione con film in ETFE ed EVA coestrusoverde per il contenimento di Sclerotium cepivorum Berk.In: Atti del Convegno su “Tecniche alternative alla fumi-gazione dei terreni con bromuro di metile in fragola eortive”. Metaponto, MT, Italy 30 ottobre 2001 (abstract).

Cartia G. and R. Causarano, 1996. Possibilità di conteni-mento di Fusarium oxysporum f. sp. radicis-lycopersicimediante inoculazione di Trichoderma harzianum a gio-vani piante di pomodoro. Tecnica Agricola 2/3, 25–29.

Cartia G., N. Greco and P. Di Primo, 1997. Soil solarizationfor the control of Fusarium oxysporum f. sp. radicis-lyco-persici and Meloidogyne incognita on tomato grown inplastichouse. Tecnica Agricola 19(3), 13–18.

Cartia G. and A. Minuto, 1998. Vegetable production with-out methyl bromide in Italy. In: Proceeding from the“Workshop on Methyl Bromide Alternatives for NorthAfrican and Southern European Countries”. Roma, Ita-ly, 26–29 May, 1998, 17–32.

Cascone G., G. Polizzi, C. Arcidiacono and A. D’Emilio, 2001.Influenza sulla solarizzazione sotto serra di un film dicopertura in ETFE. Colture Protette 3, 77–86.

Cascone G., G. Polizzi, A. D’Emilio and R. Grillo, 2000. Ef-fectiveness of greehouse soil solarization with differentplastic mulches in controlling corky root and root-knoton tomato plant. In: Proceedings IS Chemical and Non-Chemical Soil and Substrate Disinfestation. Acta Horti-culturae 532, 145–150.

Di Primo P., C. Arcidiacono and A. D’Emilio, 1999. Inno-vazioni per contenere gli agenti ipogei di malattia dellepiante. Colture Protette 8, 21–28.

Di Primo P. and G. Cartia, 1998. La “Biofumigazione” delterreno per il contenimento di Sclerotium cepivorum eSclerotium rolfsii. In: Atti Giornate Fitopatologiche 1998,677–680.

Di Primo P and G. Cartia, 1998. Use of Soil Solarizationand Biofumigation for the Control of Sclerotium cepivo-rum in Southern Italy. Phytoparasitica 26(3), 171.

Di Primo P. and G. Cartia, 2001a. Contenimento del mar-ciume del colletto e della radice su pomodoro in Calab-ria e Sicilia. Colture Protette. 12, 89–94.

Di Primo P. and G. Cartia, 2001b. Solarizzazione e biofu-migazione del terreno per il contenimento di Fusariumoxysporum f. sp. melonis su melone nel sud-italia. In:Atti del Convegno “Tecniche alternative alla fumigazi-one dei terreni con bromuro di metile in fragola e ortive”.Metaponto, MT, Italy, 30 ottobre, 2001. L’InformatoreAgrario 3–6.

Garibaldi A. and G. Cartia, 1991. Soil solarization for con-trolling soilborne pathogens in closed greenhouse. In:

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International Symposium on New Applications of SolarEnergy in Agriculture. Syracuse, Italy 11–14 December1989. Tecnica Agricola 43(3–4), 188 (abstract).

Greco N. and G. Cartia, 2000. Impiego combinato delfenamiphos e della solarizzazione nella lotta contro Dity-lenchus dipsaci su carota in Sicilia. Supplement Nemat-ologia Mediterranea 28, 141–147.

Gutkowski D. and S. Terranova, 1991. Dependance of thetemperature regime on the form of the exposed surfacein mulched soil: tests of scale invariance. InternationalSymposium on New Applications of Solar Energy in Ag-riculture. Syracuse , Italy, 11–14 December 1989. Tecni-ca Agricola (43) 3–4, 172–174.

Polizzi G., G.E. Agosteo and G. Cartia, 1995. Contenimen-to di patogeni ipogei della cipolla mediante impiego del-la solarizzazione. Tecnica Agricola 47(2), 3–8.

Polizzi G. and A. Catara, 2001. Esperienze di lotta biologi-ca contro Fusarium oxysporum f. sp. radicis-lycopersiciin coltura protetta. In: Atti del Convegno “Tecniche al-ternative alla fumigazione dei terreni con bromuro dimetile in fragola e ortive”. Metaponto, MT, Italy, 30 otto-bre, 2001. L’Informatore Agrario 7–10.

Oral and poster presentations

Integrated disease management in greenhouse-grown vegetables in Cyprus. N. IOANNOU, M. IOAN-NOU and P. POLYKARPOU. Agricultural Research Institute,1516 Nicosia, Cyprus.

This work is part of a broader interdisciplinary projectthat was initiated in 1998–99 at the Zygi ExperimentalStation with the primary objective to develop IPM/ICMprograms for tomato, cucumber and pepper grown ingreenhouses. The study is carried out in two identicalgreenhouses with the same cropping and disease histo-ry. One greenhouse receives conventional plant-protec-tion treatments, including: soil fumigation with methylbromide, preventive fungicide treatments applied onschedule, and manual regulation of the greenhouse en-vironment. The other greenhouse receives IPM practic-es, including soil solarization, fungicide treatmentsbased on disease monitoring and on prescribed econom-ic or action thresholds, automatic mechanical regula-tion of the greenhouse environment, removal of infect-ed plants or plant parts, and the use of insect-proof net-ting and yellow sticky traps to control virus vectors. Bothgreenhouses are equipped with a heating system main-taining a min. temperature of 14oC, and both use healthyplanting material and standard phytosanitary meas-ures. Results so far show that diseases were effectivelycontrolled under both regimes and that fruit yields wereabout equal in both greenhouses, for all three crops.However, IPM enabled a reduction of fungicide applica-tions by about 50% in tomato and cucumber, and byabout 30% in pepper. The only major problem in the IPMgreenhouse was powdery mildew, which appeared to be

favoured by the lower relative humidity. This problemmay be resolved by the installation of electric sulfurapplicators in the IPM greenhouse.

Integrated management of soil-borne diseases ingreenhouse-grown cherry tomato. G. NEOPHYTOU1,2,N. IOANNOU1 and D.J. WRIGHT2. 1Agricultural ResearchInstitute, 1516 Nicosia, Cyprus. 2Imperial College, Sil-wood Park, Ascot, Berks, SL5 7PY, UK.

This study was undertaken in 2000–01 in order to in-vestigate the effectiveness of solarization in combina-tion with various soil amendments to control root-knotnematodes (RKN) and other soil-borne pathogens ingreenhouse-grown cherry tomato (cv. Bar 138-8). At theend of the crop season, root galling caused by RKN wasreduced by approximately 47% and 66% with the 4 and8-wk solarization treatments respectively (applied in ahermetically-sealed greenhouse). In contrast, no sig-nificant differences were observed between soil amend-ments and the control treatment. Both solarization treat-ments significantly reduced Fusarium oxysporum pop-ulations and eliminated most soil-borne fungal diseas-es, including corky root rot, Fusarium root and crownrot, and Fusarium and Verticillium wilts. The total fruityields increased by ~ 25%, compared to that obtainedfrom uncovered soil. In a preliminary study at the samegreenhouse site, the effectiveness of the organic ferti-lizer Agrobiosol (based on dry mycelium of Penicilliumchrysogenum) alone or in combination with 4-wk solari-zation was investigated. No significant differences inthe control of RKN were observed between P. chrysoge-num and the control treatment. According to the study,solarization is an important plant protection tool for thecontrol of RKN and other soil-borne diseases of green-house grown tomato, under the climatic conditions ofCyprus.

Alternatives for the control of Fusarium oxyspo-rum f. sp. niveum in watermelon crops in Cyprus.C. POULLIS1, N. IOANNOU2 and J. HEALE3. 1Department ofAgriculture, Lefkosia, Cyprus. 2Agricultural ResearchInstitute, Lefkosia, Cyprus. 3King´s College, LondonUniversity, London, UK.

Fusarium wilt of watermelon, caused by Fusarium ox-ysporum f. sp. niveum, FON), is a limiting factor forwatermelon production in Cyprus. Trials carried outin recent years with 45 reportedly resistant or toler-ant cultivars showed that all were susceptible to localisolates of FON. In order to identify the prevailing racesin Cyprus, laboratory pathogenicity tests were carriedout with 20 local isolates of unknown race designation,comparing them with isolates from USA and Israelknown to belong to races 0, 1 and 2. Three watermeloncultivars, Sugar baby (susceptible), Crimson sweet (par-tially resistant), and P.I. 296341–FR (resistant) were


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used as differentials. The majority of local isolates be-longed to race 2, a highly aggressive race that attacksall commercial resistant cultivars produced so far. Inview of these results, efforts to control the disease weredirected towards the use of resistant rootstocks onwhich the susceptible watermelon cultivars would begrafted. A number of native cucurbits and several im-ported rootstocks were evaluated in the laboratory forresistance to FON and for compatibility with the mainwatermelon cultivars. These tests resulted in the pre-selection of 10 promising rootstocks which were thenevaluated in five field trials, with the following results:(a) All rootstocks resistant to FON provided 100% pro-tection from the disease. Some rootstocks, however,notably Cucubita maxima and C. ficifolia were suscep-tible to other soil-borne pathogens, such as Rhizocto-nia solani and Pythium spp. (b) Growth and yield ofgrafted plants were double that of the non-grafted con-trols, even in soils not infested with FON. The qualityof watermelons was not affected in any way by graft-ing. (c) The most promising rootstocks were RS 841 andEarly M among the imported ones and Lagenaria sice-raria “clavata” and Cucurbita pepo “melopepo” amongthe native ones.

Isolation of vine epiphytic yeasts and evaluationof their antagonistic activity against fungi of theAspergillus niger group, causing the sour rot ofgrapes. M.G. DEMAKOPOULOU, E.C. TJAMOS, S.E. TJAMOS,P.P. ANTONIOU and S.C. CHATZINICOLAOU. AgriculturalUniversity of Athens, Department of Plant Pathology, 75Iera Odos str., 11855 Athens, Greece.

Sour rot of grapes caused by fungi of the Aspergillusniger group is a serious problem in Greece and world-wide both in wine production and raisin grape varie-ties. Besides crop losses due to sour rot several speciesof the A. niger group produce ochratoxin A, a well knownnefrotoxic and carcinogenic substance with additive ac-tion in ppb. Research on methods to control the patho-gen is therefore needed. Some fungicides may be effec-tive in reducing disease severity but an interesting al-ternative strategy is the collection of vine epiphyticyeasts and using them as antagonists against fungi ofthe A. niger group to reduce or eliminate the pathogenand the infection it causes. Yeasts were collected fromthe phyllosphere of various grape cultivars from sever-al parts of Greece, grown under certain plant protectionsystems. Leaf and cane sampling was conducted duringspring 2002 from the vine varieties Fraoula (at AtticaRodhes), Athiri (Santorini), Asyrtiko (Limnos), Limnio(Naoussa), Muscat of Alexandria Debina (Chalkidiki),and Cabernet Sauvignon (Epirus). The sampling meth-od consisted in washing the leaves and canes and trans-ferring the washings to yeast malt agar (YMA). We re-port on preliminary experiments to develop methods of

evaluating the antagonistic activity of yeasts against A.carbonarius. Berries of the varieties Fraoula andSultanina were wounded by making a hole in them 2mm in diameter and 3 mm in depth. The berries wereembedded in yeast suspension of 5�106–107 cfu ml-1, andafter 24 hrs were inoculated with a 104 cfu ml-1 conidialsuspension of A. carbonarius. The rate of the growth ofthe pathogen, the density of sporulation and the disper-sal of rot were determined daily for 4–5 days. Prelimi-nary data indicated that epiphytic yeasts restricted ordelayed rot development. More research is needed for amore detailed evaluation and taxonomic identificationof the antagonistic yeasts.

Control of Clavibacter michiganensis subsp. mich-iganensis with the antagonistic rhizospheric bac-teria Bacillus subtilis 5-127, Paenibacillus alveiK-165 and fluorescent Pseudomonas sp. PF-17. P.P.ANTONIOU, S.M. CHRISTOGLOU, and E.C. TJAMOS. Agricul-tural University of Athens, Laboratory of Plant Patholo-gy, 75 Iera Odos str., 11855 Athens, Greece.

Bacterial canker is one of the most serious diseases oftomato. An attempt was made to control the soil phaseof this disease with biological agents. Experiments wereconducted both in a glasshouse and in the field. Dur-ing 1999–2001 glasshouse experiments were carried outto induce systemic resistance in tomatoes to Clavibactermichiganensis subsp. michiganensis (Cmm) withrhizospheric bacteria belonging to Pseudomonas sp. flu-orescent isolates Pf-17 and Pf-34, and the K-165 strainof Paenibacillus alvei. The tomato hybrid NOA F1 wasdrenched at the stage of four fully expanded leaves.The Pseudomonas isolates Pf-17 and Pf-34, and P. alveiK-165 were applied as a soil drench (30 ml 107–109 cfuml-1). Seven to ten days after application the plantswere inoculated with Cmm. Inoculum at a concentra-tion of 108 cfu ml-1 was introduced by injection into thestem and the wound was protected with vaseline. Theexperiment was repeated three times and 20 plants pertreatment were used. K-165 significantly reduced dis-ease symptoms (down to 52.89% compared with 72.78%for the untreated control), while Pf-17 (63.02%) andPf-34 (65%) were rather ineffective. Several antagonis-tic bacteria were also evaluated in the field againstCmm. The rhizosphere Bacillus subtilis strain 5-127,the P. alvei strain K-165 and the fluorescent Pseu-domonas sp. strain Pf-17 were tested against strain4007 of Cmm. Tomato stems 2–3 cm long infiltratedwith bacterial inoculum were buried at sites to whichthe Garnet F1 tomato hybrid was transplanted. Plantswere drenched with 30 ml 108 cfu ml-1 Cmm per plant.Two weeks later the plants were drenched for a secondtime and then again after further ten and twenty days.The percentage of the dead plants was 5% in the un-treated control and 0% with the treatments. Further

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symptom assessment of the percentage of diseasedleaves revealed that K-165 significantly reduced dis-ease symptoms, down to 18.8% compared with 31.5%in the untreated control, while Pf-17 (24.65%) and 5-127 (29.8%) were less effective. However, when fruitsetting and the mean number of marketable size to-mato fruits were determined, it was found showed thatcontrol plants carried only 3.55 fruits per plant, whileK-165-treated plants carried 10.17; 5-127-treatedplants 11; and Pf-17-treated plants 9.88. In generalstrain K-165 was the most promising biocontrol agentagainst bacterial canker of tomato.

Control of bacterial canker of tomato by BABA orthe antagonistic bacterium Paenibacillus alvei K-165. C. ARAMPATZIS1, P.P. ANTONIOU1, E.C. TJAMOS1 and P.KATINAKIS2. 1Agricultural University of Athens, Labora-tory of Plant Pathology, 75 Iera Odos str., 11855 Athens,Greece. 2Agricultural University of Athens, Laboratoryof Biochemistry, 75 Iera Odos str., 11855 Athens, Greece.

Systemic resistance in tomato plants to Clavibactermichiganensis subsp. michiganensis, induced with therhizospheric Paenibacillus alvei isolate K-165 and thechemical factor BABA. The tomato hybrid NOA wasdrenched at the stage of two fully expanded leaves. P.alvei K-165 was applied as soil drench (100 ml 108 cfuml-1). After seven days the plants were inoculated withC. michiganensis subsp. michiganensis. The inoculumwas injected into the stem at a concentration of 106 cfuml-1 and the wound was protected with vaseline. Theexperiment was repeated three times and 15 plants pertreatment were used. K-165 reduced disease by 30–40%,compared to the untreated control. Statistical analysisrevealed significant differences between the biocontrolagent and the untreated control. These data were con-sistent with previous results showing that K-165 induc-es systemic resistance against other vascular pathogens.In the second approach, systemic resistance was inducedusing the chemical β-aminobutyric acid (BABA) againstC. michiganensis subsp. michiganensis. The tomato hy-brid NOA was drenched at the stage of two fully expand-ed leaves with 60 ml water solution of BABA at a con-centration of 2 mg ml-1. Seven days after the applica-tion of BABA the plants were inoculated with C. michi-ganensis subsp. michiganensis at a concentration of 106

cfu ml-1. The inoculum was injected into the stem andthe wound was also protected with vaseline. The exper-iment was repeated three times and 15 plants per treat-ment were used. BABA reduced disease by 50–60% com-pared to the untreated control. Statistical analysisshowed significant differences between the chemicalagent and the untreated control. Although after rootdrenching with BABA plants developed stress symptomssuch as chlorosis, necrotic spots and wilting, they re-covered later.

Effect of the biocontrol agents Pseudomonas fluo-rescens WCS365, P. chlororaphis PCL1391 andFusarium oxysporum Fo47 on the colonization oftomato roots by the fungus F. oxysporum f. sp. radi-cis-lycopersici. A.L. LAGOPODI1, G.V. BLOEMBERG2, A.F.J.RAM2, A.H.M. WIJFJES2, G.E.M. LAMERS2, C.A.M.J.J. VAN

DEN HONDEL2 and B.J.J. LUGTENBERG2. 1Aristotle Univer-sity of Thessaloniki, Faculty of Agriculture, Plant Pa-thology Laboratory, 54006 Thessaloniki, Greece. 2Insti-tute of Molecular Plant Sciences, Leiden University,Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.

Crown and root rot of tomato caused by the soil-bornefungus Fusarium oxysporum f. sp. radicis-lycopersici canbe reduced by Pseudomonas fluorescens WCS365, P. chlo-roraphis PCL1391 and the non-pathogenic Fusariumoxysporum strain Fo47 in the rhizosphere. Using differ-ent auto-fluorescent proteins to mark these micro-or-ganisms allowed simultaneous study of the pathogenand each biocontrol agent on the tomato roots, by confo-cal laser scanning microscopy. The gfp-labelled patho-genic Fusarium were easily distinguished from the mor-phologically identical, non-pathogenic strain of Fusari-um marked with cfp. Microscopic examination revealedthat competition for colonization sites on the roots andniche exclusion is a feature of the biocontrol affect inthis biocontrol pair. Microscopic analysis of the interac-tions between the pathogenic Fusarium marked withgfp and the Pseudomonas biocontrol strains marked withcfp revealed that the fungus and the bacteria competedalso for specific colonization sites on the root. In addi-tion, the biocontrol bacteria form microcolonies in thespace between the root tissue and the hyphae prevent-ing the pathogenic fungus from penetrating and colo-nizing the hyphae directly. The interaction between thepathogenic fungus and the biocontrol agents revealedimportant aspects of the biocontrol mechanism and canhelp in the development of more effective biocontrol sys-tems.

Three weeks of soil solarization with impermea-ble plastics singly or together with soil fumigantsor nematocides is effective against soilborne path-ogens. E.C. TJAMOS1, P.P. ANTONIOU1, S.E. TJAMOS1, N.P.FATOUROS2, J. GIANNAKOU3, A. PARASKEUOPOULOS4 and K.PAPACHRISTOS2. 1Agricultural University of Athens, De-partment of Plant Pathology, 75 Iera Odos str., 11855Athens, Greece. 2Plant Protection Service of Preveza coun-ty, Greece. 3Aristotelian University of Salonica, Saloni-ka, Greece. 4Plant Protection Service of Tryfilia region,Greece.

The effect that three weeks of soil solarization withimpermeable plastics, singly or in combination with me-thyl bromide 25 g m-2, condor or rugby, had on soil-borne pathogens of tomato in plastic houses at Preve-za and on soil-borne pathogens of cucumber in plastic


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houses at Kyparissia was studied in 2000–2001. Preve-za: All combinations controlled the major tomato path-ogens in a winter plantation of the tomato variety Jum-bo: Clavibacter michiganensis subsp. michiganensis,Pyrenochaeta lycopersici, Phytophthora sp. and Me-loidogyne sp. No nematode infection was noticed, pos-sibly due to the low temperatures. During the sameperiod other tomato plantations in Preveza county suf-fered from early or late P. lycopersici infections in plas-tic houses where only chemical soil disinfection wasapplied. In these plantations crop production was alsoonly to 2–2.5 kg per plant compared with 4 kg per plantin the experimental plastic houses studied here. InAugust 2001 a summer plantation of the Meloidogynesp.-resistant tomato hybrid 622 established in the sameplastic houses without any further soil disinfestationhad a root knot infection of only 2.5–5%, regardless ofthe application followed during summer 2000. The low-er rate of infection should be attributed not only to theuse of the resistant cultivar but also to a long-termeffect of soil solarization. Indeed, summer plastic houseplantations with the same tomato hybrid establishedin soil chemically disinfected two years before devel-oped very strong root-knot symptoms on 80% of allplants. Kyparissia: The effectiveness of three weeks ofsoil solarization with impermeable plastics singly orin combination with methyl bromide 25 g m-2, or con-dor against soilborne pathogens of cucumber (Fusari-um oxysporum f. sp. radicis-cucumerinum and Meloido-gyne sp.) was evaluated in plastic houses at Kyparis-sia in 2000. The susceptible cucumber cultivar Z 14were planted or grafted on Power, a Fusarium-resist-ant rootstock. Plants grafted on the resistant rootstockshowed no Fusarium symptoms while in the non-graft-ed plants disease incidence ranged from 2 to 22%. Norootknot symptoms were observed in any of the exper-imental plots. It seems that soil solarization can be avaluable alternative to methyl bromide fumigation inGreece and elsewhere if impermeable plastics are usedsingly or in combination with nematicides.

The fumigant Condor (1,3-dichloropropene) as analternative to methyl bromide. C. MAVROTAS1, C. FO-TIADIS2 and N. TSIBOUKIS2. 1Dow AgroSciences ExportS.A.S., Athens, Greece. 2K+N Efthimiadis SAS, Thessa-loniki, Greece.

The gradual phasing out of methyl bromide has createda gap in the protection of crops, especially greenhousecrops. The main problems with greenhouse soil in Greeceare (in order of importance): nematodes, soil fungi, weedsand soil insects. Condor is a broad-spectrum soil fumi-gant that can be applied as a water dilution in a dripirrigation system. It acts on contact, controlling all nem-atode species and soil insects, and it indirectly controlssoil fungi, bacteria, viruses and weeds as well. K+N

Efthymiadis S.A. in cooperation with Dow AgroScienc-es has for the past three years developed a program tocontrol these pest problems. The program is based main-ly on the nematicide Condor (1,3 dichloropropene), whichcontrols nematodes completely and on Condor in combi-nation with other pesticides, in which nematodes, fun-gi, and weeds were controlled completely. By means ofCondor in Greece it was possible: (a) to cultivate againthe areas that had been heavily infested with nematodes(Chrissoupoli-sugarbeets, Ierapetra-greenhouses); (b) toreduce both the population of the easy weeds and theneed for soil fungicides. (c) to contol nematodes, fungiand weeds when this gumigant was combined with so-larization. Finally, Condor in a protective program wasan alternative to methyl bromide.

Control of tomato plant pathogens with antago-nist fungi and a commercial biological product.F.T. GRAVANIS1, N. PAGIDAS1, S. XIFILIDOU1 and I.K. VAGE-LAS2. 1Technological Education Institution of Larissa,41110 Larissa, Greece. 2The University of Reading, Ear-ley Gate, P.O. Box 236, Reading, RG6 6AT, Berkshire, UK.

Two fungal isolates acting as biological agents, Gliocla-dium virens (Gv) and Trichoderma viride (Tv) and a com-mercial biological product (BCC), derived from plant sub-stances and recommended as a plant defense promoteragainst plant pathogens and averse abiotic factors, weretested singly as was a combination of Gv+BCC, to deter-mine their effectiveness against tomato soil-borne dis-eases. Two fungal isolates, Fusarium oxysporum f. sp.lycopersici (Fol) and Verticillium dahliae (Vd) and twobacterial strains, Pseudomonas syringae pv. tomato (Pst)and Clavibacter michiganensis subsp. michiganensis(Cmm) were employed in this study. Twenty-day-old to-mato seedlings were transplanted to 9-cm-diameter potsfilled with a mixture of equal volumes of the commercialsubstrate Potgrond P and peat. Seedlings were inoculat-ed with the pathogens at transplanting and the biologi-cal agents were applied afterwards with the irrigationwater. The fungi were applied once only, as spore sus-pensions (106 spores ml-1, 20 ml plant-1). BCC was ap-plied in two doses, each of 40 mg plant-1, diluted in wa-ter; the first dose upon transplanting and the second aweek later. Treatments were given in all possible combi-nations with pathogens, biological agents and controls,with 10 replicates per treatment. The plants were keptin a greenhouse and the disease assessment was made21 days after transplanting. The height, net weight, dryweight, stem diameter and overall appearance of plantswere assessed. All biological agents and their combina-tions controlled all pathogens as far as the plant dryweight was concerned, with no significant differences be-tween treatments. The combination Gv+BCC, however,gave the best results in terms of the overall appearanceof plant stem diameter and plant net weight.

51Vol. 45, No. 1 April, 2006


Control of Leveillula taurica (Lev.) Arnaud in pep-per by soil incorporation of sulphur combinedwith Thiobacillus spp. V.A. BOURBOS1, E.A. BARBOPOU-LOU1 and K. VENETIS2. 1NAGREF, Institute of Olive Treeand Subtropical Plants of Chania, Laboratory of PlantPathology, Agrokipio, 73100, Chania, Greece. 2IntrachemHellas LTD, 31 Kiffisias str., 11523 Athens, Greece.

The work examined whether powdery mildew (Leveil-lula taurica) in a greenhouse pepper crop (cv. Drago)could be controlled with the soil-improving product bear-ing the commercial name Acidam AVC 50 at a dose of100 g m-2. Acidam contains 50% sulphur and microor-ganisms of the genus Thiobacillus. The product was in-corporated into the soil two weeks before the plantletswere transplanted to their final position. The fungicidetriadimenol was used as a reference product, at a doseof 175 ml hl-1 of the commercial product Bayfidan 050EW. Acidam effectiveness was evaluated by counting thelesions per leaf. In experimental plots where Acidam wasapplied, the pathogen was controlled with 93.98–96.42%effectiveness, which was significantly different from thatof the reference product (99.22–99.71%).

Control of soil-borne plant pathogens with thenematode symbiont entomopathogenic bacteriumPseudomonas oryzihabitans. I.K. VAGELAS1, F.T. GRA-VANIS2 and S.R. GOWEN1. 1The University of Reading,Earley Gate, P.O.Box 236, Reading, RG6 6AT, Berkshire,UK. 2Technological Education Institution of Larissa,41110 Larissa, Greece.

Pseudomonas (Flavimonas) oryzihabitans, a symbiontbacterium of the entomopathogenic nematode Stei-nernema abbasi, significantly inhibited the mycelialgrowth of Fusarium oxysporum f. sp. lycopersici (Fol),F. oxysporum f. sp. radicis-lycopersici (Forl), Rhizocto-nia solani (Rs) and Pythium ultimum (Pu), in vitro. Stud-ies using bacterial filtrates confirmed the fungistaticeffect of the cell-free solutions on mycelia of Fol, Forl,and Rs, and on spores of Fol and Forl. In a glasshouseexperiment P. oryzihabitans suppressed Fol and Rs inthe soil. In the tomato rhizosphere, P. oryzihabitans col-onized mycelia of Fol. The population of P. oryzihabi-tans increased from Log10 3.52±0.124 (in the absence ofFol) to Log10 4.82±0.011 (in the presence of Fol). P. oryzi-habitans also controlled Fusarium wilt of tomato, en-hanced plant growth and increased stem height andfresh and dry weight, acting as a plant growth promot-ing rhizobacterium.

Effect of compost and compost extracts on damp-ing-off caused by Rhizoctonia solani on tomato.G.S. KARAOGLANIDIS1, K. KLONARI1, N. BARBAYIANNIS2 andG. DIAMANTIDIS3. 1Plant Pathology Laboratory, 2Soil Lab-oratory, 3Laboratory of Agricultural Chemistry, Aristo-

telian University of Thessaloniki, Faculty of Agriculture,P.O. Box 269, 54006, Thessaloniki, Greece.

Damping-off caused by Rhizoctonia solani is a majorseedling disease in nurseries, greenhouses and outdoorcrops. Although damping-off and the yield losses it caus-es are controlled by various fungicides, these are notalways economically or environmentally sustainable.One alternative to fungicides used for disease manage-ment may be the amendment of soils with composts. Theobjective of this study was to evaluate how well damp-ing-off was suppressed by a compost prepared from grapemarcs, cotton residues and spent mushroom substrates,and by extracts of this compost also containing humicand fulvic acid. Compost and compost extracts were test-ed for their control of tomato damping-off in growth-chamber experiments, while compost extracts were alsotested in vitro for their inhibition of the mycelial growthof R. solani. Compost extracts containing humic acid sig-nificantly inhibited (P<0.05) mycelial growth of the fun-gus, but extracts containing fulvic acid at any of the con-centrations tested did not show any inhibitory effect(P>0.05) on mycelial growth of R. solani. Amendment ofthe container media with solid compost significantly low-ered Rhizoctonia damping-off severity on tomato plants.Compost extracts containing humic acid also suppressedthe disease to some degree, but no suppression was ob-served with treatments containing fulvic acid. A combi-nation of compost amendment and cultural or evenchemical control measures may provide approaches tointegrated disease management of damping-off.

Natural volatile substances of grapes of the Isa-bella variety for the control of Botrytis cinerea.E.K. KULAKIOTU1, C.C. THANASOULOPOULOS1 and E.M.SFAKIOTAKIS2. 1Aristotle University of Thessaloniki, Fac-ulty of Agriculture, Laboratory of Plant Pathology, P.O.Box 269, 54006 Thessaloniki, Greece. 2Aristotle Univer-sity of Thessaloniki, School of Agriculture, Laboratoryof Pomology, 54006 Thessaloniki, Greece.

Volatile substances produced by grapes of the Isabellavariety (sp. Vitis labrusca) were studied as providing apossible alternative method for the post-harvest controlof the fungus Botrytis cinerea. A bioassay method, thatof the closed Mariotte system, was used to quantify thebiological action of these volatile substances on thegrowth of B. cinerea. The following treatments were test-ed in vitro: (i) volatile substances excreted by grapes ofthe resistant variety Isabella, (ii) volatile substancesexcreted by grapes of the susceptible variety Roditis,and (iii) no volatile substances at all. Volatile substanc-es of the resistant variety of grapes had a fungistaticeffect on the sporulation of the fungus and on the crea-tion of sclerotia, and volatile substances from the sus-ceptible variety stimulted sporulation of the fungus. Inthe in vivo experiments, an interactive model was used


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consisting of volatile substances from grapes of the Isa-bella variety and B. cinerea-clusters of the Roditis vari-ety. The results confirmed the antibiotic action of thevolatile substances from the grapes of the Isabella vari-ety as they limited the incidence of infection, by consid-erably reducing both the amount of inoculum and theactivity of the pathogen.

Control of powdery mildew of tomatoes (Leveillu-la taurica) with a formulation of a plant extractfrom Reynoutria sachalinensis. N.E. MALATHRAKIS1,E. MARKELLOU2, E. SIRANIDOU4, M.N. FANOURAKI1, A-M.KASSELAKI1, A. SCHMITT3, N. PETSIKOS-PANAYOTAROU2 andS. KONSTANTINIDOU-DOLTSINIS4. 1School of AgriculturalTechnology, Technological Education Institute of Crete,71500 Heraklion, Greece. 2Benaki Phytopathological In-stitute, 8 St. Delta str., 14561 Kifissia, Athens, Greece.3BBA Institut für biologischen Pflanzenschutz Heinrich-str. 243 D-64287 Darmstadt Germany. 4National Agri-cultural Research Foundation, Institute of Plant Protec-tion, Amerikis and National Road, 26004 Patras, Greece.

In the framework of an EU-funded project, a study wascarried out to determine the effectiveness of Milsana®

(liquid formulations of Reynoutria sachalinensis extract)applied as a stand-alone treatment or in combinationwith other biocontrol agents, against tomato powderymildew (Leveillula taurica), in four large-scale green-house trials conducted in Peloponessse and Crete (1999–2001). In two of the trials, the effectiveness of Milsana®

was studied in relation to its rate of application, whilstin the others, Milsana® was studied in combination withthe fungus Pseudozyma flocculosa (former Sporothrixfloculossa) and the bacterium Brevibacillus brevis. Theabove-mentioned biocontrol agents were applied aloneor in double combinations and were compared with wet-table sulfur or other fungicides as reference treatments.Milsana® significantly reduced the percentage of infect-ed leaf area in comparison to the controls. Its efficacywas similar to that of sulphur and the fungicide penco-nazole, but lower than that of the fungicide treatments(applied on an as needed basis). A combination of Milsa-na® with the microbial antagonists did not significantlyimprove the effectiveness of Milsana®. Despite the levelof disease reduction on the leaves in all treatments, yield(number and weight of harvested fruits) was not affect-ed. This was attributed to the ability of tomatoes to pro-duce abundant new phylloplane, which enables them totolerate infections by L. taurica without significant im-pact on yield. Comparative analysis of the results indi-cated that the efficacy of Milsana® against powdery mil-dew was influenced by the time of onset of the epidemicin relation to the host growth stage and the infectionrate. These results along with other results from othertrials conducted in the framework of the above-men-tioned EU Project, showed that Milsana® can be a use-

ful tool to control L. taurica in organic agriculture and/or integrated crop systems of tomatoes.

Effect of the growth medium on the in vitro sensi-tivity of Botrytis cinerea strains to pyrimethanil.Lowered sensitivity of the pathogen to pyrimeth-anil after repeated applications to tomatoes grownunder cover. E. MARKELLOU1,3, D. KYRIAKOPOULOU1, V.MAVROIDIS1, A.E. KALAMARAKIS1,2, N.E. MALATHRAKIS3 andN. PETSIKOS-PANAYOTAROU1,2. 1Benaki PhytopathologicalInstitute, Department of Pesticides Control and Phytop-harmacy, 8 St. Delta str., 14561 Kifissia, Athens, Greece.2National Agricultural Research Foundation(N.AG.RE.F.), Plant Protection Institute, Athens, Greece.3School of Agricultural Technology, Technological Edu-cation Institute of Crete, 71500 Heraklion, Greece.

The effect of the anilinopyrimidine fungicide pyrimeth-anil on one wild-type and four strains of Botrytis cine-rea resistant to the benzimidazoles and/or the dicarbox-imides and with wild-type insensitivity to diethofencarbwas studied in vitro and in vivo. In vitro, when pyrimeth-anil was incorporated into media containing asparag-ine and inorganic salts, or glucose and agar, it was veryeffective against the mycelial growth of the B. cinereastrains (EC50 from 0.005 to 0.04, and from 0.02 to 0.19µg ml-1 respectively). In more complex media (such asMEA) B. cinerea activity was reduced. In vivo, preven-tive applications of pyrimethanil completely protectedyoung cucumber plants and fruits that were inoculatedwith a conidial suspension (1�106 spores ml-1) or myce-lial plugs (17 h old) of the B. cinerea strains. The effec-tiveness of pyrimethanil against grey mould was alsotested in greenhouse tomatoes in relation to a) the typeof infection on different plant parts and b) the responseof the naturally occurring B. cinerea population to theselection pressure caused by eight successive applica-tions of this fungicide. Before any treatment, the B. cine-rea population was found to be resistant to the benzim-idazoles and the dicarboximides, and the wild-type wasinsensitive to diethofencarb, and sensitive to dichloflu-anid and anilinopyrimidines (phenotype BenHRDicMR-

PcmRDichlSAniS). Pyrimethanil effectively controlledgrey mould on the leaves, fruits and stems but it did notsignificantly reduce the number of dead plants or fruitswith ghost-spot symptoms. The effectiveness of py-rimethanil on the leaves declined progressively duringthe spraying period and was inferior to that of the ref-erence mixture after the 6th application. This was at-tributed to the development of a low level of resist-ance in the B. cinerea population (Rf=7.7) which wasdetected in vitro after the 8th application. With the useof linear models (logistic) pyrimethanil delayed the on-set of the mould but it did not reduce the rate of infec-tion. The risk of Botrytis developing resistance to theanilinopyrimidines in practice is discussed.

53Vol. 45, No. 1 April, 2006


Integrated control of cucumber damping-off withbacterial peat inocula and reduced rates of fungi-cides. D.G. GEORGAKOPOULOS1 and N.E. MALATHRAKIS2.1Agricultural University of Athens, Dept. of Agricultur-al Biotechnology, Laboratory of General and AgriculturalMicrobiology, 75 Iera Odos str., 11855 Athens, Greece.2Technological Educational Institute of Crete, School ofAgricultural Technology, P.O.Box 140, 71500 Heraklion,Greece.

The bacteria Pseudomonas fluorescens B5 and X and P.corrugata R117 satisfactorily protect cucumber seed andseedlings against damping-off caused by the pathogenicfungus Pythium ultimum. These bacteria were formulat-ed into peat inoculum for seed treatment with good re-sults. Peat inoculum was combined with seed treatmentwith fungicides (thiram, propamocarb, mancozeb, fosetyl-Al, captan and metiram) at lower dosages to achieve bet-ter protection against damping-off. The fungicides test-ed were not toxic to the bacteria in vitro in experimentswith plates and broth, nor after seed coating, except forcaptan. The initial rate of application was 70 mg g-1 seed,equal to the commercial rate. In the integrated treat-ments, fungicide levels were reduced from 1/4 to 1/256 ofthe commercial rate, which drastically reduced the effec-tiveness of most of them. Integrated seed treatment withthiram at 1/16 the commercial rate and mancozeb at 1/64 the commercial rate were comparable in effectivenessto commercial fungicide treatments. However, no repro-ducible results were obtained with metiram. WhenPropamocarb was reduced even to as little as 1/256, itremained almost as effective as at the commercial rate,showing that the effectiveness of this integrated treat-ment was due to the fungicide. Fosetyl-Al was not effec-tive, and captan was not used because of its toxicity.This work was partly supported by the grant FAIR-CT96-1373 from the European Union to the T.E.I. ofCrete and other partners.

Biological and integrated control of sugar beetdamping-off in the field. D.G. GEORGAKOPOULOS1, R.TILCHER2 , U. FISCHER2, F. IOANNIDIS3, K. DOULIAS3 and N.E.MALATHRAKIS4. 1Agricultural University of Athens, Dept.of Agricultural Biotechnology, Laboratory of General andAgricultural Microbiology, 75 Iera Odos str., 11855 Ath-ens, Greece. 2KWS Saat AG, Grimsehlstrasse 31, D-37555, Germany. 3Hellenic Sugar Industry S. A., Facto-ry of Platy, 590 32 Platy, Greece. Hellenic Sugar Indus-try S. A., Factory of N. Orestiada, 682 00 N. Orestiada,Greece. 4Technological Educational Institute of Crete,School of Agricultural Technology, P.O.Box 140, 71500Heraklion, Greece.

The bacteria Bacillus subtilis B6 and Pseudomonas flu-orescens B5 and X were selected on the basis of small-scale experiments as biological antagonists of the path-ogenic fungus Pythium ultimum, causing damping-off

of sugar beet seed and seedlings. Bacterial inoculum wasincorporated into sugar beet seed pellets in accordancewith the commercial method of production. Pellets ei-ther contained bacteria alone (biological treatment) orthe bacteria combined with the fungicides thiram andhymexazol reduced to 50% of the commercial dose, andwith the insecticide imidacloprid at the full dose (inte-grated treatment). Initial populations per pellet were1�105 cfu seed-1 for B. subtilis and 2.9�104 cfu seed-1 forP. fluorescens B5 and X, which were reduced to 33% (B.subtilis) and 10% (P. fluorescens) because of pellet dry-ing during production. The biological and integratedtreatments along with the appropriate controls (com-mercial pellets, pellets with reduced fungicide, pelletswith no protectants at all) were evaluated in field trials(randomized complete block design with 4–6 blocks) inMavrodendri (Kozani) and N. Orestiada (Evros). Due tohigh temperatures, infection occurred only in the N.Orestiada trial, where the biological treatments werenot effective, although P. fluorescens X showed a posi-tive trend. The integrated treatments were not differ-ent from the commercial treatment, since the lower fun-gicide dose was as effective as the commercial dose. P.fluorescens X (alone and combined at the lowered dose)was also evaluated in trials at Platy (Imathia). Lack ofinfection did not allow proper evaluation. It seems prob-able that pellets were ineffective because of the low bac-terial populations in the pellets, since 107 bacteria perseed are reported to be necessary for infection to occur,and this population size is difficult to achieve with thecurrent method of pellet production.The work was supported by the grant FAIR-CT96-1373from the European Union to the T.E.I. of Crete and oth-er partners.

Study on the effect of combinations of biocontrolagents with a plant extract against powdery mil-dew of cucumber (Sphaerotheca fuliginea) inGreece and The Netherlands. S. KONSTANTINIDOU-DOLTSINIS1, E. MARKELLOU2, A. KALAMARAKIS2, E. SIRANIDOU1,A. SCHMITT3, A.J. DIK4 and N. PETSIKOS-PANAYOTAROU2. 1Na-tional Agricultural Research Foundation (N.AG.RE.F),Plant Protection Institute, Amerikis and National Road,26004 Patra, Greece. 2Benaki Phytopathological Institute,8 St. Delta str., 14561 Kifissia, Athens, Greece. 3BBA In-stitut für biologischen Pflanzenschutz, Darmstadt, Ger-many. 4Applied Plant Research (PPO) Section Horticul-ture, Naaldwijk, The Netherlands.

In the framework of an EU-funded project, the effec-tiveness of combinations of Milsana® (a plant extractfrom Reynoutria sachalinensis, which enhances plantresistance) and the biological control agents Brevibacil-lus brevis and Pseudozyma flocculosa (ex Sporothrix floc-culosa) (that have a direct effect on the pathogen) wasstudied in planta. Five greenhouse trials were conduct-


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ed in Greece and the Netherlands during 2000 and 2001,along with preliminary in vitro trials in Greece. In thegreenhouse trials all treatments with Milsana® (wheth-er alone or combined) reduced disease severity on theleaves as compared with the water controls. In Greece,the effectiveness of Milsana® was not enhanced whenthe biocontrol agents were combined with it or used in-stead of it. In the Netherlands, all agents significantlycontrolled powdery mildew and in one trial the combi-nation of B. brevis with Milsana® was significantly bet-ter than either agent acting alone. Applications of Mil-sana® alone or of P. flocculosa alone (only in 1 trial, inThe Netherlands) significantly increased yield (numberand weight of harvested fruits) as compared with thecontrol. The combination of Milsana® and S. flocculosa(Greece) or B. brevis (The Netherlands) also led to in-creased yield. In conclusion, Milsana® is a useful tool forthe control of cucumber powdery mildew, but the effec-tiveness of the antagonists B. brevis and P. flocculosa wasnot consistent, and their combination with Milsana®

mostly did not improve the efficacy of Milsana® alone.

Effectiveness of plant extracts against powderymildew of cucumber (Sphaerotheca fuliginea) andcereals (Erysiphe graminis). S. KONSTANTINIDOU-DOLTSINIS, E. ROEHNER2, E. SIRANIDOU1, E. MARKELLOU3 andH. BUCHENAUER2. 1National Agricultural Research Foun-dation, Plant Protection Institute, 26004 Patras, Greece.2Institut für Phytomedizin, University of Hohenheim,Stuttgart, Germany. 3Benaki Phytopathological Institute,8 St. Delta str., 14561 Kiffisia, Athens, Greece.

In the framework of a Greek-German collaboration pro-gramme, plant extracts from a) Reynoutria sachali-nensis leaves, b) Cassia septentrionalis pods and c) Ca-lendula officinalis flowers were studied for their effec-tiveness against powdery mildew of cucumber and ce-reals (wheat and/or barley), in small-scale experimentsin Greece and Germany. Aqueous extracts of R. sacha-linensis (0.5% w:v), C. septentrionalis (1%) and C. of-ficinalis (3%), on cucumber plants with only one trueleaf reduced powdery mildew severity by >70% whenapplied two days before inoculation. Mixtures of theseextracts or the addition of saponin did not further in-crease their effectiveness. Extracts from R. sachali-nensis and C. septentrionalis were highly effective whenapplied prophylactically (-2 to 0 day before inoculation).On the contrary, C. officinalis was most effective (ca.60%) when applied up to 2 days after inoculation. Ingreenhouse trials (with potted cucumber plants), ex-tract effectiveness ranged from 34–65% when they wereapplied at 7-day intervals. At longer intervals extractswere less effective. In preliminary trials, extracts fromC. septentrionalis and R. sachalinensis had no system-ic activity. Indications of translaminar movement ofthese extracts were initially obtained. In wheat and

barley, preventive application of C. septentrionalis ex-tract (2.5% w:v) reduced the leaf area infected with E.graminis. The severity of powdery mildew was inverse-ly correlated with the length of time between treat-ment and inoculation (-4 to -1 d.). A high effectivenessof 97% and 80% against powdery mildew was obtainedonly with the Cassia extract applied 1 and 2 days be-fore inoculation respectively, while the effectiveness ofthis extract decreased rapidly with increasing pre-in-fectional lengths of time. No curative effect againstpowdery mildew in either was obtained when Cassiaextract was applied 1–4 days after inoculation. BothC. septentrionalis and R. sachalinensis extracts (2.5%)completely controlled E. graminis (effectiveness >95%)on barley when applied 1 day before inoculation. C.officinalis (3%) did not reduce disease severity. Noneof the extracts tested on either host produced systemicactivity. Effective concentrations that would reducepowder mildew by 50% (EC50) were estimated for thetwo effective extracts. The potential of these extractsunder commercial conditions should be investigatedfurther in large-scale trials.

Biological activity of Gliocladium virens and Tri-choderma spp. against soil-borne plant pathogens.I.K. VAGELAS1, F.T. GRAVANIS2 and S.R. GOWEN1. 1The Uni-versity of Reading, Earley Gate, P.O. Box 236, Reading,RG6 6AT, Berkshire, UK. 2Technological Education In-stitution of Larissa, 41110 Larissa, Greece.

An isolate of Gliocladium virens taken from disease-af-fected soil in a commercial tomato greenhouse in theLarissa region (Greece) was highly antagonistic in vitroto the soil-borne fungi Fusarium oxysporum f. sp. lyco-persici (Fol) and Rhizoctonia solani in dual-cultures. Asignificant improvement in tomato yield was obtainedwhen G. virens and other fungal isolates active as bio-logical agents were added to the soil of potted tomatoesartificially infected with Fol., G. virens, Trichodermaharzianum and T. viride significantly reduced tomatowilt caused by Fol and significantly increased stem freshweight and yield as compared with infected and untreat-ed control. G. virens and T. harzianum were most effec-tive against Fusarium wilt of tomato. T. viride was some-what less effective.

Potential use of ionized water in the spraying so-lution to control Spilocaea oleagina (Cast.) Hugh.E.A. BARBOPOULOU and V.A. BOURBOS. National Agricul-tural Research Foundation (N.AG.RE.F), Institute ofOlive Tree and Subtropical Plants of Chania, Laborato-ry of Plant Pathology, Agrokipio, 73100, Chania, Greece.

Peacock spot of olive tree, attributed to the fungus Spilo-caea oleagina (Cast.) Hugh., causes leaf-drop, and whensevere, may lead to complete fruitlessness. This work ex-amines whether the pathogen can be controlled using

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water ionized with Cu ions in the spraying solution anda reduced dose of fungicide. The trial was conducted on23-year-old olive trees of the Kalamon variety. BouillieBordelaise 20 WP, a copper sulphate-based fungicide wasused as the reference product and applied at the recom-mended dose of 500 g hl-1. The same fungicide was alsoused with ionized water in the “Superior Aqua” system,at fungicide doses of 250 and 125 g hl-1. To determine theeffectiveness, the number of spots per leaf in a sample of100 leaves was counted, and the percentage of leaf-dropin 8 shoots per tree was calculated. Ionized water alonecontrolled the pathogen with an effectiveness of 28.7 to34.8%. The fungicide at the recommended dose with nat-ural water had an effectiveness from 89.6 to 99.5%. Ion-ized water with the fungicide at half and at 1/4 of therecommended dose had an effectiveness from 82.9 to98.5% and from 80.0 to 96.1% respectively.

Biological control of Penicillium spp. by the useof the biocontrol yeast Pichia anomala: ecophys-iological studies and their impact on inoculumquality and shelf-life. S. MOKIOU and N. MAGAN. Ap-plied Mycology Group, Biotechnology Centre, CranfieldUniversity, Silsoe, Bedford MK45 4DT, UK.

For BCAs to be successfully used, several aspects of theirdevelopment, including the economic mass productionof ecologically effective inoculum and elucidation of theirmode of action need to be understood. Pichia anomalais being assessed as a commercial agent to control Peni-cillium roqueforti and the mycotoxigenic P. verrucosumin moist cereals. Manipulating yeast physiology bychanging the water stress [water activity (aw), 0.98/0.96]of cane molasses and rich defined (NYDB) media usingdifferent compatible solutes/sugars and NaCl markedlyaffected yield and quality of cells. The endogenous wa-ter potential of cells (Ψc), and the sugar/sugar alcoholcontents underwent significant changes. The accumu-lation/synthesis of trehalose or sugar alcohols was af-fected by the type of medium and the solutes used. In-terestingly, there was an intracellular accumulation ofthe desiccation protectant trehalose when proline, glu-cose and sorbitol were added to media for the first time.When compatible solutes/sugars and NaCl were addedto the media, there was an accumulation/synthesis ofglycerol and varying amounts of arabitol. The ecologi-cal competence of the yeast treatments was examinedby plating on non-stressed (0.995 aw) and water-stressedmedia (0.96 aw). Adding proline or NaCl to molassesmedia improved viability. Subsequently, the best celltreatments were suspended in isotonic PEG 200 (0.98/0.96 aw) solutions and then stored as wet pastes at 22oCand 4oC for up to six months. Cells with high trehaloselevels had better viability; wet pastes stored at 4oC gavethe best results. Studies on the mode of action of thebiocontrol yeast P. anomala are in progress.

Biological control of root knot nematodes (Mel-oidogyne spp.). G. NEOPHYTOU1,2, N. IOANNOU1, D. KLEAN-THOUS1 and D.J. WRIGHT2. 1Agricultural Research Insti-tute, 1516 Nicosia, 22016 Cyprus. 2Imperial College, Sil-wood Park, Ascot, Berks, SL5 7PY, UK.

In a pot trial, selected bio-nematicides were evaluatedfor control of root knot nematodes (RKN) on the sus-ceptible tomato cultivar Graziella. All bio-nematicides,especially bionem (Bacillus firmus) and DiTera (My-rothecium verrucaria), alone or in combination, reducedthe galling index compared with untreated plants.Though significant control of RKN was achieved, plantstreated with bionem also showed a significant reduc-tion in growth, suggesting that bionem had some phy-totoxic properties. A second study was a small-scalegreenhouse trial carried out in naturally RKN-infest-ed soil, to evaluate selected bionematicides for controlof RKN disease on the susceptible cherry tomato ‘Bar138-8’. At the end of the crop season, root galling causedby RKN was reduced by ~21% and ~9% with bionemand DiTera respectively, while Intercept (Burkholde-ria cepacia) applied alone had no effect on the gallingindex. In contrast, bionem+Intercept at lower dosesreduced the galling index by 26%, suggesting a syner-gistic effect between these two bio-nematicides. Nophytotoxic symptoms were observed. Plant yield in-creased with all bio-nematicide treatments, comparedto the control. The increase in yield may result fromthe reduction in RKN infestation, as well as fromchanges in the nutrient status of the soil, and the stim-ulation of beneficial microorganisms. The results of thisstudy showed a partial reduction in RKN galling se-verity and an increase in plant yield, indicating thatbio-nematicides have an important role in integratedcrop management.

Biological control of the weed Convolvulus arven-sis (L) with the fungus Erysiphe convolvuli de Can-dolle ex St. Amans in planta. V. SALTZIS1, A. TZAVELLA-KLONARI2 and P. LOLAS3. 1Technological Education Insti-tute of Epirus, Faculty of Agricultural Technology, De-partment of Plant Production, P.O.Box 110, 47100 Arta,Greece. 2Aristotle University of Thessaloniki, Faculty ofAgriculture, Laboratory of Plant Pathology, P. O. Box269, 54006 Thessaloniki, Greece. 3University of Thessaly,Department of Agriculture Plant Production and Agri-cultural Environment, Laboratory of Weed Science, Fy-toko, 38446 Volos, Greece.

In the spring and summer of 1999 and 2001, Convol-vulus arvensis plants were inoculated with the fungusErysiphe convolvuli, in order to determine its efficacyas a biological control agent of the weed. The inoculumwas obtained from field-grown C. arvensis naturallyinfected with the fungus. Seedlings of C. arvensis weregrown outdoors in pots. The first inoculation was car-


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ried out when the C. arvensis seedlings were at the 2–6 true-leaf stage. The above-ground parts of the plantswere sprayed with a conidial suspension of E. convol-vuli at a concentration of 106 spores ml-1 and the inoc-ulated plants were placed in a growth chamber with atemperature of 19–20oC, RH 91–92% and a 15-hour dayfor 25 days. After 5–6 days the white mildew appeared.A second inoculation was performed 7–8 days after thefirst, using conidial suspensions at the same concen-tration and covering the newly expanded foliage whilethe plants were kept in the same condition. The inocu-lated plants were totally covered with mildew 10–11days after the first inoculation and more than 75% ofthe leaves were dead after 18–20 days.

Production of the entomopathogenic biocontrolagent Metarhizium anisopliae in liquid culture:the effect of environmental parameters on sporeproduction and quality. I. YPSILOS and N. MAGAN.Applied Mycology Group, Biotechnology Centre, Cran-field University, Silsoe, Bedford, MK45 4DT, UK.

One of the major bottlenecks in the development of bio-control agents is the sensitivity of these agents tochanges in relative humidity and temperature, whichlimit their control potential. For optimal ecological fit-ness these limitations need to be addressed. In thisstudy production of Metarhizium anisopliae in liquidculture was examined in a range of environmental re-gimes and nutrient statuses. After the optimal physic-ochemical conditions for blastospore production hadbeen determined different water stress conditions wereimposed using ionic solutes (NaCl, KCl). With inter-mediate water stress (0.98 aw=-3.0 MPa water poten-tial) the production of blastospores significantly in-creased. Analysis of the endogenous reserves in blast-ospores showed that there were also greater amountsof glucose and erythritol. Greater water stress (0.96aw=-5.6 MPa) further increased in endogenous glucoseand erythritol levels, but blastospore production de-creased. Erythritol can act as a compatible solute re-ducing the intracellular water potential and enablingfaster germination at a wider range of environmentalrelative humidity. Subsequent viability assays simu-lating different water stresses showed that germina-tion of such characterized blastospore treatments wasfaster and germ tube extension was improved. Wash-ing the blastospores in water caused a significant lossof endogenous sugars and sugar alcohols with a con-comitant loss in germinability. However, if isotonic so-lutions were used for washing, the blastospores re-tained their endogenous reserves (sugars and sugaralcohols) and this may enable better germination andestablishment. These studies could have significantimplications for developing formulations with longerviability and shelf life.

NEW TECHNOLOGIES IN PLANT PROTECTION

Invited lecture

Systemic acquired resistance: frontline news andprospects for applications. J-P. MÉTRAUX. Départe-ment de Biologie, Université de Fribourg, 1700 Fribourg,Switzerland.

Plants protect themselves against pathogens by con-stitutive barriers and by a number of inducible defensemechanisms deployed after contact with a pathogen. Afirst infection with a fungal, bacterial or viral patho-gen often induces resistance towards subsequent in-fections. To a certain extent, plant immunization of thissort is analogous to immunization in animals and hu-mans. Induced resistance may be expressed locally atthe site of infection, or systemically, in uninfected partsof the plant. This latter phenomenon is termed sys-temic acquired resistance (SAR) to denote the capacityof the plants to acquire resistance after an initial in-fection even in plant parts remote from the first infec-tion. Many plants express this type of resistanceagainst a wide variety of pathogens, including patho-gens unrelated to the initial causing agent. SAR is ex-plained as being caused by the production of a signalreleased from an infected leaf and translocated to oth-er parts of the plant where it induces a defense reac-tion. Some non-pathogenic root-colonizing bacteria alsoinduce a SAR in the leaves. When the biochemical na-ture of the changes induced in infected plants was close-ly examined, it led to the discovery of a number of pro-teins termed pathogenesis-related proteins (PRs). Itwas also observed that a simple phenolic compound,salicylic acid (SA), induced PRs in tobacco and protect-ed the plant against TMV. Plants accumulate SA local-ly, at the site of infection, but also in the phloem sapand in uninfected systemic leaves. It was suggestedthat SA was an endogenous signal for SAR. This sug-gestion opened the way for molecular investigationsinto induced resistance. A further advance was markedwhen SAR was found to operate in the genetically trac-table system Arabidopsis thaliana.

Physiological changes associated with SAR

Generally, the success of an induced defense mecha-nism depends on the outcome of a race between theinvading pathogen and the reaction of the plant. Incompatible interactions, the virulent pathogen is of-ten recognized too late and the plant is infected. Inthe case of incompatible interactions, plants rapidlyrecognize the avirulent pathogen and resistance mech-anisms efficiently block the invader. A first infectionleads to many changes, some of which eventually leadto the formation of barriers that block the invadingpathogen. These barriers include modifications of the

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cell wall such as by the deposition of lignin or papil-lae at sites of attempted penetration. In addition, an-timicrobial metabolites or phytoalexins are produced.Phytoalexins have a broad unspecific activity and forma toxic barrier to invaders. The activation of geneti-cally determined programmed cell death, similar toapoptosis in animals, also prevents the spread of in-vaders. The synthesis of novel proteins after patho-gen attack is perhaps the most fully documented re-action. These host-encoded, pathogenesis-related pro-teins are induced locally and systemically after path-ogen infection. They have been found in most plantswhere they have been looked for and have various bi-ochemical activities. Some PRs were enzymes such asβ-1, 3-glucanases, chitinases or proteinases capableof hydrolyzing the cell wall of invading fungal patho-gens, while the function of others, for example PR-1,is still unclear. Combinations of PRs (for example glu-canase and chitinase) tend to be more efficient anddifferent types of PRs target different pathogens. Withthe advent of genome-wide analysis of gene expres-sion using gene chips in the model plant Arabidopsisthaliana, it has become apparent that the expressionof many genes is modified by pathogen infection. Thusthe family of PRs is likely to be larger than hithertothought. The question now is to understand how thesegenes function and are regulated.The sequence of reactions taking place in a leaf at-tacked for the first time by a pathogen has been ex-tensively studied using various mutants of Arabidop-sis. After initial recognition of the pathogen by theplant, a cascade of early events is induced that in-clude ion fluxes, phosphorylation events, and the gen-eration of nitric oxide and active oxygen species. SAacts as a secondary signal molecule and is requiredfor increased expression of resistance and variousdefense-related proteins such as the PRs. Dependingon the inducing microorganism, the signal transduc-tion pathway takes a different course depending onthe nature of the initial interaction (virulent versusavirulent pathogen, rhizobacteria). Resistance to agiven pathogen may be activated via different signaltransduction pathways. For example, the infectionwith leaf pathogens that induce resistance to P. sy-ringae depends on a pathway involving SA, whilerhizobacteria-induced SAR act via the plant hormoneethylene and jasmonic acid. The complexity of thesesignaling pathways is further illustrated by the oc-currence of crosstalk or interferences between path-ways. For instance, both the induction of PR-1 andthe resistance to P. syringae are strongly dependenton the light signal transduction pathway.Given the central role of SA in pathogen-induced sig-naling for induced resistance, studies have been fo-cused on the regulation of its production and on itsmolecular mode of action. In tobacco, cucumber and

potato, evidence indicates that SA is produced fromphenylalanine via coumaric and benzoic acid. In Ara-bidopsis a different pathway operates and SA is madefrom chorismic acid via an isochorismate synthase.More work is needed to understand the regulation ofSA biosynthesis and its localization after pathogenattack, both locally and systemically.The mode of action of SA was investigated by search-ing for SA-binding proteins. SA was found to bind toH2O2-scavenging enzymes such as catalase and ascor-bate peroxidase, and this inactivation it was suggest-ed could lead to the formation of a radical involved inlipid peroxidation. Lipid peroxidation products canactivate defense gene expression providing a link be-tween SA and defense. It remains to be shown thatsuch phenolic radicals form sufficient lipid peroxidesin the right time frame for the defense response totake place. More work is needed to complete our un-derstanding of how SA acts at the molecular level.The responses induced by SA include the transcrip-tional activation of genes. This is controlled by pro-tein phosphorylation events that eventually lead tothe regulation of proteins involved in the control ofgene expression.The importance of SA for SAR was shown by variouscorrelative studies, but most compellingly by trans-genic plants overexpressing a bacterial salicylate hy-droxylase gene (the NahG gene). When it is expressedin the plant, this enzyme degrades SA. Transgenicplants carrying the NahG gene do not display a SAR.Similarly, mutants of Arabidopsis with an impairedcapacity to synthesise SA are also less able to cause aSAR. A variety of experiments suggest that SA has arole as a systemic signal but do they not exclude thepossibility that another putative systemic signal be-sides SA might be involved in systemic signaling dur-ing a SAR.The systemic responses could be clearly separatedfrom the reactions taking place in the infected partsof the plant, and it was clear that the systemic signaltriggered defense-related reactions before contact withthe challenging pathogen. In contrast, other reactionssuch as changes in cell wall lignification were onlydetected after challenge infection of the upper leaf butwith faster induction kinetics: the systemic signalconditioned the tissue to respond faster. Future ex-periments should investigate more closely how thesystemic signal conditions the induced leaves.

Prospects

Substantial progress has been achieved in the study ofSAR in the last few years. An increasing number of newcomponents in the signal transduction pathway for in-duced resistance responses has been discovered andtheir number will undoubtedly increase still further withthe advent of large-scale investigations of gene expres-


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sion. Fascinating questions about SAR include thoseconcerning its systemic signal, its regulation and itsmode of action. SA has been implicated in this processinitially, and its role as a key signal in pathogen-inducedSAR is well documented. Its function as a translocatedsystemic signal remains a matter of debate. In the nearfuture, more will be learned about the regulation andlocalisation of SA synthesis and its mode of action. Thesignal transduction involved in the regulation of the SARresponse turns out to be far from a linear chain of eventsand can more correctly be described as a network. Itappears that several pathways interact in part, leadingto sets of responses targeted to specific pathogens.Compounds such as SA are interesting model structuresthat can be used to develop non-antibiotic crop protect-ants, which trigger the natural potential for resistancein various plants. Such compounds can be compared toimmunostimulants by analogy to certain drugs used inhumans. A good example is BION®

, which has recentlybeen released on the market. Further study is requiredon the effect of various environmental parameters onthe plant response to pathogens. An example is the roleplayed by light in SA-dependent defense responses. Thistype of knowledge will be of great importance in improv-ing the transfer of SAR to field conditions.

Acknowledgements

Work in my laboratory is financially supported by theSwiss National Science Foundation (grant 31-055662.98).

Further readingGenoud T., and J.P. Metraux, 1999. Crosstalk in plant cell

signaling: structure and function of the genetic network.Trends in Plant Science 4, 503–507.

Glazebrook J., 1999. Genes controlling expression of de-fense responses in Arabidopsis. Current Opinions inPlant Science 4, 280–286.

Grant M., and J. Mansfield, 1999. Early events in host-pathogen interactions. Current Opinions in Plant Biolo-gy 2, 312–319

Hammerschmidt R., 1999. Phytoalexins: what have welearned after 60 years? Annual Review of Phytopatholo-gy 37, 285–306.

Jakab G., V. Cottier, V. Toquin, G. Rigoli, L. Zimmerli, J.P.Metraux. and B. Mauch-Mani, 2001. β-Aminobutyricacid-induced resistance in plants. European Journal ofPlant Pathology 107, 29–38.

Pieterse C.M.J and L.C. van Loon, 1999. Salicylic acid-in-dependent plant defence pathways. Trends in Plant Sci-ence 4, 52–58.

Metraux J.P., 2002. Recent breakthroughs in the study ofsalicylic acid biosynthesis. Trends in Plant Science 7,332–334.

Metraux J.P., 2001. Systemic acquired resistance and sali-cylic acid: current state of knowledge. European Jour-nal of Plant Pathology 107, 13–18.

Sticher L., B. Mauch-Mani, and J.P. Métraux, 1997. Sys-temic acquired resistance. Annual Review in Plant Pa-thology 35, 235–270.


Molecular investigation of the defense pathwayof a rhizobacterium Paenibacillus sp. antagonis-tic to a virulent fungus and a bacterium. A.S. VEN-IERAKI1,2, E.C. TJAMOS1 and P. KATINAKIS2. 1AgriculturalUniversity of Athens, Laboratory of Plant Pathology, 75Iera Odos str., 11855 Athens, Greece. 2Agricultural Uni-versity of Athens, Laboratory of Molecular Biology, 75Iera Odos str., 11855 Athens, Greece.

The capacity of the antagonistic rhizobacterium Paeni-bacillus sp. (strain K-165) to induce systemic resist-ance against the wilt pathogen Fusarium oxysporum f.sp. raphani (For) and the foliar pathogen Pseudomonassyringae pv. tomato DC3000 (Pst) was investigated. Themodel plants tested were the Arabidopsis ecotype Col-0 (wild-type), the mutants etr-1 (ethylene response mu-tant), npr-1 (non-expressed pathogenesis related pro-teins, PRs), jar-1 (jasmonate response mutant), and thetransgenic NahG (that cannot accumulate salicylicacid). All these plants were individually sown accord-ing to a system ensuring spatial separation of the in-ducing agent and the challenging root pathogen. Symp-tom development in the K-165 treated plants was re-duced by up to 50% compared with the control (untreat-ed Col-0 plants), 30 days after inoculation. Similar re-ductions were observed with the etr-1 and the jar1mutants and the NahG transgenic plants. With thenpr1 ArabidoPs mutant on the other hand there wereno significant differences between plants treated withK-165 and untreated plants. The absence of resistancein the npr1 mutants showed that the resistance causedby K-165 depends on NPR1 regulator protein. The sec-ond part of the experiment investigated the capacityof K-165 to induce resistance to Pst by studying thegene expression of the ISR-involved genes PR-1, PR-2,PR-5, Hel, Atvsp, Pdf1.2 and Tub, which were ampli-fied with PCR, subcloned and sequenced. Leaf tissueswere harvested on different days after inoculation withPst, for RNA analysis. Gene expression was analyzedby RT-PCR. The RT-PCR results showed that thesegenes were potentiated, leading to enhanced expres-sion after challenge inoculation with Pst, and that PRgenes had a vital role in the development of inducedresistance. It is therefore concluded that pathogen-in-duced systemic resistance is associated with a potenti-ation of PR genes. The results with both host-patho-gen systems studied showed that the pathogenesis-re-lated proteins caused the induced resistance of Paeni-bacillus sp. strain K-165 to Fusarium oxysporum f. sp.raphani and Pseudomonas syringae pv. tomato.

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Detection of βββββ-avr genes in Pseudomonas savasta-noi pv. phaseolicola. D.S. TSALTAS, G. TSIAMIS and J.W.MANSFIELD. Molecular Plant Pathology Laboratory, Im-perial College at Wye, University of London, Ashford,Kent, TN25 5AH, UK.

A 154-kb plasmid was cured from race 7 strain 1449Bof the phytopathogen Pseudomonas savastanoi pv. pha-seolicola (Pph). The cured strain, named RW60, lostvirulence towards bean, causing a hypersensitive re-action (HR) in the previously susceptible cultivars Ca-nadian Wonder and Tendergreen. Removal of the 154-kb plasmid revealed the presence of a second categoryof avr genes. Jackson et.al. (1999, PNAS 96, 10875–10880) suggested that the function of virulence factorsis to suppress the HR induced by this new category ofavr genes, which they called β-avr genes. Our aim wasto identify such β-avr genes. We made use of transpo-son mutagenesis with a mini-Tn5 construct. Mutantswere infiltrated into leaves of the cv. Tendergreen (108

cfu ml-1) and any changes from the RW60 HR pheno-type were recorded. Of a total of 987 putative mutants,15 non-auxotrophic strains generated altered pheno-types; nine of these caused a weaker HR and six a nullresponse. The six null mutants also caused null reac-tions on the leaves of the cv. Canadian Wonder andRed Mexican, but they caused a variable HR on cv. A43.The genes disrupted by the Tn5 insertions were char-acterized. Sequencing revealed that one mutation wasin hrpZ while the other disrupted genes were similarto fabB (β-ketoacyl-ACP synthase), to the GTP-bind-ing protein in thiophene and furan oxidation (x2), totrpB (tryptophane synthase), and to phosphoserinephosphatase. None of these genes showed any charac-teristics of known avr genes and they most likely con-tributed to basic physiological mechanisms. Recent ex-perimental data on the role of the β-avr genes will bediscussed.

Quantification of Verticillium dahliae microscle-rotia in the soil using competitive PCR. E.J. PA-PLOMATAS, G.D. DIMOU and A. TZIMA. Agricultural Uni-versity of Athens, Laboratory of Plant Pathology, 75 IeraOdos str., 11855 Athens, Greece.

Knowing the number of Verticillium dahliae microscle-rotia in the soil is very important to be able to predictthe amount of disease and to decide on control meas-ures. The most popular methods that have been devisedfor this purpose are wet sieving and dry plating of thesoil on semi-selective culture media. Comparative stud-ies have concluded in favor of either one or other of thesemethods, indicating methodological flaws such as thefailure to distinguish between colonies of V. dahliae andof the saprophytic or non-pathogenic V. tricorpus, thefailure of all microsclerotia to germinate equally on amedium, and the bias of individual researchers in the

application of the method. As a result, the estimates ofV. dahliae density in the soil published so far are unsat-isfactory. The aim of the present study was to develop amolecular method of V. dahliae quantification in the soilthat would overcome the weaknesses of the classicalmethods. The method chosen was competitive PCR withthe ribosomal RNA gene of V. dahliae as a target. Anadvantage of this method was that the rDNA gene ispresent in a high copy number in the cell, somethingthat would increase the sensitivity of the method. Aftera search in an electronic database, primers amplifyinga 347 bp fragment of V. dahliae rDNA were designed.Since competitive PCR requires the simultaneous am-plification of an internal standard (a competitor frag-ment) with the target DNA, a “linker primer” withinthe target sequences was also designed. In this way, aninternal standard fragment of 257 bp (90 bp smaller thanthe target DNA) could be co-amplified. The method de-veloped was evaluated on ten genomic DNA samplesfrom an increasing number of V. dahliae microsclerotiain the soil (from 10 to 100). It was found that competi-tive PCR showed quantitative differences among thesamples that were not detectable with simple PCR. Ap-plication of this method to V. dahliae DNA isolated fromthe soil will be discussed.

Molecular study of the resistance of Botrytis cine-rea to new botrycides. A. TZIMA1, E.J. PAPLOMATAS1 andV.N. ZIOGAS2. Agricultural University of Athens, Labora-tories of 1Plant Pathology and 2Pesticide Science, 75 IeraOdos str., 11855 Athens, Greece.

The RAPD technique was employed to study the genet-ic diversity and relatedness among nine isolates of Bo-trytis cinerea that differed in their sensitivity to the fun-gicides cyprodinil, fludioxonil and fenhexamid. The den-drogram that resulted after phylogenetic analysis of thedata showed that resistance to these fungicides was as-sociated with genetic changes that could be detected bythis technique, and that specifically enabled the mutat-ed resistant strains to be distinguished from the parentwild-strain. In order to develop molecular markers thatdifferentiated B. cinerea strains resistant to the fungi-cides tested, four RAPD-PCR DNA fragments producedfrom strains representing each of the fungicide groupswere cloned. The clones obtained will be evaluated ei-ther as DNA probes or to design specific DNA primers.Finally, the cystathionine-β-lyase gene from both B. ci-nerea strains that were sensitive to cypronidil andstrains that were resistant to it was cloned. This en-zyme is considered a putative target site of anilinopyrmi-nidines. Comparison of gene sequences of strains sensi-tive and resistant to cyprodinil could give further in-sight into the mode of action of anilinopyrimidines andthe mechanism of resistance of B. cinerea to this groupof fungicides.


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Agrobacterium tumefaciens–mediated transforma-tion of Verticillium dahliae with the enhancedgreen fluorescent protein (EGFP) gene. E.J. PAPLO-MATAS1, E.C. TJAMOS1, D.F. ANTONOPOULOS1, P. ANTONIOU1

and S. KANG2. 1Agricultural University of Athens, De-partment of Plant Pathology, 75 Iera Odos str., 11855,Athens, Greece. 2The Pennsylvania State University,Department of Plant Pathology, University Park, PA16802, USA.

Agrobacterium tumefaciens–mediated transformationhas long been used to transfer genes to plants and hasrecently also served to transform fungi. As regards fun-gi, A. tumefaciens can transform protoplasts, spores,hyphae and blocks of mushroom mycelial tissue. Thepurpose of this study was to transform the soilborneplant pathogenic fungus Verticillium dahliae with theenhanced green fluorescent protein (EGFP) gene. EGFPwill be used as a cytological marker in future studies ofV. dahliae pathogenesis and the penetration and rami-fication of the fungus in the plant tissue. The EGFP-labeled pathogen will also be used to study the interac-tion between the fungus and several potential biocon-trol agents. For this purpose, binary vectors for fungaltransformation have been constructed. The vectors har-bor the bacterial hygromycin B phosphotransferase gene(hph) under the control of the Aspergillus nidulans trpCpromoter as a selectable marker and the EGFP or EYFP(enhanced yellow fluorescent protein) gene under thecontrol of the Cohliobulus heterostrophus GAPD promot-er. Vectors were inserted into strain AGL–1 of A. tume-faciens. Fungal transformation was carried out by co-cultivating A. tumefaciens strain AGL-1 carrying anappropriate vector with V. dahliae conidia at a concen-tration of 106 ml-1. An attempt was made to transformisolates of V. dahliae from various hosts, including to-mato (race 1 and 2), cucumber, cotton and eggplant.Transformed fungal cells expressing the green or yel-low fluorescent protein were visualized under a micro-scope with UV light at 450–490 nm. Fluorescence wasobserved in the conidia, mycelia and microsclerotia ofV. dahliae. When a GFP-transformed V. dahliae isolatewas used to inoculate eggplants, it was found to be equal-ly pathogenic as the wild-type strain. Moreover, underfluorescence microscopy the vascular bundles of smalleggplant roots were observed to emit green fluorescentlight.

Expert systems in plant protection. B.N. ZIOGAS1 andA.E. KALAMARAKI2. 1Laboratory of Pesticide Science, Ag-ricultural University of Athens, 75 Iera Odos str., 18855Athens, Greece. 2Benaki Phytopathological Institute,Department of Pesticides Control and Phytopharmacy,8 St. Delta str., 14561 Kifissia, Athens, Greece.

Expert systems are artificial intelligence applicationsthat emulate the logic and problem-solving proficiency

of a human expert. They are most successful in address-ing problems in which the knowledge and the experi-ence of a human expert is required. The prediction, di-agnosis, control and classification of plant phytopara-sites are suitable areas for developing an expert sys-tem. The basic components of an expert system are: theuser interface, the knowledge base, the database andthe inference mechanism. The development of an expertsystem proceeds through several stages, including prob-lem selection, knowledge acquisition, knowledge repre-sentation, programming, testing, evaluation and releaseinto agricultural production. The power of an expertsystem depends on the knowledge of the experts. Theknowledge that the expert uses to solve a problem iswritten in dependency networks, which are coded intothe computer. Expert systems can offer solutions withdiffering certainty values even when an incomplete setof data is given. The conclusion process, which is usedby the system to resolve a problem, is recorded and pro-vides the user with an explanation for how the conclu-sion was reached.

An empirical model for prognosis of loss assess-ments in eggplant crops (Solanum melongena L.).C.C. THANASSOULOPOULOS1, F.A. BLETSOS2 and A.M. MOUS-TAFA1. 1Aristotelian University of Thessaloniki, Facultyof Agriculture, Plant Pathology Lab., 54006 Thessalo-niki, Greece. 2National Agricultural Research Founda-tion (N.AG.RE.F), Agricultural Research Center of Mac-edonia and Thrace, 57006 Thermi, Thessaloniki,Greece.

A model of Verticillium wilt prognosis in the early stag-es of eggplant crops has been made based on the resultsof a three-year experiment. Eggplants were planted insoil infested with Verticillium inoculum, and in soil fu-migated with methyl bromide used as control, for threeconsecutive years. Disease incidence was determined atthe end of the first month after transplanting and thetotal yield at the end of the cultivating period was cor-related. The model resulted from the integration of theareas line below the regression curve and the ordinatesaxis on axis X from X0 to X5 which are the limits of %yield losses. According to these computations the finalproposed model is:

f(y) dy =A= (0.5/n (0�n0 + 2�n1+ 4�n2 + 6�n3 + 8�n4

+5�n5)

where n is the total number of counted plants, n = n0 +n1 + n2 + n3 + n4 + n5, and the area A has limits 0 to 5.The algorithm which is the results of the model is inter-polated in the data of yield losses of each rank of thesymptom index found in the experimental work, and theresulting number is the final prognosis for future loss-es. The model was verified with several results fromother experiments, and was highly credible.

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61Vol. 45, No. 1 April, 2006


The sequence of phases during the feeding cycleof the plant-parasitic nematode Paratrichodorusanemones, and its significance for successful to-bravirus transmission. E. KARANASTASI. Departmentof Entomology and Plant Zoology, Laboratory of Nema-tology, Benaki Phytopathological Institute, 8 St. Deltastr., 14561 Kifissia, Athens, Greece.

The plant-parasitic, soil-inhabiting nematode Paratri-chodorus anemones is one of the most efficient vectorsof Tobacco rattle virus (TRV). Nevertheless, its efficien-cy as a TRV vector has always been perplexing, since ithas been shown that the majority of root cells becomeirreversibly disorganised when attacked by these nem-atodes, and since all viruses require living tissues toestablish an infection. Cells attacked by P. anemonesshould therefore be no longer suitable for tobravirusestablishment. To investigate this contradiction, livingP. anemones specimens feeding on tobacco seedlings wereexamined in real-time using video-enhanced interfer-ence light microscopy. It was concluded that the feedingcycle of this nematode may be divided in four distinctphases: (i) root exploration, (ii) cell exploration, (iii) cellsampling, and (iv) cell feeding, followed by a period ofquiescence. Before commencement of phase (iv), an av-erage of four cells were perforated, but then immediate-ly rejected, so that these cells remained fully function-al. Additionally, during phase (iv) 5% of the attackedcells remained alive, providing conditions that are suit-able for virus infection. In the study, it was also observedthat each feed on a single cell is divided into four phas-es in a way similar to what has been reported for Tri-chodorus similis, i.e. (i) cell wall perforation, (ii) saliva-tion, (iii) ingestion and (iv) withdrawal.

Mapping the biogeographical distribution and de-velopment of electronic databases for the macro-fungi of Greece. G. ZERVAKIS1, D. DIMOU2 and E. PO-LEMIS1,2. 1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute of Kalamata, 85Lakonikis str., 24100 Kalamata, Greece. 2AgriculturalUniversity of Athens, Laboratory of General and Agricul-tural Microbiology, 75 Iera Odos str., 11855 Athens, Greece.

In recent years the detailed study of the diversity of mac-rofungi in Greece has increased the number of recordedspecies to ca. 1300; even so, however, this seems to beless than 25% of the existing number of species. Dataon the geographical distribution of almost all mushroomgroups are still limited, and there is practically no in-formation to permit a chronological assessment of theirabundance in specific regions. The mapping of the fun-gal diversity is performed by dividing the map of Greeceinto square grids each with an area of 100 square kil-ometers; these grids are based on a scale modificationof the Chorological Grid Reference System (CGRS); ini-tially only a limited number of selected species are

mapped in them. The establishment of an organizedmonitoring network of macromycetes together and theevaluation of the data collected enables their frequencyof occurrence to be estimated, risk assessments to bemade, red-data lists to be compiled and measures forthe protection of biodiversity to be taken. In parallel,all the information collected is inserted in an electronicdatabase (Claris, FilemakerPro software) which includesfields on the taxonomic arrangement of the fungus, thelocation of its occurrence, the nature of the host andsubstrate, photographs, encoding for the exsiccata keptin herbaria, ecomorphological data, etc. Of great impor-tance is the ex-situ conservation of fungal genetic re-sources and their evaluation for purposes of environ-mental biotechnology and organic farming programs.

Control of sporulation and germinability of Bot-rytis cinerea Pers. in tomato greenhouses coveredwith the plastic film TUV 3935. V.A. BOURBOS1, E.A.BARBOPOULOU1 and M.V. KYKRILIS2. 1National Agricultur-al Research Foundation (N.AG.RE.F), Institute of OliveTree and Subtropical Plants of Chania, Laboratory ofPlant Pathology, Agrokipio, 73100 Chania, Greece. 2Plas-tika Kritis S.A., P.O. Box 1093, 71110 Heraklion, Greece.

Botrytis cinerea may cause serious losses of tomato grownin unheated greenhouses. The aim of this work was tostudy the effect of TUV 3935, a new plastic film for roofcovering with a special UV permeability, on the sporula-tion and viability of Botrytis cinerea in comparison withcommon PE plastic film. Trials were carried out in 0.2-hectare greenhouses in Crete, during two consecutive cul-tivation periods. Botrytis spores in the greenhouse werecollected using Petri dishes containing maltose nutrientmedium. After incubation for 16 hours at 23oC, the totalnumber of spores, and spores having a germination tube,were counted. There were 10 spore collections at 15 daysintervals. The number of infected fruits per plant werealso counted before each spore collection. The number ofBotrytis spores was lower in greenhouses covered withTUV 3935 plastic film (10.3–14.4 per Petri dish, vs. 20.1-26.2 in greenhouses covered with PE plastic film) andthe germination of spores was also markedly reduced(12.8–14.7%, vs. 89.9–97.8%). The percentage of infectedfruit per plant under TUV 3935 ranged from 13.4 to 16.5%and was statistically lower than that under PE film, whichwas from 23.8 to 27.0%.

Phytoalexin accumulation during hypersensitivereaction in Phaseolus vulgaris challenged by Pseu-domonas savastanoi pv. phaseolicola. D.S. TSALTAS,M. BENNET and J.W. MANSFIELD. University of London,Imperial College, Department of Agricultural Sciences,Wye, Ashford, Kent UK.

The resistant reaction of French bean to strains of Pseu-domonas savastanoi pv. phaseolicola (Psph) is visibly


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different in terms of the timing of the plant cell collapse,and the browning of the tissue during the hypersensi-tive reaction (HR). We developed HPLC-based methodsto quantify the isoflavonoid phytoalexins (PAs) phase-ollin, phaseollinisoflavan and phaseollidin in pod tis-sue. Analysis of PA accumulation provided a biochemi-cal phenotype of the reactions. Changes occurring in PAswere used to characterise reactions to race 7 of Psphand the plasmid-cured variant RW60. In the cultivarRed Mexican the slow development of the HR in responseto RW60 was associated with very high levels of each ofthe isoflavonoids. The PAs were only detected in cellsundergoing the HR, not in the surrounding green tis-sue. The HR elicited by RW60 differed from that acti-vated during gene-for-gene interactions, indicating thatit may not be induced by proteins commonly recognisedas the product of avr genes.

DISEASES OF FRUIT TREES AND GRAPEVINE

Invited lecture

The future of plant quarantine in Europe. I.MSMITH. European and Mediterranean Plant ProtectionOrganization, 1 Rue Le Nôtre, 75016 Paris, France.

The European plant quarantine system, which is enshrinedin the relevant EU Directives and in the current regula-tions of the EU Member States arose as a synthesis of thephytosanitary regulations of individual European coun-tries. Its main purpose remains to exclude from the wholeEU region a long list of what EPPO calls ‘A1 quarantinepests’, and to ensure that the European countries take acommon approach in both their regulatory systems andtheir operational work. These measures taken in relationto so-called third countries have had considerable success.However, the EU system also has to operate in its ownsingle market, so that member states can no longer pro-tect themselves in the same way as before from quaran-tine pests already present in the EU. In particular, it is nolonger possible to take control measures at the bordersbetween member states. Instead, the emphasis now is onproducing healthy plant material, certified by ‘plant pass-ports’, in all parts of the EU. Material moving into certainareas, known as ‘protected zones’, may be made subject tomore stringent requirements before a plant passport canbe issued. This internal EU system has the advantage ofimproving the phytosanitary quality of planting materialwithin the EU, whether traded within or between themember states, and ensuring that nurseries, which haveto be duly registered, work to high standards. Neverthe-less, it also increases the volume of material moving be-tween member states and thereby increases the possibili-ty for certain quarantine pests to spread that were con-tained by the old system. However, the problems which

have arisen since the new system was introduced havemainly concerned ‘EU’ pests that are new to certain partsof Europe (Liriomyza huidobrensis, Bemisia tabaci, toma-to yellow leaf curl, Ralstonia solanacearum, pepino mosa-ic), and that therefore require new internal measures anddifficult negotiations between the member states. These‘domestic’ disputes tend to obscure other important aspectsof plant quarantine.The future of the European system will depend on thesuccess with which such internal measures are handledin future. The problems that arise are illustrated byErwinia amylovora (fireblight) a very dangerous pestthat is subject to the protected zone system, and by var-ious citrus pests entering Europe from outside, or mov-ing within Europe. The EU system will also in futurehave to justify itself in relation to international agree-ments under the World Trade Organization and the In-ternational Plant Protection Convention. In general, theEuropean countries can claim that their phytosanitarymeasures are technically justified and non-discrimina-tory to other countries. However, in a world of increas-ingly frequent trade disputes, countries have to be readyto justify and defend themselves, and this includes per-forming Pest Risk Analyses according to internationalstandards. National Organizations are accordingly facedwith an increasing demand for information on pests, onthe losses caused by them, and on the effectiveness ofcontrol measures. The fact that at least 10 other Euro-pean countries are likely to become members of the EUin the next few years also means that they have to ac-cept the EU approach (which thus becomes virtually thestandard for Europe, whether wider organizations likethe EPPO like it or not), and that the EU system has toconsider the concerns of these new members. At presentthe main concern on the part of the EU is that the newmember states should be able to take key phytosani-tary measures so as to control high-profile, recently ar-rived pests. However, the success of the system will alsobe judged by the degree to which it ensures the futurephytosanitary security of the new members.Finally, it should be pointed out that plant quarantineservices in Europe are increasingly concerned with newbiological problems that are not a traditional plant quar-antine but which now have a high political profile: theseproblems concern genetically modified organisms, inva-sive species, and organisms that are harmful to biodi-versity. What used to be simply the exclusion of plantpests at the border thus becomes a more general policyto regulate the global movement of organisms, in whichplant quarantine services are involved because they havethe structure in place to certify and inspect plant mate-rial moving in trade, which is one of the principal path-ways by which micro-organisms are spread. The pur-poses of plant quarantine used to be to protect agricul-ture, but now it also has to protect the environment,and the human consumer.

63Vol. 45, No. 1 April, 2006



Trifloxystrobin ® – the new dimension in plant dis-ease control. A. WITZENBERGER. BCS – PM – Fungicides.

Trifloxystrobin is a second-generation strobilurin fun-gicide of the chemical class of oximino-acetates and of-fers a very broad and balanced spectrum of plant dis-ease control. It is very effective against ascomycetes,basidiomycetes, deuteromycetes and oomycetes in alarge number of temperate, tropical and subtropicalcrops. Trifloxystrobin inhibits mitochondrial respiration.In general it has a strong fungicidal effect on spore ger-mination and other early stages of fungal development;only in a few specific cases (e.g. Venturia spp.) does itinhibit later stages of the pathogen. Consequently tri-floxystrobin can be viewed as a foliar fungicide for pre-ventive use. Trifloxystrobin exhibits a unique patternof distribution and re-distribution on and in plants forwhich the term ‘mesostemic’ has been proposed. In shortthat means that: it has a high affinity to plant surfacesgiving it excellent persistence and rainfastness; smallamounts of it penetrate the plant tissues and displaytranslaminar and curative activity against specific path-ogens; its re-distribution by superficial vapour activityand by surface water ensures significant protection ofuntreated tissue; and its lack of transport in the vascu-lar system of the plant prevents the dilution of its activ-ity. Trifloxystrobin has a very favourable toxicologicaland ecotoxicological profile and is safe to operators, con-sumers and the environment. Consequently it has beengiven reduced-risk status in the USA, allowing fast-trackregistration and label extensions. Tailor-made formula-tions have been prepared to meet specific requirements.These include various solo-products and co-formulationswith active ingredients from other chemical classes.Global registration of trifloxystrobin is expected for 2003.

Tristeza in Greece: the progress of eradication. P.E.KYRIAKOPOULOU. Agricultural University of Athens, Lab-oratory of Plant Pathology, 75 Iera Odos str., 11855 Ath-ens, Greece.

With the further expansion of the EU in 1994, and theconsequent abolition of phytosanitary regulations be-tween EU countries, the danger that the destructiveCitrus tristeza virus (CTV) would be introduced intoGreece appeared immediate. As a result, in 1995 theMinistry of Agriculture, in collaboration with the cit-rus-growing districts and prefectures, started indexingcitrus for a possible occurrence of the virus in Greece.As part of this effort, it was discovered in 2000 that in1994 Lane Late orange seddlings had been illegally in-troduced from Spain into the Argolis and Chanea pre-fectures, three of these seedlings were CTV-infected,while their scions had been used for topworking someorchard trees to provide some variety. Since then, an

anti-tristeza campain was started in Greece, to elimi-nate the virus from the country. In Argolis, the battle issystematic and intensive and seems to be successful.Extensive surveys for new cases are being pursued ag-gressively, and any trees (there have been very few sofar) found to be infected are immediately destroyed.Unfortunately, however, some CTV-infected citrus prop-agation material from Spain and Italy have managed toevade the controls and entered the country. One case, inwhich such material was introduced illegally throughthe port of Patras is especially alarming. Attentionshould therefore be given by the port customs authori-ties to find and destroy any such attempted introduc-tions. Nevertheless, in new citrus orchards recently es-tablished in Argolis with introduced ‘certified’ citrusseedlings from abroad, some trees were found to be CTV-infected. Unfortunately ‘Certified’ in this context doesnot mean ‘absolutely virus-free’, but only: ‘with very lim-ited infection’. However, this grade of less-than-abso-lute certification is not satisfactory for Greece, which isat the moment basically CTV-free. There is therefore astrong need for EU legislation to be changed so as toprohibit the introduction of any citrus propagation ma-terial from other EU countries into Greece. Citrus grow-ers should scrupulously avoid buying any citrus propa-gation material imported from outside Greece, whetherlegally or illegally. Fortunately, the rest of the Pelopon-nese, apart from Argolis, has not shown any cases ofCTV. In Crete, however, the situation is very different.A significant number of trees were topworked with in-fected scions from the two original Lane Late trees im-ported into the Chanea prefecture in 1994, and some ofthe growers are objecting to their infected orchards be-ing destroyed. In Crete, besides the Chanea prefecture,the virus has been detected in isolated cases in theRethymnon prefecture and in one orchard of the Hera-clion prefecture. Special phytosanitary measures forCrete to counter the threat should be taken immediate-ly. The Ministry of Agriculture should also proceed withthe certification of citrus propagation material.

Tristeza in Argolis and problems arising from theintroduction of citrus propagation material. D.DIMOU1, I. DROSSOPOULOU2, E. MOSCHOS2, K. SPANOU1 andP. DERMATAS1. 1Argolis Direction of Agricultural Devel-opment, Department of Plant Protection, 21100 Nauplion,Greece. 2Aspropyrgos Control Station of Vegetative Prop-agation Material, 19300 Aspropyrgos, Greece.

Tristeza, the most destructive disease of citrus, was de-tected for the first time in Greece (Argolis, June 2000),in young orange trees, variety Lane Late, of Standardquality (C.A.C.), illegally imported from Spain in 1994.Of those trees, (originally 50 in number), 18 survived, 7of which were infected with Citrus tristeza virus (CTV).Those trees served as inoculum sources, both as mother


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trees for obtaining budsticks, and through aphid vec-tors, for orchards of the Argolic plain (Argos, Dala-manara, Argoliko, Aghia Triada, Aghios Adrianos, Iria,Kallithea Drepanou). All trees of the initial introduc-tion, as well as trees that subsequently became infectedthrough aphids in the field (18) or through grafting, wereuprooted when they were identified (74 in all). The sur-veys were carried out during the spring and autumnand the specimens collected were tested with DAS-ELI-SA and immunoprinting. During the last surveys (May–June 2002), eight additional trees were found to be CTV-positive in an orchard in Kallithea Drepanou, as well asseven more in a neighbouring orchard; these are addi-tional data providing evidence of aphid transmission.Unfortunately, problems caused by the introduction ofcitrus propagation trees from other EU countries contin-ue. In the spring of 2001, 1126 duly certified trees of anew clementine clone (Clemenpons) were introduced fromSpain and established, but despite certification seven ofthese trees turned out to be CTV-infected. In April of 2002,there was an attempt to introduce some trees illegallyfrom Italy (Sicily). These trees were of C.A.C. quality, buttheir accompanying certification label was blue insteadof yellow, which was misleading, since blue labels are for‘certified’ stock material. These data make clear that CTV-testing of all citrus propagation material introduced fromEU countries is imperative. Citrus certification withinGreece is also of course vital.

Evaluating the resistance of young olive cultivarsand rootstock suckers cv. Amfissis and LianoliaKerkyras to Verticillium dahliae. P.P. ANTONIOU1, S.E.TJAMOS, E. MARKAKIS, E.J. PAPLOMATAS and E.C. TJAMOS.Agricultural University of Athens, Laboratory of PlantPathology, 75 Iera Odos str., 11855 Athens, Greece.

While investigating rapid methods of evaluating the re-sistance of olive varieties to Verticillium wilt, differenc-es were found between two-year-old trees of the mostsusceptible cv. Amfissis and the tolerant cv. Kalamon,after the branches were inoculated with a V. dahliaeconidial suspension (100 µl of 108 spores ml-1). Applica-tion of this method in suckers of established orchards ofthe cv. Amfissis in Fthiotis county demonstrated theoccurrence of Verticillium-resistant olive rootstocks (4trees out of 65 inoculated). Since several trees of thecorresponding rootstock suckers were infected with thepathogen while the rootstocks remained healthy, a de-tailed study was conducted by injecting a conidial sus-pension of the pathogen into two-year-old trees of theVerticillium-susceptible cv. Amfissis and the tolerant cv.Kalamon to elucidate the observed phenomenon. Culti-vars were inspected at 10–90 day intervals. The distri-bution of the pathogen several cm above and below thesite of infection was determined. The pathogen was iso-lated more often and farther from the injection site in

the susceptible than in the tolerant cultivar. Differencesin symptom development between the cultivars may bedue not so much to the difficulty in conidial movement inthe vessels of Kalamon variety as to variations in thecontainment of the pathogen in that variety. A downwardmovement could be due to the negative pressure observedin the suckers and this would explain why the pathogenascends further up in those cv. Amfissis trees that devel-oped Verticillium wilt. Similar resistance evaluation ex-periments were also carried out on olive rootstock suck-ers on established olive groves of the Verticillium-toler-ant variety Lianolia Kerkyras in Preveza county. Of 15suckers that were injected twice over a period of two years,one remained symptomless and the pathogen was notisolated, but from all the other 14 the pathogen was iso-lated. Rooted cuttings from suckers of the resistant root-stock will soon be inoculated with V. dahliae microsclero-tia to evaluate the resistance through soil infection.

Comparative study of isolates of the genus Ca-marosporium from pistachio and olive usingRAPDs. E.J. PAPLOMATAS1, I. PANTELIDES1, A. TZIMA1 andA. CHITZANIDIS2. 1Agricultural University of Athens, Lab-oratory of Plant Pathology, 75 Iera Odos str., 11855 Ath-ens. 214 Kanari str., 10674 Athens, Greece.

The fungus Camarosporium pistaciae attacks pistachiopanicles, shoots and leaves. The anamorph stage Ca-marosporium is rare: usually, the pycnidiospores are ofthe Macrophoma or Fusicoccum type. Olive fruits areattacked by the fungus Macrophoma dalmatica, whichunder certain conditions also produces spores of theCamarosporium type. This polymorphism, which is usu-al in Coelomycetes, causes a confusion in taxonomy andnomenclature of these two fungi. Their perfect stage isnot known, but it is believed that it belongs to the ge-nus Botryosphaeria. The aim of the present study wasto compare 10 isolates of the fungus from pistachio and7 from olive using molecular markers. RAPDs markerswere selected based on random decamer primers in PCRreactions. After preliminary screening, five primers thatgenerated highly polymorphic bands of the RAPD-PCRproducts were selected for testing. The electrophoreticprofiles of the RAPD bands were scored on a binary ba-sis, where the presence of a band was recorded as ‘1’,and its absence as ‘0’. These data were used as input ina software package to infer phylogenetic relations. Iso-lates were divided into two groups that differed by 20%.Each group mostly contained isolates of one host. Onegroup included 7 of the 10 pistachio isolates. The sec-ond group was split into two sub-groups, a main sub-group that contained 6 of the olive isolates, and a small-er one that hosted the seventh olive isolate plus the 3remaining pistachio isolates. It is concluded from thesefindings that fungal isolates exhibit genetic variationwithin and between the two plant hosts.

65Vol. 45, No. 1 April, 2006


Cypress canker and its control in Greece. P. TSOPE-LAS and S. XENOPOULOS. National Agricultural ResearchFoundation (N.AG.RE.F), Institute of MediterraneanForest Ecosystems, Terma Alkmanos, 11528 Athens,Greece.

Cypress canker, caused by the fungus Seiridium cardi-nale (Wag.) Sutton and Gibson, is a very important dis-ease in many Mediterranean countries. In Greece, thedisease is widespread. It is most severe in areas with ahumid climate, but its incidence is very low in dry cli-mates. A high incidence of cypress canker has been re-corded on the west coast, and in the Ionian islands andmany areas of the Peloponnese, Sterea Hellas and theisland of Euboia. The incidence of cypress canker is alsovery high in many parts of northern Greece. The mainhost of the disease in Greece is Cupressus sempervirensL., which has been devastated by the pathogen in cer-tain areas. The disease also infects other species of Cu-pressus as well as species of Thuja and Cupressocypa-ris, which are used as ornamentals, and lately it hasalso been found to infect species of Juniperus in naturalforests of Greece. The most susceptible host is Cupres-sus macrocarpa Hartweg; the variety Goldcrest of thisspecies has recently been widely planted in Greece, con-tributing to the spread of the disease. Sanitation meas-ures are beneficial in areas with a very low incidence ofthe disease. Nurseries for the production of Cupressace-ae should not be established in areas with high levels ofcypress canker. Chemical control with benzimidazolicfungicides (benomyl, carbendazim), can be applied onlyin nurseries, in spring and in autumn. In areas with ahigh incidence of the canker, planting resistant trees isthe only method of control. In Greece there are morethan 100 resistant clones of C. sempervirens, of both thepyramidalis and the horizontalis varieties, which aremainly reproduced by grafting. The species Cupressusglabra Sud. and C. arizonica Gr. can also be used, sincethey too have shown resistance to the disease.

Vegetative compatibility groups of Cryphonectriaparasitica in Greece. C. PERLEROU and S. DIAMANDIS.National Agricultural Research Foundation(N.AG.RE.F), Forest Research Institute, 57006 Vassili-ka Thessaloniki, Greece.

Cryphonectria parasitica, the causal agent of chestnutblight, has caused considerable damage in orchards andchestnut forests in several European countries over thelast five decades. In Greece the disease was first detect-ed in 1963, on Mount Pelion. A recent survey has re-vealed that the blight has now spread to almost thewhole country. An investigation was carried out into thevegetative compatibility (vc) groups of Cryphonectriaparasitica in Greece. Twelve widely separated subpop-ulations distributed all over the country were sampledand a total of 611 isolates were tested. Each isolate was

assigned to a single vc group. Four vc groups were iden-tified. Pairing with European vc group testers revealedthat the most common vc group in Greece is EU-12,which occurred in all the subpopulations and comprised88.4% of all isolates. EU-2 was detected in 4 subpopula-tions and the other two groups, EU-1 and EU-10, werefound only in one subpopulation each. Vc group EU-12has already been reported as the dominant vc group insouthern Italy and eastern Europe, while in centralEurope the dominant groups are EU-1 and EU-2. In viewof the quite small number of vc groups in Greece, webelieve that biological control of chestnut blight is pos-sible. The state however should take all possible pre-cautions to pevent the introduction of new vc groups.

Occurrence, distribution and ochratoxigenic ca-pacity of fungi of the Aspergillus niger group caus-ing the sour rot of grapes of wine-producing cul-tivars in Greece. E.C. TJAMOS, S.E. TJAMOS, P.P. ANTO-NIOU, D.F. ANTONOPOULOS and E.J. PAPLOMATAS. Agricul-tural University of Athens, Department of Plant Pathol-ogy, 75 Iera Odos str., 11855 Athens, Greece.

Sour rot of grapes, caused mainly by Aspergillus niger,A. carbonarius and A. ochraceum is a serious disease bothin Greece and worldwide in both wine-producing and rai-sin grape varieties. Besides crop loss due to sour rot, sev-eral species of the A. niger group also produce ochratoxinA, which negatively affects the quality of fresh grapes,wine and raisins. Within the framework of an EU researchproject to examine sour rot in general, we decided to in-vestigate the occurrence, distribution and ochratoxin-pro-ducing capacity of some fungi of the A. niger group col-lected from wine-producing grape varieties all overGreece. As a side project the sour-rot situation with Cor-inth raisins was also studied. During 2001 we sampledthe wine-producing varieties Xinomauro of Naousa rodi-tis, Cabernet Sauvignon and Sauvignon Blanc fromChalkidiki, Athiri and Cabernet Sauvignon from Moun-tain Atho, Limnio and Muscat of Alexandria from Lim-nos and Agiorgitiko and Corinth raisins from Nemea. In2002 the varieties Mandilaria, Athiri, Cabernet Sauvi-gnon and Grenache were also sampled from Rhodes andthe varieties Aidani, Athiri, and Asyrtiko from Santorini.Grapes sampled during fruit setting, veraison and har-vesting in selected vineyards of the above-mentioned re-gions showed that the prevailing Aspergillus spp. isolat-ed in the selective media (DRBC and CZ) belonged to theA. niger group, and that A. niger and A. carbonarius werethe dominant species. The frequency of isolation of indi-vidual Aspergillus spp. and the composition of the popu-lation depended on the stage of sampling, the variety,the applied control measures and to a lesser extent tothe latitude of the vineyard region. Data obtained fromochratoxin A quantification using ELISA and HPLCshowed that A. carbonarius isolates were very strong


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ochratoxin A producers, whereas A. niger isolates pro-duced little ochratoxin A.

The role of fungal pathogens associated with sub-standard grapevine propagation material of cul-tivars and rootstocks in Greece. I.C. RUMBOS, I. AD-AMOPOULOS, A. TOURTOURI and A. CHATZAKI. National Ag-ricultural Research Foundation (N.AG.RE.F), Plant Pro-tection Institute of Volos, 38001 Volos, Greece.

Decline symptoms associated with pathogenic fungi areoften observed in young grapevines during the first fewyears after planting. It was hypothesized that declinemay be caused by contaminated or sub-standard grapepropagation material produced in Greece or importedfrom abroad. In the period January–May 2002, 15,000samples from Greek and foreign nurseries were sent toour laboratory for phytopathogenic fungal identification.Four types of vine material were examined: (a) non-root-ed cuttings of rootstocks from both Greek and foreignmother plantations, (b) rooted rootstock cuttings fromGreece and abroad, (c) canes of various cultivars usedfor grafting, and (d) rooted grafted plants ready for plant-ing out. In total, 20,000 petri dishes were used and80,000 isolations were carried out. Isolations yielded alow incidence (up to 3%) of the fungi Botryosphaeriadothidea, Cylindrocarpon destructans, Phaeomoniellachlamydospora and Phaeoacremonium spp. In the root-stock mother blocks a basidiomycete sp. was isolatedwith a high incidence (7–30%); its role is under study.The most prominent result of this study was the lowincidence of the pathogenic fungi isolated, althoughbrown wood discoloration was frequent (2–100%). It wasassumed that abiotic causes, such as cuts made in thenursery during preparation processes as well as improp-er storage and transportation of the propagated mate-rial, may have contributed to worsening the decline.

Production of standard grapevine propagationmaterial from local cultivars in Cephalonia. I.C.RUMBOS, A. CHATZAKI, S. MILLA, G. DRACOPOULOS and A.I.RUMBOU. National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute of Volos, 38001Volos, Greece.

The lack of certified grapevine propagation materialof Greek cultivars combined with the need for healthypropagation material to establish new plantations (EUdirective 1493/99) makes it interesting to developquick procedures for the production of such material.In some grapevine-growing areas of Greece (Cephalo-nia, Rodos, Paros, Santorini, Samos) attempts havebeen made to carry out the clonal and sanitary selec-tion of genotypes. In Cephalonia the selection of gen-otypes from the most important cultivars (Robola, Ma-vrodafni, Muscat of Cephalonia, Vostilidi) was carriedout in the summer of 2000. Sanitary control was di-

rected against viruses, fungi and bacteria that couldbe disseminated by the propagation material. Virusdetection was done by ELISA and the following sixviruses were found: Grapevine fanleaf virus (GFV),Grapevine leafroll associated virus (GLRaV) 1 and 3,Grapevine virus A and B, and Grapevine fleck virus(GFkV). In total, 855 vines from 81 vineyards and 22local cultivars were tested. One hundred and ten vines(13%) were virus infected. The most widespread vi-rus was GLRaV-1 (92 vines; 11%); seven vines wereinfected with GVA, five with GLRaV-3, four with GFVand four with GFkV. Canes from vines that were freeof the above six viruses were used to establish moth-er plants in the local nursery.

A new virus of the genus Closterovirus in grape-vine. C.I. DOVAS and N.I. KATIS. Aristotle University ofThessaloniki, Faculty of Agriculture, Plant PathologyLaboratory, P.O. Box 269, 54124 Thessaloniki, Greece.

Grapevine leafroll disease is widespread all over the worldand causes serious crop losses. The production of virus-free propagative material is the only way to combat thisdisease. However, the fact that at least eight viruses areimplicated in its etiology does not allow a reliable andlow-cost detection method to be applied. For this reason,we developed a nested-spot polymerase chain reaction(PCR), using degenerate primers, for the simultaneousdetection of grapevine closteroviruses. This method wassuccessfully applied in 80 grapevine samples resultingfrom clonal selection for the detection of viruses associ-ated with grapevine leafroll disease. The same sampleswere also tested serologically by ELISA using polyclonalantibodies prepared against Grapevine leafroll-associat-ed virus -1,-2,-3,-5,-6,-7 (GLRaV -1,-2,-3,-5,-6,-7). In grape-vine plants cv. Debina from the area of Zitsa (Epirous), anew closterovirus was detected by PCR only. The ampli-con of 482 bp was cloned and sequenced, showing a se-quence similarity of 80% with GLRaV-5, and of 74% withGLRaV-4. Two new primers were designed and specifi-cally detected the new virus by PCR, in grapevine sam-ples of the cultivars Debina, Repsodebina and Kontokla-di from the area of Zitsa.

The new virus disease ‘grapevine angular mosaic’is caused by the homonymous new ilarvirus. S.M.GIRGIS1 and P.E. KYRIAKOPOULOU2. 1National Agricultur-al Research Foundation (N.AG.RE.F), Athens GrapevineInstitute, 1 S. Venizelou str., 14123 Lycovryssi, Attica,Greece. 2Agricultural University of Athens, Laboratoryof Plant Pathology, 75 Iera Odos str., 11855 Athens,Greece.

A new virus-like symptomatology of grapevine wasdescribed in 1994 on the grapevine hybridBaresana�Baresana in the ampelographical collectionof the Grapevine Institute of Athens, in Lykovryssi of

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Attica. This symptomatology consisted of a striking an-gular mosaic on the leaves, connected with the veinsand the vein angles, leaf malformation, small leaves andreduction of vine size, flower drop, small berries, smallwrinkled and non-germinating seeds, and fruitlessness.Since then, symptoms have gradually become more se-vere, and two of the original seven symptomatic vineshave now died, whereas the remaining five are gradual-ly declining and have become completely fruitless. Fromall these vines a virus was isolated by mechanical inoc-ulation, originally on Gomphrena globosa, and fromthere to other indicators. On the basis of its seed andpollen transmissibility, its parasphaerical particles andtheir diameter (29 nm), and the cloning and sequencingof the part of its genome that was characteristic for ilar-viruses, the virus was identified as a species of the ge-nus Ilarvirus (data presented at the 10th Hellenic Phy-topathological Congress). Using pollen of Chenopodiumquinoa infected with pure culture of the virus as inocu-lum, the virus was transmitted mechanically to healthygrapevine seedlings from tissue culture, which reactedshowing the original field-symptoms on the leaves. Inthese leaves the virus was detected by DAS-ELISA, us-ing homologous antiserum. With these experiments, thefull cycle of Koch’s postulates was fulfilled for this vi-rus, so that we can state that it is the cause of the dis-ease originally described. Based on these data, we calledthis virus Grapevine angular mosaic virus (GAMV), af-ter the disease. In preliminary graft-transmission ex-periments of the virus on grapevine (Vitis) indicatorswe obtained a positive reaction on V. rupestris du LotSt. George, with leaf symptomatology similar to the orig-inal field symptomatology.

Susceptibility to iron deficiency of some peachrootstocks grown in nutrient solutions. A. ASSIMA-KOPOULOU1 and C.D. HOLEVAS2. 1Benaki Phytopathologi-cal Institute, 8 St. Delta str., 14561 Kifissia, Athens,Greece. 24 Kanari str., 15121 Athens, Greece.

The susceptibility of eleven peach rootstocks to iron de-ficiency was studied in synthetic nutrient solutions (hy-droponics). Young rootstocks were grown with iron dep-rivation (no iron added to the nutrient solution), as wellas with reduced iron availability (nutrient solution con-tained: 3 mM FeEDDHA, 10 mM NaHCO3 and 0.5 gCaCO3 l-1). On the basis of the intensity of leaf chloro-sis, three experimental peach-almond hybrids, PR 204/84, Stylianidis K and K.I.D 2, from the Pomology Insti-tute of Naoussa (Greece), showed the same or even great-er tolerance to iron deficiency than the French peach-almond hybrid GF 677. The Greek peach-almond hy-brid Retsou�Nemaguard, the plum-almond hybridMyrandier 617 and the peach seedlings GF 305, ID S 37and Wild Greek Seedling, showed the greatest suscepti-bility, whereas the plums St Julien GF655/2 and M29C

ranked intermediate. Chemical analysis of the leavesshowed that iron deprivation decreased concentrationsof N and Fe, and increased concentrations of P, K, Mg,Mn, Zn, Cu and B, whereas the addition of bicarbonatesdecreased not only concentrations of N and Fe but alsothose of P, Mn and Zn, and increased concentrations ofK, Mg, Cu and B. The synergistic and antagonistic rela-tionships observed between leaf nutrient elements bothunder real iron deprivation, and under low levels of ironcaused by a relatively high concentration of bicarbonates- as is usual in calcareous soils - can be a means to de-tect the early stages of iron deficiency.

Control of Mg deficiency in cherries by using theclones ‘F.12/1’ (Prunus avium L.) and ‘Colt’ (P.avium�P. pseudocerasus). Y. TROYANOS1 and N.A.HIPPS2. 1Benaki Phytopathological Institute, 8 St. Deltastr., 14561 Kifissia, Athens, Greece. 2HRI, East – WestMalling, ME19 6BJ, Kent, UK.

Cherries are susceptible to Mg deficiency which causesdefoliation and may reduce plant growth. Differencesin susceptibility to Mg deficiency between different cher-ry varieties have been found in field experiments wherePrunus avium seedlings showed Mg deficiency symp-toms, whereas hardwood cuttings of ‘Colt’ did not. Inorder to investigate whether these differences are dueto genetic differences, ‘F.12.1/’ and ‘Colt’ cherry rootstocksfrom tissue culture were grown in a flowing solutionculture system. The results of the experiments showedthat ‘F.12/1’ required a three times greater minimuminflow rate of Mg than did ‘Colt’. The minimum inflowrate of Mg in ‘F.12/1’ was supported by a smaller rootsystem (weight and length) and a greater concentration(500 µM) of Mg in the nutrient solution than with ‘Colt’(50 µM). The inhibitory effect of an increased concen-tration of K in the nutrient solution was also investi-gated. ‘F. 12/1’ had a higher concentration of K in theleaves and a greater inflow rate of K. The absorption ofMg in both species was affected similarly by the increas-ing concentration of K in the nutrient solution. Fromthe results of these experiments it seems that the greatersusceptibility of ‘F.12/1’ to Mg deficiency is due to genet-ic differences in the size of the root system; ‘Colt’ conse-quently had a larger root system than ‘F.12/1’. Thesedifferences could be used as a guide for the genetic im-provement of ‘F.12/1’ towards larger root system in or-der to increase its resistance to Mg deficiency.

A serious Pseudomonas syringae complex out-break in apricot trees cv. Early Tirynth and Bebe-cou in the Argolis region of Greece. P.P. ANTONIOU,S.M. CHRISTOGLOU, E.C. TJAMOS and A.C. GREGORIOU. Ag-ricultural University of Athens, Laboratory of Plant Pa-thology, 75 Iera Odos str., 11855, Athens, Greece.

An unusual bacterial outbreak on apricot trees was ob-


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served in the Argolis region of Greece during the springof 2002. The outbreak was fairly extensive along astream running through apricot orchards of the villag-es Sterna and Lyrkia. The damage was serious in thewhole area of orchards regardless of the cultivar (EarlyTirynth or Bebecou) but it was more severe along or nearthe stream banks. Heavy rainfall in March, 2002, cre-ated very humid conditions. Major symptom develop-ment resembled a brown rot epidemic: however, closerfield surveys and laboratory examinations showed thatMonilia laxa was not involved, and in any case fungi-cide sprays (but no copper compounds) had been exten-sively used against brown rot. The most striking symp-tom was necrosis of the leaves and branches as a resultof short or elongated cankers and dead areas that formedon the twigs, the bark of the large branches and thetrunks. Gummosis was evident in most of the cankers,although in some instances little or no gum was presentand the bark became brown, moist and sour smelling.Shriveled flowers and unopened buds were also ob-served, as were brown necrotic flowers, as a result ofwhich fruit setting was drastically affected. Althoughthe apricot tree was highly susceptible to this disease,records of bacterial infection on apricot are rare inGreece. As regards the identification of the causal agent,laboratory tests (cell morphology, gram stain, oxidasereaction, catalase reaction, acid from glucose, hypersen-sitivity reaction) as well as fatty acid analysis and Bi-olog (GN2) tests carried out by BCCM/LMG, showed thatmost of the isolated bacteria were members of the Pseu-domonas syringae complex. Two pathovars of Pseu-domonas syringae (pv. syringae and pv. morsprunorum)are thought to cause bacterial canker on prunus trees.To check the pathogenicity of the main bacterial isolatesobtained, Koch postulates will be applied in the springof 2003. Previous reports of this disease attributed it toPseudomonas sp., so this is the first report of the epi-demic appearance of the Pseudomonas syringae complexin Greece.

Molecular detection and characterization of Cit-rus tristeza virus in Greece. C. VARVERI. Benaki Phy-topathological Institute, Laboratory of Virology, 8 St.Delta str., 14561 Kifissia, Athens, Greece.

In Greece, Citrus tristeza virus (CTV, family Clostero-viridae, genus Closterovirus) was first identified in Arg-olis, in 2000, on young Lane Late navel orange trees ofSpanish origin. The virus was graft-transmitted to theindicators, sweet orange cv. Madame Vinous and Mexi-can lime, where CTV was detected by immunoprintingand by IC-RT-PCR, amplifying a 520 bp fragment of thevirus 3’ end genome between the p20 and p23 genes.RFLP analysis of the PCR product gave the same pro-file as that produced by the known T385 isolate whichbelongs to the virus group III strains. The same results

were also obtained in the other cases where the viruswas isolated: a 20-year-old navel orange tree in Argolis,some Lane Late navel trees in Chania, Crete, and somecertified Clemenpons mandarin trees, also of Spanishorigin. The phylogenetic nucleotide sequence analysisof the PCR product of the first isolate confirmed its closesimilarity to the Spanish strains, which is consistentwith the historical background of the disease.

Grapevine chlorotic vein banding and grapevineyellow mottle, two new virus diseases. S.M. GIRGIS1

and P.E. KYRIAKOPOULOU2. 1National Agricultural Re-search Foundation (N.AG.RE.F), Athens Grapevine In-stitute, 1 S. Venizelou str., 14123 Lycovryssi, Attica,Greece. 2Agricultural University of Athens, Laboratoryof Plant Pathology, 75 Iera Odos str., 11855 Athens,Greece.

In 1996, in the Amaroussion annex (Sygrou farm) of thegrapevine variety collection of the Athens Grapevine In-stitute, the following two new diseases were observedand were named as follows: i) Grapevine chlorotic veinbanding. All ten vines of the white wine grapevine vari-ety Petsariko (origin Kozani, Greece) showed uniformchlorotic bands on both sides of the leaf veins. Usingtissue from these vines as a scion, a series of grapevineindicators were grafted (R110, 41B, 5BB, and SO4) inMarch 2000 and maintained in the greenhouse. In Au-gust 2001, the grafted indicator 5BB showed an extend-ed bright-yellow discoloration, in the form of sectors withthe veins as axes. To our knowledge this reaction is newto grapevine virology. It should be mentioned that 5BB(Vitis berlandieri�V. riparia) is a known indicator ofgrapevine Kober stem grooving disorder, but that dis-ease has a completely different symptomatology. Wetherefore consider this case as new and worth furtherstudy, since 5BB may be a new indicator of a knowngrapevine virus, or of a new virus of grapevine. In 2002,a virus was mechanically transmitted from the sympto-matic vines of the vineyard to the indicator Chenopodi-um quinoa, which showed intense local and systemicmottle, top leaf necrosis and top necrosis. This was there-fore a well reacting herbacous host that will help in thestudy of the virus. ii) Grapevine yellow mottle. In May–June, all ten vines of the coloured wine grapevine vari-ety Lehonitis (origin Magnesia prefecture, Greece)showed a clear yellow mottle in the form of an oak-leafpattern, which became diffuse later in the summer. InMarch 2000, using tissue from these vines, the grape-vine indicators R110, 41B, 5BB and SO4 were grafted,of which SO4 showed wrinkling and chlorosis of theleaves after one year, in May 2001. One year later, inMay 2002, the symptomatology of this indicator wasmore intense. To our knowledge, this indicator reactionis here reported in grapevine virology for the first time.The infection is under further study.

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Influence of grape volatiles cv. Isabella in forma-tion of sclerotia by Botrytis cinerea. E.K. KULAKIO-TU1, C.C. THANASOULOPOULOS1 and E.M. SFAKIOTAKIS2. 1Ar-istotle University of Thessaloniki, Faculty of Agriculture,Laboratory of Plant Pathology, P.O. Box 269, 54006 Thes-saloniki, Greece. 2Aristotle University of Thessaloniki,School of Agriculture, Laboratory of Pomology, 54006Thessaloniki, Greece.

The influence of volatile substances produced by grapes(Vitis labrusca) cv. Isabella, on the formation of sclero-tia by Botrytis cinerea was studied in vitro. Since thiswas a bioassay method, use was made of the closedMariotte system. To determine the anti-fungal action ofthe volatile substances the following tests were carriedout: (i) using volatile substances excreted by grapes ofthe resistant cv. Isabella, (ii) using volatile substancesexcreted by grapes of the susceptible variety Roditis (V.vinifera), and (iii) using no volatile substances at all.The action was studied at various temperatures (21, 10and 0oC). Volatile substances from cv. Isabella inhibitedsclerotium formation. This indicates that the resistanceof cv. Isabella to B. cinerea is related to the volatile sub-stances secreted by the grape berries, although otherfactors may also be involved. It also indicates that vola-tile substances can have an important role as inhibitorsor stimulators of an organism.

First report of Alternaria spp. as a foliar patho-gen of the strawberry tree (Arbutus unedo) inEurope. I.A. LAIDOU and C.C. THANASSOULOPOULOS. Ar-istotelian University of Thessaloniki, Faculty of Agricul-ture, Plant Pathology Laboratory, P.O.Box 26, 54006Thessaloniki, Greece.

Leaves of strawberry trees were found to be heavily spot-ted in the spring of 1999, 2000 and 2001 in the area ofThessaloniki, northern Greece. Small, brown necroticspots were observed on the leaves, and, where spottingwas intense, extensive defoliation. The same symptomswere also noted on the leaves of plants in florists’ shopsthroughout the year. Isolations from the leaves on PDAconsistently yielded three isolates that belonged to thegenus Alternaria. Pathogenicity tests were carried outon healthy plants, which were inoculated with a sterilewater suspension containing 6.4�103 conidia ml-1 plus0.05% Tween 20. Control plants received only steriledistilled water and 0.05% Tween 20. After incubation inplastic bags for 24 h, the plants were returned to thegreenhouse. Symptoms identical to those originally ob-served appeared ten days after inoculation, on all plantsexcept the control plants. The frequency of reisolationof those fungi from leaf spots was more than 65%. Fromthe three isolates, one belonged to group 4, one to group6 (according to Simmons’ taxonomy), and one did notsporulate. This is the first report of Alternaria spp. caus-ing leaf spotting of strawberry tree in Europe.

Infection of olive trees with the fungus Fomiti-poria punctata (Phellinus punctatus). E.J. PAPLO-MATAS1, K. ELENA2, P. TSOPELAS3, A. TZIMA1, A. PAR-ASKEVOPOULOS4 and A. PAPANIKOLAOU5. 1Agricultural Uni-versity of Athens, Laboratory of Plant Pathology, 75 IeraOdos str., 11855 Athens, Greece. 2Benaki Phytopatho-logical Institute, Department of Plant Pathology, Lab-oratory of Mycology, 8 St. Delta str., 14561 Kifissia,Athens, Greece. 3National Agricultural Research Foun-dation (N.AG.RE.F), Institute of Mediterranean ForestEcosystems, Alkmanos str., 11528 Athens. 4Prefectureof Messinia, Directorate of Agriculture and Husbandryof Trifyllia, Department of Plant Protection, 24500Kyparissia. 5Prefecture of Messinia, Directorate of Ag-riculture and Husbandry of Messinia, 24100 Kalama-ta, Greece.

In the last few years, a serious disease of olive treeshas been observed mainly in the areas of Messini-aki Mani and Kyparissia. The disease seems to bespread by chainsaws used in olive pruning. The in-fection is mostly located at the trunk wood and inthe main twigs. The most common symptoms of theattack are a brown discoloration leading to wood rotand necrosis of the bark, usually on one side of thetrunk, and resulting in cankers. Wood eventually be-comes soft and shows symptoms similar to thosecaused by esca on grapevine. In many cases, fruit-ing bodies of Fomitiporia punctata Murrill [Phelli-nus punctatus (P. Karst.) Pilat] appear on the sur-face of the trunk and the main twigs. Fruiting bod-ies are perennial, woody, with a porous, brown,smooth (velvet) surface that expands superficially(resupinate). The fungus has been isolated from theinfected wood of several samples as well as from thefruiting bodies, and formed orange brown colonieson PDA. Identification of the pathogen was con-firmed by Dr. M. Fischer, Weinbauinstitut, Freiburg,Germany, (personal communication). The patho-genicity of the isolates was tested with artificial in-oculations on three-year old olive trees cv. Koroneiki,Amphisis and Kalamon. About three months afterinoculation, the infection progress was checked insome of the inoculated trees by removing the bark,inspecting for wood discoloration and re-isolating thefungus. On a trunk cross section, browning of thewood was observed extending 4–5 cm from the inoc-ulation site and F. punctata was isolated from woodat 1 and 2.5 cm from the infection site. Because ofthe slow growth of this fungus, disease developmentin the rest of the inoculated trees is still continu-ing. Although this fungus had been recorded on ol-ive trees in Greece before (Plank 1980, Ann. Inst.Phytop. Benaki; F. Kotlaba and J. Klan 1994, CzeckMycol.), this is the first report of F. punctata on ol-ive trees causing economic damage.


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Grapevine downy mildew epidemics in the Ioni-an islands: what has population genetics taughtus? A.I. RUMBOU1,2, C. GESSLER2, I.C. RUMBOS1 and I. ADA-MOPOULOS1. 1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute of Volos, 38001Volos, Greece. 2ETH-Zentrum, Institute of Plant Scienc-es, Universitätstr. 2, 8092, Zürich, Switzerland.

The role of sexual and asexual reproduction of theoomycete Plasmopara viticola in causing the spread ofgrapevine downy mildew, as well as the distance andmeans of sporangia dispersal were studied with thehelp of population genetics. During the period of Mayto November 2001, leaf lesion samples were collectedin non-treated plots in Cephalonia, Lefkada and theZakynthos islands. Initially, all the primary lesions ineach plot were collected first. Every time the environ-mental conditions favored a new spread of the disease,further sampling was done. In total, three samplingswere carried out in Cephalonia (147 lesions), four inLefkada (640 lesions) and two in Zakynthos (210 le-sions). Each leaf lesion was deemed to have derivedfrom a single zoospore and to constitute a single ‘iso-late’ of the population. Microsatellite markers wereused to characterize each isolate genetically. Isolatespresenting the same allele pattern were taken to beclones while isolates with different allele patterns wereassumed to derive from different oospores. In all threeepidemic cases, there was a gradual decrease of thepopulation genotypic diversity (Gst); this was becauseof the clonal reproduction of some isolates. At the sametime the populations were enriched with new geno-types, due to sporangia migration from nearby vine-yards or late oospore germination (until the middle ofJuly). The three island populations did not share anygenotype, meaning there was no sporangia exchangeamong them. On the other hand, the populations sharedthe majority of their alleles.

Identification, detection and transmission of avirus associated with the Strawberry Pallidosisdisease. I.E. TZANETAKIS1, W.M. WINTERMANTEL2 and R.R.MARTIN1,3. 1Molecular and Cellular Biology Program,Department of Botany and Plant Pathology and Centerfor Gene Research and Biotechnology, Oregon State Uni-versity, Corvallis, OR 97331. 2USDA-ARS, Salinas, CA93905, USA. 3USDA-ARS, Corvallis, OR 97330, USA.

Pallidosis is a disease of strawberry first identified in1957. Graft transmissibility and the existence of dsR-NA in infected plants indicate the viral nature of thedisease. DsRNA was extracted from 22 infected plantsand cloned. After sequencing about 100 clones we iden-tified eight genes of a virus with homology to Lettuceinfectious yellows virus, the type member of the Crini-virus genus. The major coat protein is most closely re-lated to Cucurbit yellows stunt disorder virus, another

member of the group. Using the sequence informationwe developed a sensitive RT-PCR test that detected 37of the 38 isolates of the virus available to us. We clonedand expressed the major coat protein of the virus andobtained antibodies to the virus for developing a relia-ble immunological test. We have also started transmis-sion studies with the whitefly species known to trans-mit criniviruses and we have identified one of them asbeing a vector, while we are continuing to study theremaining species.

DISEASES OF VEGETABLE, INDUSTRIAL AND

ORNAMENTAL CROPS


Phytophthora sp. causing stem rot of lettuce. A newdisease in Greece. A. GRIGORIOU1 and K. ELENA2. 1Agri-cultural University of Athens, Department of Plant Pa-thology, 75 Iera Odos str., 11855 Athens, Greece. 2BenakiPhytopathological Institute, 8 St. Delta str., 14561 Ki-fissia, Athens, Greece.

A severe disease of lettuce (Lactuca sativa L.) was ob-served for the first time in Greece, on February andMarch 2000 in a one-acre field at Marathona, Attikacounty. The percentage of infected plants was 60%. Thisdisease was observed in the same and in neighboringfields in January, February and March of 2001 and 2002with similar percentages of diseased plants. Wilt ini-tially affected the lower leaves, then progressed alongthe upper leaves as well. The plants became chlorotic,declined and died. A brown-black rot appeared along theinfected stems starting from the root area and progress-ing upwards in the stem. A fungus of the genus Phy-tophthora was isolated from all infected plants. On sol-id and liquid culture media the fungal strains formedsemipapillate sporangia, hyphal swellings and coilingsof mycelium. Aplerotic oospores formed abundantly withamphigynous and paragynous antheridia. The maxi-mum growth temperature was less than 25°C. Classifi-cation of the fungus and pathogenicity tests are inprogress. The morphological and physiological charac-teristics resembled those of Phytophthora porri Foister.To our knowledge, this is the first report of the genusPhytophthora infecting lettuce in Greece.

Classification of fungi of the genus Alternaria. I.A.LAIDOU and C.C. THANASSOULOPOULOS. Aristotelian Uni-versity of Thessaloniki, Faculty of Agriculture, PlantPathology Laboratory, P.O.Box 26, 54006 Thessaloniki,Greece.

Different nomenclatures of species of the genus Alter-naria and different systems of taxonomy have createdproblems in the taxonomy of this genus. The taxonomi-

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cal systems of Saccardo, Elliott, Neergaard, Joly, Ellis,and Simmons have been consulted. The system of Sim-mons is the best as it distinguishes between species ofthe genus Alternaria, which it divides into six groupsbased on morphological characters and it defines thesimilarities and differences between taxa. Isolations offungi of the genus Alternaria in Greece from field-in-fected cotton, beetroots, parsley and celery plants wereclassified according to Simmons and assigned to differ-ent groups. In this way, of the 31 isolations from cotton,32.22% belonged to group 1, 9.67% to group 2, 19.35%to group 4, 3.22% to group 5, and 29% to group 6, while6.45% did not produce any spores. Of the 23 isolationsfrom celery, 26% belonged to group 1, 30.43% to group2, 4.35% to group 4, 34.78% to group 6, and 4.34% didnot produce any spores. Of the 5 isolations from beet-roots, one belonged to group 1, one to group 4 and threeto group 6. Lastly, of the 4 isolations from parsley, onebelonged to group 1 and one to group 5, while two ofthem did not sporulate.

Characterization of iprodione-resistant isolates ofAlternaria solani. I. VLOUTOGLOU, I. ASPROMOUGOS andT. KATSAMAKIS. Benaki Phytopathological Institute, PlantPathology Department, 8 St. Delta str., 14561 Kifissia,Athens, Greece.

Four Alternaria solani isolates from naturally infect-ed tomato, and seven from naturally infected potato,all growing in southern Greece (Peloponnese) were ex-amined for sensitivity to iprodione. All isolates wereiprodione-sensitive (IS) with EC50 values ranging from0.40 to 2.40 µg ml-1. None of the isolates grew on V-8agar amended with 10 or 100 µg ml-1 of iprodione. How-ever, when mycelial plugs of these isolates were incu-bated for 3 to 10 days beyond the standard 4 days ofthe sensitivity test, some of them developed resistantsectors. Isolates randomly selected from these sectorsand designated as in vitro-derived iprodione-resistantisolates (IR) grew well on V-8 agar amended with 100µg ml-1 of iprodione. The EC50 values for these IR iso-lates ranged from 182 to 614 µg ml-1, whereas the EC50

values for their original wild-type IS counterparts wereless than 1 µg ml-1. The linear growth as well as thecolony morphology of the IR isolates differed from thoseof their corresponding wild-types, when grown on una-mended V-8 agar. The in vitro sporulation and patho-genicity of the IR isolates on tomato plants were com-pared with those of the IS isolates. Studies on a possi-ble genetic differentiation between IR and IS isolatesare in progress.

Bacterial bract spot of artichoke caused by Xan-thomonas cynarae. D.E. GOUMAS1, A. TSAGKARAKOU1 andS. SPLINI2. 1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute, P.O. Box 2228,

71003 Heraklion, Crete, Greece. 2Technological Educa-tion Institute, P.O. Box 1939, 71003 Heraklion, Crete,Greece.

A bacterial disease of the bract leaves has recently beenobserved on artichoke in Crete. Symptoms of the dis-ease are restricted to the bracts of the capitulum. Smallwater-soaked spots appear mainly on the external bractswhich progressively coalesce to form larger lesions thatare dark-green to brown. The symptoms occur duringwarm and humid periods and mainly in crops irrigatedwith an overhead system. The disease reduces the qual-ity of the market crop. Circular, yellow and glisteningbacterial colonies are consistently isolated on NAG me-dium. Morphological, physiological and biochemicaltests, coupled with a specific PCR test, identified theeight tested isolates of the bacterium from Crete asmembers of the species Xanthomonas cynarae. Koch pos-tulates have been fulfilled on a bract of a detached ca-pitulum of the plant.

Transmission of Tomato spotted wilt virus (TSWV)in relation to Thrips tabaci Lindeman host spe-cificity. E.K. CHATZIVASSILIOU1, P.C. BRUNNER2, D. PETERS3,J.E. FREY2 and N.I. KATIS1. 1Aristotle University of Thes-saloniki, Faculty of Agriculture, Plant Pathology Labo-ratory, P.O. Box 269, 54124 Thessaloniki, Greece. 2SwissFederal Research Station, P.O. Box 185, CH-8820Waedenswil, Switzerland. 3Wageningen AgriculturalUniversity, Department of Virology, Binnenhaven 11,6709 PD Wageningen, The Netherlands.

Thrips tabaci Lindeman is the most erratic vector ofTomato spotted wilt virus (TSWV). Although it is a poly-phagous species, its scientific and trivial names (tobac-co and onion thrips, respectively) imply that within itswide host range, tobacco and onion/leek are probablyits preferred host plants. T. tabaci samples were collect-ed from tobacco and leek in different countries, in orderto study the vectoring ability and host specialization ofeach. Thrips populations, from tobacco and leek, showedsignificant differences in the transmission of TSWV: leekpopulations did not transmit or transmitted inefficient-ly, while tobacco populations were highly efficient vec-tors. In choice tests, the thrips population preferred itsnatural host, while in infection tests leek populationsfailed to infect tobacco plants. In preliminary tests, atobacco and a leek population did not interbreed. Addi-tionally, phylogenetic analysis of mitochondrial cyto-chrome oxidase I (COI; ~450bp) sequences separated T.tabaci populations by their host plant preferences. Whenother thrips species from previous studies were added,both types of T. tabaci formed a monophyletic group.COI sequence variation within different types was low.In contrast, genetic variation between the two host typeswas high, being comparable to distances among speciesand representing convincing evidence for genetic struc-


AA.VV.

turing within T. tabaci, according to host-plant type. Sofar, results from the biological experiments and geneticanalysis suggest a strong polymorphism among T. taba-ci populations. The question whether T. tabaci variantsis a cryptic group of sibling species (i.e. whether T. ta-baci is a species complex) is discussed.

A nested multiplex polymerase chain reactionmethod for the detection of Tomato infectious chlo-rosis virus and Tomato chlorosis virus and theimplication of these viruses in epidemics of a yel-lowing disease of tomato in Greece. C.I. DOVAS1, V.MALIOGA1, A.D. AVGELIS2, P.E. KYRIAKOPOULOU3 and N.I.KATIS1. 1Aristotle University of Thessaloniki, Faculty ofAgriculture, Plant Pathology Laboratory, P.O. 269, 54124Thessaloniki, Greece. 2National Agricultural ResearchFoundation (N.AG.RE.F), Crop Protection Institute,Plant Virology Laboratory, 71110 Heraklion, Crete,Greece. 3Agricultural University of Athens, Faculty ofCrop Sciences, Department of Plant Pathology, 75 Ieraodos str., 11855 Athens, Greece.

Since 1997, a yellowing disease has been observed ingreenhouse tomato crops in Greece. By 2001, this dis-ease was widespread throughout the country and alsoaffected tomato field crops; in most cases disease inci-dence was 80–90%. Epidemics were mainly associatedwith high populations of the whitefly species Trialey-rodes vaporariorum and Bemisia tabaci (Homoptera:Aleyrodidae). The main symptoms were severe yellow-ing, downward leaf rolling and brittle leaves. Samplesfrom symptomatic plants were analysed by RT-PCR andwere found to be infected with Tomato infectious chloro-sis virus (TICV) and Tomato chlorosis virus (ToCV) (fam-ily Closteroviridae, genus Crinivirus). TICV was foundin 164 of 183 symptomatic samples, while ToCV was lesscommon (25/183). Sequence comparisons of the ampli-fied 229 bp and 466 bp products revealed 99 and 100%identity with the reported sequences of TICV and ToCVrespectively. Multiplex RT-PCR using a simple sample-preparation procedure was developed to allow rapid,specific, and simultaneous detection of both ToCV andTICV sequences in two steps. The method involves aone-tube RT-PCR step, in which a pair of degenerateprimers amplifies part of the heat shock protein (HSP)region from both ToCV and TICV, and is followed bymultiplex nested PCR amplification. This is the firstreport of TICV and ToCV in Greece, and of TICV in Eu-rope.

Study of a severe NTN isolate of Potato virus Y. C.VARVERI and N. VASSILAKOS. Benaki PhytopathologicalInstitute, Laboratory of Virology, 8 St. Delta str., 14561Kifissia, Greece.

Biological, serological and molecular characterization of

an NTN isolate (PVY-24) of Potato virus Y (PVY, familyPotyviridae, genus Potyvirus), originating from potatoof the Psahna area, Evia, was undertaken. PVY-24 in-duced severe vein necrosis in Nicotiana tabacum cv.Samsun, indicative of PVYN strains. It also induced se-vere symptoms of the potato tuber necrotic ringspot dis-ease (PTNRD) on tubers of Solanum tuberosum cv.Hermes, indicative of PVYNTN strains, which are consid-ered to form a subgroup inside the PVYN strain group.Serologically, however, PVY-24 reacted positively withmonoclonal antibodies specific to the PVYO group; thisbeing the first time such a reaction is reported for aPVYNTN isolate. At the molecular level, in RT-PCR PVY-24 reacted positively with specific primers from the 5’region of PVYNTN strains. However, nucleotide sequenc-ing of a fragment of the same region and comparisonwith those of other isolates led to its clustering withPVYO strains. By contrast, when nucleotide sequencesof a coat protein fragment at the 3’ virus genomic regionwere compared, they were similar to those of the PVYN

strains. PVY-24 is therefore a variant isolate that prob-ably emerged after recombination between differentstrains. Cloning of its genome for sequence determina-tion is under way.

The effect of infection by a third virus to the spe-cific resistance exhibited by transgenic plants andthat induced through cross protection. N. VASSILA-KOS, C. VARVERI and F. BEM. Benaki PhytopathologicalInstitute, Department of Phytopathology, Laboratory ofVirology, 8 St. Delta str., 14561 Kifissia, Greece.

Gene silencing is a general regulation mechanism asso-ciated with the inherent ability of plants to control vi-rus infections and transposon DNA elements. In partic-ular, the branch that involves the sequence-specific RNAdegradation mechanism (post-transcriptional gene si-lencing-PTGS or RNA silencing) has been used to pro-duce transgenic plants resistant to viruses. Plant virusesfor their part counter by encoding proteins that are sup-pressors of gene silencing. The present work investigat-ed the effect that infection with two viruses has on re-sistance to Tobacco rattle virus (TRV) exhibited by Nico-tiana tabacum transgenic plants carrying the 59K re-gion of the replicase gene of TRV. The two viruses usedwere Potato virus Y (PVY) and Cucumber mosaic virus(CMV) both of which encode well-characterized gene-silencing suppressor proteins. In these experiments theresistance of the transgenic plants was overcome, sincethey were infected with TRV at least on the inoculatedleaves. There is also evidence that gene silencing is in-volved in the resistance that is induced through crossprotection. In correspondence to the transgenic plants,PVY infection was examined in a cross-protectionscheme, in which the mild CMV isolate S (CMV-S) con-ferred resistance on the virulent CMV-4. N. tabacum

73Vol. 45, No. 1 April, 2006


plants were inoculated with CMV-S, and 15 days laterinoculated with PVY, and then with CMV-4, and a smallnumber of these plants became infected with CMV-4.The significance of these observations for the effective-ness of the above strategies, which are basic for the con-trol of virus diseases, as well as a possible explanation,are discussed.

Incidence of insect-borne viruses of cucurbits inCyprus. L.C. PAPAYIANNIS1, I.N. BOUBOURAKAS1, C.I. DO-VAS1, N. IOANNOU2 and N.I. KATIS1. 1Aristotle Universityof Thessaloniki, Faculty of Agriculture, Plant PathologyLaboratory, P.O.Box 269, 54124 Thessaloniki, Greece.2Agriculture Research Institute, 1516 Nicosia, 22016Cyprus.

A survey was carried out in order to determine the inci-dence of cucurbit viruses in Cyprus. About 3000 sam-ples were collected, tested with ELISA, and samples withyellowing symptoms also with RT-PCR. In watermeloncrops, the main viruses detected were Zucchini yellowmosaic virus (ZYMV) (90%) and Papaya ringspot virus(PRSV) (19%). ZYMV was likewise the most prevalentin melon (77%), followed by Cucurbit aphid-borne yel-lows virus (CABYV) (20%) and PRSV (7%). In squash,the predominant viruses identified were ZYMV (59%),PRSV (40%), CABYV (38%) and Watermelon mosaic vi-rus 2 (WMV-2) (19%). In cucumber crops, Cucurbit yel-low stunting disorder virus (CYSDV) and Beet pseudo-yellows virus (BPYV) were associated with yellowingsymptoms with CYSDV being the most widespread,whereas ZYMV, PRSV and Cucumber yellow vein virus(CVYV) were detected in only a limited number of sam-ples. Cucumber mosaic virus (CMV) and Squash mosa-ic virus (SqMV) were not detected in this survey. Nucle-otide sequence analysis of a 791-bp capsid protein frag-ment from a Cypriot ZYMV zucchini isolate revealed99% sequence similarity with the Austrian isolate Ber-lin-1. Finally, sequence analysis of heat shock protein-70 from a cucumber isolate of CYSDV revealed a highsimilarity with isolates from Spain and Israel.

Criniviruses associated with a yellowing diseaseof cucurbits in Greece and development of a mul-tiplex polymerase chain reaction for their detec-tion. I.N. BOUBOURAKAS1, A.D. AVGELIS2, P.E. KYRIAKOPOU-LOU3 and N.I. KATIS1. 1Aristotle University of Thessaloni-ki, Faculty of Agriculture Plant Pathology Lab. 54124Thessaloniki, Greece. 2National Agricultural ResearchFoundation (N.AG.RE.F), Plant Protection Institute,Plant Virology Laboratory, 71003 Heraklion, Crete,Greece. 3Agricultural University of Athens, Faculty ofCrop Sciences, Department of Plant Pathology, 75 IeraOdos str., 11855 Athens, Greece.

Since 1992, a novel disease has been observed in glass-house cucumber crops. The disease consists of chlorotic

angular spots on the leaves that evolve to interveinalyellowing. Large whitefly populations of Trialeyrodesvaporariorum (Westwood) and Bemisia tabaci (Genna-dius) were observed in affected plants, suggesting thepossible involvement of whitefly-transmitted crinivirus-es. Moreover, similar symptoms are also caused by Cu-curbit aphid-borne virus (CABYV). In 2000 and 2001the disease was widespread with an incidence of 50–80%, and sometimes as much as 100%. Identification ofBeet pseudo-yellows virus (BPYV) and Cucurbit yellowstunting disorder virus (CYSDV) was carried out withthe reverse transcription-polymerase chain reaction (RT-PCR) and the sequencing part of the HSP70 gene ana-logue. A simplified method of multiplex RT-PCR was alsodeveloped, using as template crude plant extract, forthe rapid, sensitive and simultaneous detection of BPYV,CYSDV and CABYV. The results showed that BPYV wasthe predominant virus in cucumber and melon crops,whereas CYSDV was isolated only in Rhodes and Leo-nidio. This is the first report of CYSDV in Greece. CA-BYV was detected only in four cucumber crops. BPYValso infected arable weeds such as Amaranthus retro-flexus, Selosia cristata and Sonchus oleraceus surround-ing cucurbit crops.

Relationships between Aphis spiraecola Patch andCucumber mosaic virus (CMV). K.E. KAPETANOPOU-LOU and P.E. KYRIAKOPOULOU. Agricultural University ofAthens, Laboratory of Plant Pathology, 75 Iera Odos str.,11855 Athens, Greece.

In 1998, in an extended epidemiological investigationon Cucumber mosaic virus (CMV) in the tomato-processing area of Eleia prefecture, a high correlationwas found between twice-weekly catches of the aphidAphis spiraecola in Moericke traps and the frequencyof CMV infection. The correlation prompted us to studythis aphid species as a vector of CMV, using analyticaltransmission experiments in the laboratory. In a pre-vious study of this aphid species as a vector of CMV inIsrael, field catches of the aphid were used as experi-mental vectors. Three clones of the aphid, two fromAttica (from sweet orange and Spiraea media) and onefrom Corinthia (from sweet orange), raised in the lab-oratory on Spiraea media, were used. Three isolates ofCMV, one from Gastouni, Eleia and two from Corin-thia were used in the transmission experiments, andthe Nicotiniana tabacum cv. Xanthi and Samsung werethe virus-sources and indicator plants. The classicalaphid virus vector Myzus persicae Sulzer (raised onpepper) was used for comparison. The experiment wascarried out in an air-conditioned greenhouse. DAS-ELI-SA was used for virus detection in the experimentalplants. The results showed that A. spiraecola was avector of CMV. Transmissibility ranged from 2.8 to 28%in the various treatments. The highest values were


AA.VV.

obtained with acquisition feeding periods of 1–6 min-utes as compared with the longer periods of 60, 120and 240 minutes. This was expected, considering thenon-persistent transmission of CMV (optimum, a fewminutes). No differences in transmission efficiencywere observed between aphid clones and virus isolates,but efficiency differed depending on the number ofaphid individuals per indicator plant: transmissionnever occurred with one aphid/plant, but increased withincreasing number of aphids/plant. Five to sevenaphids/plant gave an easily measurable transmissionrate (12.5–28% of experimental plants infected). Thetransmission efficiency of A. spiraecola was slightlylower than that of M. persicae. Transmission of the vi-rus by A. spiraecola occurred as an erratic phenome-non with low reproducibility, this was also the case withM. persicae. In a comparative experiment on tobacco,with mixed infections of CMV and Potato virus Y (PVY)as the virus source, A. spiraecola (5–7 individuals/testplant) was a much more efficient vector of PVY (92%)than of CMV (14%). The present work showed that A.spiraecola participated in the natural transmission ofCMV, as well as in epidemics of this virus, dependingon its populations and their movements. The epidemi-ological significance of A. spiraecola in the particularexperimental field of 1998 in Savalia of Eleia was ob-vious, since the twice-weekly aphid captures carriedout at the height of tomato plants (Moericke traps)yielded individuals of this species almost exclusivelythroughout the season and in very high numbers.

Resistance of rice to Fusarium oxysporum f. sp.oryzae. M. PAPADOPOULOU. Technological Education In-stitute of Kalamata, Faculty of Agricultural Technology,Antikalamos 24100 Kalamata, Greece.

Root rot is a very serious and widespread disease ofrice. A study of rice plantations in Kazakhstan showedthat parasites belonging to the genus Fusarium (es-pecially F. oxysporum Schlecht f. sp. oryzae Bilai)cause root rot of rice. Among the measures to protectthe plants we singled out the use of varieties resist-ant to the disease. To determine the resistance of riceto root rot in the laboratory, five-day-old seedlingswere tested. Inoculation was performed by dipping theplant root system into a 106 ml-1 spore suspension ofthe fungus or by placing rice seeds with the fungalculture on Petri dishes containing an agar medium.Field inoculation was performed by incorporating intothe soil 30–50 g per 100 m2 of fungal culture grown onoat seeds. A total of 1500 samples of rice were checked,850 of which came from the collection of the NationalBank of Genetic Material of the N. Vavilov Instituteof Plant Growth (Petersburg, Russia). None of the ricevarieties proved immune to the fungus, only 19 vari-eties proved highly resistant, 10.6% of varieties

showed low sensitivity to root rot, 33.5% medium sen-sitivity, and 30.4% high sensitivity. The resistant va-rieties were tested in various kinds of crossings. Thosevarieties tolerant to cold were also resistant to thefungus. It was also found that disease resistance wascytoplasmically inherited.

The fungus Dematophora necatrix on cotton. K.ELENA. Benaki Phytopathological Institute, 8 St. Deltastr., 14561 Kifissia, Athens, Greece.

In August 1994, in the Fthiotis area, cotton plants werefound with roots severely damaged by Dematophoranecatrix Hartig (perfect state Rosellinia necatrix Prill.).In August 2001 the disease appeared again on cottonplants var. Vulcano in Evia Island. Plants in the fieldhad yellow leaves and progressively wilted and died. Thedisease caused decline, sudden wilting or apoplexy ofthe plants. The central root was severely attacked, rot-ted, covered with cottony white mycelium, and infectedtissues turned brown. The symptoms appeared in themiddle of July. The disease was severe in only one field,in which cotton had been cultivated successively in pre-vious years. The aim of this study was to isolate andidentify the causal agent of the disease in Evia Islandand to investigate the pathogenicity of isolates of D. ne-catrix to cotton plants of different varieties and ages.Isolates from diseased roots did not form the conidial orthe teleomorph stage. The mycelium was white and hadpear-shaped swellings adjacent to the septa. In twoweeks, the cultures became dark-brown as the fungusformed dark-brown sclerotia-like globose structures. Thefungus was identified as D. necatrix by its morphologi-cal characteristics. Three representative D. necatrix iso-lates were used to inoculate seeds or seedlings of thecotton varieties Acala SJ2, Christina and Myrto usingtwo inoculation methods. All three strains proved to behighly pathogenic to 13 and 30-day-old plants of all va-rieties. It is clear that this disease of cotton caused se-vere damage in the field where it occurred. The fungusisolates, as was demonstrated in the greenhouse patho-genicity experiments, attack and destroy cotton seedsand seedlings, causing the same symptoms. In the sum-mer, when the disease appears in the field, it can causesevere losses.

Host plants of Ralstonia solanacearum in the Ka-lavryta area. A.S. ALIVIZATOS1, P.E. GLYNOS1, C. KARAF-LA1, C. ZIAZIARI2 and F. STATHOPOULOS2. 1Benaki Phy-topathological Institute, 8 St. Delta str., 14561 Kifissia,Athens, Greece. 2Regional Plant Protection and QualityControl Centre of Patras, 23 Zaimi str., 26221 Patras,Greece.

The quarantine bacterium Ralstonia solanacearum (Rs)was isolated from 3 out of 6 samples of tomato plantsthat had symptoms of bacterial wilt collected from the

75Vol. 45, No. 1 April, 2006


area of Petsaki Village, Kalavryta. Subsequently theoccurrence of Rs in the Kalavryta area was studied bylaboratory-testing 39 samples of ware potato tubers,collected from the areas of 11 villages, two samples ofegg-plants, one sample of pepper plant, 14 samples ofweeds from the banks of the river Selinous and 13 wa-ter samples from this river. Rs was detected in 19 pota-to tuber samples from 7 areas and in one eggplant sam-ple, but not in any samples of pepper, weeds or water.Isolates of Rs from all positive potato, tomato and egg-plant samples were identified on the basis of morpho-logical, physiological, biochemical, immunological (IFtest) and pathogenicity tests according to EC Directive98/57/EC. This is the second report of Rs on ware pota-to, and comes 40 years after the first. Instructions onmeasures to be taken for the eradication of Rs from po-tato, tomato and eggplant fields, and for avoiding theundesirable consequences to the area and the countryas a whole, were given to the growers (Directive 98/57/EC, PD 255/2000).

Watermelon fruit spot caused by Pseudomonas sy-ringae pv. syringae. P.E. GLYNOS1, A. PARASKEVOPOU-LOS2, A.S. ALIVIZATOS1 and C. KARAFLA1. 1Benaki Phy-topathological Institute, 8 St. Delta str., 14561 Kifissia,Athens, Greece. 2Messinia Prefecture, Trifilia Directionof Agriculture and Animal Husbandry, Department ofPlant Protection, 24500 Kiparissia, Greece.

Small necrotic spots surrounded by a halo, and smalloily spots were observed on watermelon fruits (Citrul-lus vulgaris Schrad.), late in May 2001 in the area ofAgrili, Filiatra, and Trifilia. When the epidermal tis-sues were cut, the oily spots were found to have pro-gressed and spread deeper into the rind. Transversallycut fruits showed a spread of the infection to the fleshas far as the center of the fruit. Infected tissues had anoily appearance and brown discoloration at the edge.These symptoms appeared in a 2-ha field located in alarger 120-ha watermelon-growing area, following fa-vorable conditions created by a hailstorm and high rel-ative humidity. Affected tissues were not watery or rot-ted. Bacteria of the genus Pseudomonas were constant-ly isolated in pure culture from these oily spots. Mor-phological, physiological, biochemical characters andpathogenicity tests (on tobacco leaves and watermelonfruits) identified four of the isolates as Pseudomonassyringae pv. syringae (Pss) (members of the Ia group ofthe LOPAT tests). To our knowledge this is the first re-port of Pss on watermelon in Greece and worldwide.

Physiological stress on clover due to tropospher-ic ozone toxicity. D. VELISSARIOU1, A. ASSIMAKOPOULOU2

and A. KOLOKOUTSAS1. 1Technological Education Institute(TEI) of Kalamata, School of Technology-Agronomy,24100 Antikalamos, Kalamata, Greece. 2Benaki Phy-

topathological Institute, 8 St. Delta str., 14561 Kifissia,Athens, Greece.

The effect of tropospheric ozone on the growth and phys-iology of clover plants was studied at an experimentalsite at Benaki Phytopathological Institute, Kifissia, Ath-ens, in the summer of 2001. Two special biotypes of clo-ver (Trifolium repens) were used. These two biotypesare broadly used as ozone bioindicators, one being sen-sitive and the other resistant to ozone damage. Twentyplants of each biotype were grown in pots and harvest-ed every 28 days, to measure the above-ground dryweight of both biotypes. The visible symptoms of ozonetoxicity were scored on each biotype before each har-vest. In addition, stomatal conductance and net assimi-lation rate were measured between harvests, using anLci 2003 (ADC) Infra Red Gas Analyzer. The resultsshowed persistent statistically significant loss of drybiomass and higher ozone toxicity scores for the sensi-tive biotype than for the resistant one. As well, stomat-al conductance of the sensitive biotype was in generallower than that of the resistant one. The net assimila-tion rate showed no differences between the two bio-types. Clover plants, as bioindicators, recorded highlyphytotoxic levels of ozone in the area, and this was con-firmed by ozone levels officially monitored by a Stateair-pollution monitoring station at a close distance tothe experimental site. The effects on growth and thedifferences in physiological responses between the twobiotypes indicate that the vegetation growing in a pho-tochemically polluted climate, is subject to long-term,stresses that are often without visible symptoms but canhave severe effects on their performance.

Identification of Phytophthora palmivora isolatesfrom Dieffenbachia and evaluation of their sensi-tivity to metalaxyl. K. ELENA1, G. MATTHAIADOU2 andA.C. PAPPAS2. 1Benaki Phytopathological Institute, 8 St.Delta str., 14561 Kifissia, Athens, Greece. 2University ofThessaly, School of Agriculture, Plant Production andAgricultural Environment, 38446 N. Ionia, Volos, Greece.

From Dieffenbachia maculata plants showing soft footrot, in the spring of 2001, a fungus of the genus Phy-tophthora was constantly isolated on PDA medium. Afungus with similar morphological and physiologicalcharacteristics had also been isolated from diseasedDieffenbachias originaing from the same glasshouse in1994. On the basis of morphological and physiologicalcharacteristics such as chlamydospores, antheridia,oospores, sporangia, maximum temperature limit formycelial growth, and the rate of colony development,the 2001 isolates were identified as P. palmivora But-ler. This species has been a severe pathogen on Dieffen-bachia in various states of the USA, since it was firstreported in 1947. This is its first report of this funguson Dieffenbachia plants outside the USA. Twelve ran-


AA.VV.

domly selected isolates were tested for their sensitivityto phenylamides. These isolates were obtained from aDieffenbachia crop where the mixture metalaxyl 7.5%+ mancozeb 56% was applied as a soil drench (1 g l-1).Applications were made at 3-month intervals, followingeach transplanting, during the period 1989–2001. Eval-uations of the mycelial growth and sporangia produc-tion on PDA media amended with various concentra-tions of metalaxyl revealed that all isolates were sensi-tive to phenylamides. Concentrations of 0.1 and 0.5 mgl-1 of metalaxyl completely inhibited sporangia produc-tion and mycelial growth, respectively. Although themixture of metalaxyl plus mancozeb has been continu-ously applied for 12 years, it still offers satisfactory con-trol of Phytophthora soft foot rot of Dieffenbachia.

Fusarium wilt, caused by Fusarium oxysporum f.sp. basilici, a new and serious disease of basil (Oci-mum basilicum) in Greece. D. BIRIS1, D.J. VAKA-LOUNAKIS2 and E. KLIRONOMOU2. 1National AgriculturalResearch Foundation (N.AG.RE.F), Plant ProtectionInstitute, 38001 Volos, Greece. 2National AgriculturalResearch Foundation (N.AG.RE.F), Plant ProtectionInstitute, 71003 Heraklion, Crete, Greece.

Fusarium wilt is a disease of basil (Ocimum basilicum)new to Greece, recognized for the first time in 1999 inVolos. In the immediately following years it was foundin all greenhouse and field crops of the small-leavedcultivars of basil around Volos, becoming epidemic, aswell as in Agrinion and Heraklion, Crete. The causalagent, Fusarium oxysporum f. sp. basilici, was identi-fied at the species level on the basis of morphologicalcharacteristics, and at the forma specialis level usingpathogenicity tests and by vegetative compatibilitygrouping (VCG). All the Greek isolates of the pathogenexamined were found to belong to VCG 040, which isthe only VCG of the pathogen found so far worldwide.This suggests that the pathogen was probably intro-duced to Greece through imported organic substratesor seeds from some other country, in which it had al-ready been established. In pure cultures on PDA, thefungus grows in a wide range of temperatures, with anoptimum close to 27°C. The fungus is a soil-borne par-asite, but it can also infect the above-ground parts ofthe host, acting as a foliage parasite and then can moveto the crown and the root of the plant. The disease canappear at all the growth stages of basil. In the seedbed and the young seedlings the disease is manifestedas damping-off. In older plants, the first symptoms arethose of drought, i.e. a dull light-green coloring of thefoliage, withering, shriveling and partial leaf fall. Wiltcan be restricted to only some branches of the plant(hemiplegia), but may gradually extend to the wholeplant, which finally dries up. The symptoms often ap-pear suddenly throughout the whole plant which with-

ers and dies very soon (apoplexy). The interior of theinfected tissues turns dark in color, while their sur-face under high-humidity conditions is covered by acream-colored mycelium and conidia of the fungus.This, and the possibility of infection of the plantsthrough the foliage facilitate the spread of the disease.The fact that the fungus can also act as a foliage path-ogen, which is unusual for a soil-borne pathogen, re-quires a new strategy for disease control.

Effect of nitrogen concentration on the levels ofmicronutrients in spinach. A. ASSIMAKOPOULOU, G.TROYANOS and A. VITORATOS. Benaki PhytopathologicalInstitute, 8 St. Delta str., 14561 Kifissia, Athens, Greece.

The effect that seven concentrations of nitrogen, 1, 3,6, 10, 14, 16 and 22 mM N, in the nutrient solutionhad on the concentrations of Fe, Mn, Zn and Cu in theleaves and roots of the curly leaved spinach cv. Viro-flay, was studied in a hydroponic system. Nitrogen wasadded as nitrate (NO3-N), except at the concentrationof 14 mM N, where it was added as ammonium at aratio 3:1 (NO3-N:NH4-N). At the 14 mM N concentra-tion the growth of leaves was significantly higher thanwith all other N concentrations. Increasing the N con-centration past 14 mM did not affect the level of Fe,but decreased that of Mn (r=-0.66), Zn (r=-0.48) andCu (r=-0.68). At the 14 mM N concentration, root con-centrations of Fe and Cu increased, whereas that ofMn decreased; no significant differences were foundamong Zn concentrations at any N concentrations. Thetotal plant dry weight was significantly correlated withtheir content in Fe (r=0.91), Mn (r=0.82), Zn (r=0.76)and Cu (r=0.85). At the 14 mM N concentration, theuptake of Fe, Mn, Zn and Cu, expressed as mg of ele-ment g-1 d wt of root, was found higher than that withother N concentrations.

Occurrence of Tomato yellow leaf curl virus on to-mato crops in Greece. A.D. AVGELIS1, N. RODITAKIS1,C.I. DOVAS2, N.I. KATIS2, C. VARVERI3, N. VASSILAKOS3 andF. BEM3. 1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute, 71003 Herak-lion, Crete, Greece. 2Aristotle University of Thessaloni-ki, Faculty of Agriculture, Plant Pathology Laboratory,P.O. Box 269, 54124 Thessaloniki, Greece. 3Benaki Phy-topathological Institute, Laboratory of Virology, 14561Kifissia, Athens, Greece.

In the late summer of 2000, tomato (Lycopersicon lyco-persicum Mill.) crops grown under greenhouses in Ier-apetra, Tympaki and Chania (Crete) showed leaf curl-ing, reduced leaf size, yellowing, short internodes andbushy appearance. More than 30 ha of tomato green-houses were affected and the disease incidence rangedfrom 15 to 60%. Similar symptoms were observed intomato samples from Marathon (Attiki) and the south-

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ern Peloponnese, while in 2001 an outbreak was ob-served in Rhodes island. All greenhouses with infectedplants were infested by dense populations of Bemisiatabaci (Gennadius), which were also observed outsidethe greenhouses on several weeds. Tomato symptomswere similar to those caused by Tomato yellow leaf curlvirus (TYLCV). The assumed virus could not be trans-mitted mechanically but successful transmission wasobtained by grafting on healthy tomato plants. Over100 samples of symptomatic tomato plants collectedfrom Crete and the southern Peloponnese gave a posi-tive reaction when tested by ELISA using monoclonalantibodies (Adgen Ltd, TYLCV-European). The sero-logical results were confirmed by PCR using two pairsof primers, amplifying different parts of the virus ge-nome. The RFLP analysis (AluI, HaeIII and TaqI) ofthe 541 bp amplicon obtained with the first primer pair,showed patterns similar to the Israeli species of TYL-CV (TYLCV-Is). The second pair of primers gave theexpected 348 bp product which was sequenced. Se-quence comparisons revealed 99% identity with TYL-CV-Is (X15656, X76319). Identity with other isolatesof the Israeli cluster (Dominician Republic-AF024715,Cuba-AJ223505, Portugal-AF105975, Iran-AJ13271,Spain-AF071228) was at least 97.7%. This is the firsttime that TYLCV-Is caused damage in tomato cultiva-tion in Greece.

Production of transgenic plants carrying part ofthe RNA polymerase gene of Cucumber green mot-tle mosaic virus (CGMMV) and their resistance tothe virus. N. VASSILAKOS, A.S. ROTA and F. BEM. BenakiPhytopathological Institute, Department of Phytopathol-ogy, Laboratory of Virology, 8 St. Delta str., 14561 Kifis-sia, Athens, Greece.

Plant transformation using parts of viral RNA polymer-ase genes has been used for the production of plantsresistant to the relative viruses. This approach was fol-lowed in the present study for the production of trans-genic plants resistant to Cucumber green mottle mosaicvirus (CGMMV, genus Tobamovirus). First, primers weredesigned according to the published sequence of CGM-MV isolate SH, for the amplification of the 57K part ofthe RNA polymerase gene of the Greek CGMMV isolateGR-7. Amplification was carried out on total RNA iso-lated from infected cucumber plant. Primers incorpo-rated XbaI and BamHI restriction sites for the inser-tion of the PCR product in pKSII and subsequently Es-cherichia coli DH5a cells were transformed. Recom-binant pKSII/57K plasmid was subcloned into the planttransformation vector pWRok2 and mobilized into Agro-bacterium tumefaciens strain LBA4404 by triparentalmating using the helper plasmid pRK2013. Nicotianabenthamiana plants were then transformed and theirresistance to CGMMV is under study.

Sudden decline of Washingctonia filifera causedby Erwinia carotovora subsp. carotovora and Er-winia chrysanthemi. D.E. GOUMAS1 and S. MAGOUFI2.1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute, P.O. Box 2228,71003 Heraklion, Crete, Greece. 2Technological Educa-tion Institute, P.O. Box 1939, 71004 Heraklion, Crete,Greece.

A sudden decline of some Washingctonia trees (Wash-ingctonia filifera) has been observed over the last fewyears in the city of Heraklion, Crete. Declined treesare usually less than ten years old. The inner newleaves initially show a pale green discoloration; laterall leaves gradually become yellowish. Yellowing startsfrom the crown periphery and moves downward. Abrown discoloration and rot of the inner tissues of leafstalk, stem and roots also occurs with the whole plantusually declining and dying within two months. Bac-teria have been consistently isolated from diseased tis-sues. So far, six isolates from declining trees have beenidentified by their morphological, physiological andbiochemical profile (tests API 20E and 50CHE): theyare members of the species Erwinia carotovora subsp.carotovora and E. chrysanthemi. The symptoms of thedisease have been reproduced on six-month to one-year-old Washingctonia seedlings, after injection of a bacte-rial suspension (106 cfu ml-1) into the crown area of theseedlings.

Characterization of Agrobacterium tumefaciensisolates from pepper. D.E. GOUMAS1 and E. PROUSANI-DOU2. 1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute, P.O. Box 2228,71003 Heraklion, Crete, Greece. 2Technological Educa-tion Institute, P.O. Box 1939, 71004 Heraklion, Crete,Greece.

During the last two years, important and unusual in-fections by Agrobacterium tumefaciens have been ob-served on pepper growing in different parts of Crete (Ier-apetra, Arvi and Tympaki). Infection is manifested bygall formation mainly on the stem, with a disease inci-dence ranging from 20 to 100%. Galls develop at thelevel of the crown and in injured parts of the plant stem.Initially tumorous tissues are formed at pruningwounds, reaching a size bigger than the stem diameter.Pepper plants with many galls are stunted and eventu-ally die. Infected plants show microelement deficiencies.The crop period was reduced to one to two months, andconsequently yield losses were estimated at 10–30%.Bacteria isolated from the galls were identified by stand-ard morphological, biochemical and physiological testsas tumorigenic strains of Agrobacterium tumefaciens.The high disease incidence is possibly correlated withthe extensive use of commercially available peppertransplants.


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Leek rot caused by the bacterium Pseudomonassyringae pv. porri. P.E. GLYNOS and A.S. ALIVIZATOS.Benaki Phytopathological Institute, 8 St. Delta str., 14561Kifissia, Athens, Greece.

Small, oily irregular, isolated spots without a halo wereobserved on leek (Allium porrum L.) leaves early inAugust 2000, in the area of Megara, Attica, Greece.These spots later coalesced to form larger spots and tospread to the whole leaf area. Fall of the affected leaveswas observed at an advanced stage of the disease, suchfall was favoured by the overhead irrigation. Observa-tion under the microscope of oily spot tissues taken fromdiseased samples of leek plants, showed large numbersof bacterial cells exuding from the diseased tissues.Spread of the bacterial suspension onto culture mediaNA and NAS, caused one type of colonies to form, whichwere identified as colonies of the bacterial genus Pseu-domonas. Based on the morphological, cultural, biochem-ical, immunological, pathogenicity characteristics andthe electrophoretic pattern of whole cell proteins of fourisolates of the bacterium, these colonies were identifiedas members of the Ia group of the LOPAT tests and asPseudomonas syringae pv. porri. This is the first reportof this pathovar for Greece.

Macromycetes associated with Alnus glutinosa inGreece. D.M. DIMOU1, E. POLEMIS1,2 and G.I. ZERVAKIS2.1Agricultural University of Athens, Laboratory of Gen-eral and Agricultural Microbiology, 75 Iera Odos str.,11855 Athens, Greece. 2National Agricultural ResearchFoundation (N.AG.RE.F), Plant Protection Institute ofKalamata, 85 Lakonikis str., 24100 Kalamata, Greece.

The genus Alnus in Greece is almost exclusively repre-sented by Alnus glutinosa (L.) Gaertner. This tree spe-cies is distributed throughout northern and centralGreece, in central and western Peloponnese and insome Aegean islands (Andros, Naxos, Ikaria). It growsin damp regions, often by lakes or rivers, solitary or instands. Although these are habitats that favor the oc-currence of macrofungi, the reports from Alnus inGreece are extremely few. During the last six yearsAlnus stands have been investigated in only two re-gions: near the mountainous village of Gardiki (Fthi-otida) by the Nisvaris torrent and in the Vori valley ofAndros island by the seaside. Approximately 50 mac-romycetes species on Alnus have been recorded on Al-nus, and almost all of them constitute new records forGreece or they were reported on/under Alnus for thefirst time. Paxillus rubicundulus is the only speciesfound which is known to be associated exclusively withAlnus, while all the others occur in diverse habitats.Some of the wood-destroying species recorded were:Bjerkandera adusta, Botryobasidium candidans, Co-niophora puteana, Crepidotus cinnabarinus, Cyathusstriatus, Exidia thuretiana, Phellinus ferruginosus,

Hyphoderma setigerum, Scutellinia scutellata, andTomentella stuposa. In addition, Conocybe siennophyl-la, Entoloma ameides, Hemimycena cuculata, H. ep-ichloë, Hypholoma polytrichi, Limacella guttata, Om-phalina philonotis, Russula subfoetens var. grata, Tu-baria conspersa, and Xerocomus truncatus were foundgrowing near A. glutinosa (on the soil, on plant remainsor among grasses and moss). Of particular interest isthe finding of Galeropsis angusticeps, which is the firstrepresentative of the family Galeropsidaceae and thesecond secotioid species ever reported in Greece.

Susceptibility of melon cv. Golden Head to racesof Fusarium oxysporum f. sp. melonis. K. ELENA1,A.C. PAPPAS2 and V. PAPAVASSILIOU1. 1Benaki Phytopatho-logical Institute, 8 St. Delta str., 14561 Kifissia, Athens,Greece. 2University of Thessaly, School of Agriculture,Plant Production and Agricultural Environment, 38446,N. Ionia, Volos, Greece.

The susceptibility of melon cv. Golden Head to Fusari-um oxysporum Schlecht. f. sp. melonis (Leach and Cur-rence) Snyder and Hansen isolates of the four knownraces was evaluated. Eight isolates from Israel, repre-senting the four races (0, 1, 2, and 1,2), 10 isolates fromGreece, originating from various commercial melon cul-tivars, and belonging to races 0, 2, and 1,2, and 9 Greekisolates of undetermined race, were tested. Pathogenic-ity was tested by dipping the roots of 12-day-old seed-lings in a conidial suspension of the fungal isolates (107

conidia ml-1). Disease severity was assessed 30 days af-ter inoculation. Melon cv. Golden Head was susceptibleto four races of F. oxysporum f. sp. melonis and to allisolates with undetermined race. The isolates of races1, 2, and 1,2 were highly virulent. Most of the inoculat-ed plants showed apoplexy. The fungus also had a nega-tive affect on the plant’s vigor. By the end of the exper-iment the diseased plants had significantly less heightand dry weight compared with the uninoculated con-trols. Fusarium wilt is the main limiting factor for thereduction of melon cultivation in some areas of north-ern Greece.

Pathogenicity of Phytophthora species to tomatohybrids and a tomato variety. K. ELENA and I. SPYRO-POULOU. Benaki Phytopathological Institute, 8 St. Deltastr., 14561 Kifissia, Athens, Greece.

The virulence of five isolates of the genus Phytophthorawas tested on hybrids or varieties of tomato. The iso-lates tested were the BPIC1131 of Phytophthora capsiciLeonian, BPIC1184 of P. cryptogea Pethybridge andLafferty, BPIC1197 of P. erythroseptica Pethybridge, andBPIC1132 and BPIC1133 of P. nicotianae Breda de Haan.Thirty-day-old tomato seedlings cv. Rio Bojo and the hy-brids Belladona F1, Electra F1, Polo F1, tomato 1028,tomato 1410, tomato 1415 and tomato 1418 were used.

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Isolates of the fungus were subcultured on Petri disheswith corn meal agar (CMA) for 15 days. Inoculum wasprepared by blending the contents of 14 inoculated dish-es in a litre of deionized water. Twenty ml of inoculumwas poured into the soil around the stem of each plant.Seven plants from each variety were inoculated witheach Phytophthora strain. The isolates most virulent toall the tomato plants were these of P. nicotianae, andspecifically P. nicotianae isolate BPIC1133. The tomatoplants most resistant to this virulent isolate were thehybrids Belladona F1 and Polo F1. The isolates of P. cap-sici and P. erythroseptica were not very virulent. Theisolate of P. cryptogea was medium virulent. The studyshowed that tomato was primarily attacked by P. nico-tianae.

First report of Alternaria spp. as a foliar patho-gen of lettuce in Greece. I.A. LAIDOU and C.C. THA-NASSOULOPOULOS. Aristotelian University of Thessaloni-ki, Faculty of Agriculture, Plant Pathology Laboratory,P.O. Box 26, 54006 Thessaloniki, Greece.

Heavily spotted leaves of lettuce were found in the win-ter of 1999, 2000 and 2001 in parts of northern Greece,Thessaloniki and Halkidiki. The brown necrotic spotswere initially rather small, with concentric cycles, andlater becamne confluent, creating extensive infectedareas. Intense spotting occurred mainly on older leaves.From the leaves, eight isolates of fungi belonging to thegenus Alternaria were isolated on potato dextrose agar.Pathogenicity tests were performed on healthy, four-week-old plants inoculated with a sterile water suspen-sion containing 5�103 conidia ml-1 plus 0.05% Tween20. Control plants received only sterile distilled waterplus 0.05% Tween 20. After incubation in plastic bagsfor 24 h, the plants were replanted near the greenhouse.Symptoms identical to the original ones were observedten days after inoculation in all plants except the con-trol plants. From the eight isolates, three belonged togroup 1; one to group 2; three to group 6 (according toSimmons’ taxonomy) and one did not sporulate. This isthe first report of Alternaria spp. causing leaf spottingof lettuce in Greece.

First report of Alternaria alternata as a foliarpathogen of a Magnolia sp. in Europe. I.A. LAIDOU

and C.C. THANASSOULOPOULOS. Aristotelian University ofThessaloniki, Faculty of Agriculture, Plant PathologyLaboratory, P.O. Box 26, 54006 Thessaloniki, Greece.

Leaves of Magnolia plants were found covered withsmall, brown to necrotic spots during a disease surveyof Magnolia (Magnolia sp.) in the spring of 1999, 2000and 2001 in northern Greece. Intense spotting was as-sociated with defoliation as well as impairment of theornamental value of the plants. Three fungal isolatesfrom symptomatic leaves incubated on PDA belonged

to the genus Alternaria. Pathogenicity tests, accord-ing to Koch’s postulates, were conducted by woundingleaves with a sterilized needle and inoculating twoplants per isolate with 5 ml of a sterile water suspen-sion of 5�103 conidia ml-1. Two control plants receivedonly sterile distilled water. Plants were placed in agrowth chamber at 25oC. A week later, necrotic lesionsdeveloped on the leaves similar to those originally ob-served and the fungi were reisolated from all exceptthe control plants. Of these three isolates, one belongedto group 1 and the rest belonged to group 4 accordingto Simmons’s taxonomy. One of those isolates that be-longed to group 4 was identified as Alternaria alterna-ta (Nees: Fries) Keissler, by its conidial and morpho-logical characteristics. This is the first report of Alter-naria alternata causing leaf spotting and defoliationof Magnolia plants in Europe.

Hosts of Verticillium dahliae race 2 in Greece andworldwide. E.K. LIGOXIGAKIS1, D.J. VAKALOUNAKIS1 andC.C. THANASSOULOPOULOS2. 1National Agricultural Re-search Foundation (N.AG.RE.F), Plant Protection Insti-tute, 71003 Heraklion, Crete, Greece. 2Aristotelian Uni-versity of Thessaloniki, Faculty of Agriculture, PlantPathology Laboratory, 54006 Thessaloniki, Greece.

To determine new hosts of Verticillium dahliae race 2in Greece, the pathogenicity of 105 isolates, collectedin several parts of Crete in 1997–2002 from 25 culti-vated and 12 weed species, was checked in artificialinoculation tests (root-dip technique) on the differen-tial tomato cultivars Earlypak No 7 (lacking the Vegene) and ACE 55 VF (possessing the Ve gene). Of the105 isolates: 50, obtained from 21 cultivated species:Brassica oleracea var. botrytis, B. oleracea var. capita-ta, B. oleracea var. italica, CaΠcum annuum, Cicer arie-tinum, Cichorium endivia, C. intybus, Citrullus vul-garis, Cucumis melo, C. sativus, Cucurbita pepo, Lac-tuca sativa var. longifolia, Lathyrus ochrus, Lycopersi-con esculentum, Olea europea, Phaseolus vulgaris, Ra-phanus sativus, Solanum melongena, S. tuberosum,Tagetes erecta and Vicia sativa, and 17, obtained fromnine weed species: Capsella bursa-pastoris, Cardariadraba, Convolvulus arvensis, Erodium sp., Raphanusraphanistrum, Senecio vulgaris, Sinapis alba, Solanumnigrum and Trifolium sp., belonged to race 2. Of theremaining 38 isolates, 12, obtained from nine cultivat-ed and one weed species, belonged to race 1; whereas26, obtained from ten cultivated and six weeds specieswere not pathogenic to tomato. Of the 30 hosts of V.dahliae race 2, nine: C. arietinum, C. melo, C. sativus,C. pepo, L. sativa var. longifolia, L. esculentum, O. eu-ropea, S. melongena and S. nigrum, belonging to fivebotanical families, are known; while the remaining 21,belonging to seven families, are newly reported world-wide.


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Pathotypes of Verticillium dahliae in Greece. E.K.LIGOXIGAKIS1, D.J. VAKALOUNAKIS1 and C.C. THANASSOULO-POULOS2. 1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute, 710 03, Herak-lion, Crete, Greece. 2Aristotelian University of Thessalo-niki, Faculty of Agriculture, Plant Pathology Laborato-ry, 540 06 Thessaloniki, Greece.

To determine the pathotypes of Verticillium dahliae inGreece, 105 isolates of the fungus, collected from sev-eral parts of Crete in 1997–2002 from 25 cultivatedand 12 weed plant species, were checked for pathogenic-ity in artificial inoculation tests using the root-dip tech-nique on four differential plant species: Brassica rapavar. rapifera, Capsicum annuum, Lycopersicon esculen-tum and Solanum melongena. Seventy-one of the 105isolates, from 21 cultivated and 10 weed species be-longing to ten families, were pathogenic to tomato, egg-plant and turnip, and classified as pathotype I; 26 iso-lates, from ten cultivated and six weed species, belong-ing to seven families, were pathogenic to eggplant andturnip, and classified as pathotype II; and eight iso-lates, from two cultivated species belonging to one fam-ily, were pathogenic to all four differential plant spe-cies and classified as pathotype III. Pathotypes I, IIand III are widely, moderately and sparsely distribut-ed respectively.

In vitro and in planta screening of a non–patho-genic isolate of Fusarium oxysporum and two Pseu-domonas strains as biocontrol agents against Ver-ticillium dahliae on eggplant. G.A. BARDAS, C.C.THANASSOULOPOULOS and A.L. LAGOPODI. Aristotle Univer-sity of Thessaloniki, Faculty of Agriculture, Plant Pa-thology Lab., 54006 Thessaloniki, Greece.

The non-pathogenic isolate Fo47 of Fusarium oxyspo-rum and the bacterial strains Pseudomonas fluorescensWCS365 and P. chlororaphis PCL1391 were tested inplanta and in vitro for their potential as biocontrolagents against the fungus Verticillium dahliae on egg-plant cv. Tsakoniki. In vitro tests were carried out onthe interactions between the bacterial strains and thepathogenic fungus in dual and triple cultures, on King’sbroth medium+2% bacteriological agar. The growth ofV. dahliae was not inhibited by P. fluorescens WCS365.In contrast, P. chlororaphis PCL1391 caused completeinhibition of V. dahliae growth. In the in planta tests,test plants were grown under the simultaneous effect ofV. dahliae and one of the biocontrol agents. The patho-genic fungus and the non-pathogenic Fo47 were mixedthoroughly in the plant growth medium in a ratio of 300microslerotia of V. dahliae and 3000 conidia-1 of Fo47per g of soil. Bacteria were applied by a seed coatingwith methyl cellulose containing 2�109 cfu ml-1. Only P.chlororaphis PCL1391 reduced disease incidence signif-icantly. This bacterial strain did not cause significant

differences of measurable plant growth characteristicsin comparison to plants that were grown with V. dahl-iae alone.

Incidence of insect-borne cucurbit viruses inGreece. C. PAPAVASSILIOU1, C.I. DOVAS1, L.C. PAPAYIANNIS1,A.D. AVGELIS2, P.E. KYRIAKOPOULOU3, K. DOULIAS4 and N.I.KATIS1. 1Aristotle University of Thessaloniki, Plant Pa-thology Laboratory, 54124 Thessaloniki, Greece. 2CropProtection Institute, Plant Virus Laboratory, Katsambas,71100 Heraklion, Greece. 3Agricultural University ofAthens, Faculty of Crop Sciences, Department of PlantPathology, 75 Iera Odos str., 11855 Athens, Greece. 4Hel-lenic Sugar Industry, Orestias Factory, Plant PathologyDepartment, 68200 Orestiada, Greece.

In 1999 and 2000, a survey was carried out to deter-mine the incidence of insect-borne viruses in cucurbitcrops in Greece. A total of 1453 and 4995 leaf sampleswere collected in 1999 and 2000 respectively, fromsymptomatic plants in 32 parts of Greece. Tests wereperformed by ELISA using antibodies against: Cucum-ber mosaic virus (CMV), Zucchini yellow mosaic virus(ZYMV), Watermelon mosaic virus-2 (WMV-2), Papayaringspot virus, (PRSV) Zucchini yellow fleck virus(ZYFV), Squash mosaic virus (SqMV) and Cucurbitaphid-borne yellows virus (CABYV). A total of 1951 ar-able weeds (38 species) were also tested by mechanicalinoculation onto indicator plants, while 834 sampleswere tested for CABYV by ELISA. Watermelon sam-ples were predominantly infected with WMV-2 (49%)and ZYMV (43%), and with PRSV (7%) in a limitednumber of samples. Melon samples were infected pre-dominantly with WMV-2 (76%), followed by CMV (50%)and CABYV (20%). A limited number of melon sam-ples was infected with ZYMV (7%), SqMV (6%) andPRSV (1%). Zucchini samples were infected with WMV-2, (78%), CABYV (40%), ZYMV (31%), CMV (11%),ZYFV (3%), PRSV (2%) and SqMV (0.15%). Cucumbersamples were infected with CMV (53%), WMV-2 (20%),CABYV (20%), ZYMV (5%) and PRSV (1%). Arable weedplants (4%) of the families Amaranthaceae, Caryophyl-laceae, Compositae, Cruciferae, Cucurbitaceae, Labia-tae, Malvaceae, Portulacaceae, Rubiaceae, Solanace-ae, Umbelliferae, Urticaceae and Zygophylaceae wereinfected with CMV, ZYMV, ZYFV and WMV-2, where-as CABYV was detected on Amaranthus retroflexus andAbutilon theophrasti.

Study of potential aerial infection of woundedstems of cucumber plants grafted onto rootstockresistant to Fusarium oxysporum f. sp. radicis-cu-cumerinum. G.C. PAVLOU1 and D.J. VAKALOUNAKIS2.1N.AG.RE.F, Olive and Horticultural Crops Institute, 85Lakonikis str., 24100 Kalamata, Greece. 2National Agri-cultural Research Foundation (N.AG.RE.F), Plant Pro-

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tection Institute, P.O. Box 2228, 71003 Heraklion, Crete,Greece.

The capacity of Fusarium oxysporum f. sp. radicis-cu-cumerinum to infect aerially wounded stems of cucum-ber plants (Brunex F1) grafted onto resistant rootstockTZ-148 F1 (Cucurbita maxima�C. moschata) was inves-tigated in an experimental unheated (Ierapetra’s type)greenhouse. Two sets of artificial-inoculation experi-ments were carried out on wounded stems and cross-sectioned petioles of adult plants, 36 and 125 days old,in autumn (Nov 27) and winter (Feb 4), respectively. Thefollowing inoculation treatments were given in five rep-licates arranged in a complete randomized block design:(A) In autumn: (i) through incisions, 2–3 cm long and2–3 mm deep, using a surgical blade. (ii) through inci-sions tangentially on the stems, using a surgical blade,where a piece of tissue1.5–2 cm long, 0.5 cm wide and2–3 mm thick was removed. Incisions were swabbed witha spore suspension of the pathogen adjusted to 7�106

spores ml-1. (B) In winter: (i) through incisions and swab-bings made as in summer treatments (i) and (ii). (ii)through incisions made as in summer treatment (i) andas well spraying with the same spore suspension. (iii)through cross-sections of petioles on the nodes, using asurgical blade and spraying them with the same sporesuspension. (iv) through cross-sections of petioles as in(iii) and then dusted with a soil/powder mixture con-taining 1.4�107 spores ml-1. All treatments in both sea-sons were applied at three heights on the stem: 20, 100and 180 cm from the ground. Wounded stems of the cu-cumber plants could be infected with the pathogen atany height. Since heavy masses of pathogenic conidiaare produced on the stems of diseased plants in com-mercial greenhouse cucumber crops, and since theseconidia can be aerially transmitted to neighbouringplants, it is concluded that F. oxysporum f. sp. radicis-cucumerinum can cause secondary infections on theabove-ground parts of cucumber, like a polycyclic foliarpathogen.

Preliminary results from the inventory of macro-fungi in mountainous areas of Naupactia (Ai-toloakarnania, central Greece). E. POLEMIS1,2, D.M.DIMOU2, L. POUNTZAS3,4, D. TZANOUDAKIS3 and G.I.ZERVAKIS1. 1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute of Kalamata, 85Lakonikis str., 24100 Kalamata, Greece. 2AgriculturalUniversity of Athens, Laboratory of General and Agri-cultural Microbiology, 75 Iera Odos str., 11855 Athens,Greece. 3University of Patras, Department of Biology,Panepistimioupoli, 26500 Rion, Greece. 4TechnologicalEducation Institute of Mesologgi, Nea Ktiria, 30200 Mes-ologgi, Greece.

A mycofloristic study of the Naupactia region was re-cently undertaken as a part of a project on the inven-

tory and mapping of Greek macromycetes. The fieldstudy started in autumn 2001, within the greater areaof the Kryoneria, Ambelakiotissa, Ano Chora and Lim-nitsa villages, at an altitude of 600–1200 m. The ex-amined sites consisted mainly of Quercus pubescens andAbies cephalonica forests, and mixed stands of A. ce-phalonica with Castanea sativa or Carpinus oriental-is, Mediterranean evergreen shrubs, streams, moun-tain meadows and pastures, etc. From the specimenscollected so far, 83 species of macrofungi have beenidentified, which belong to 61 genera. Of these, 17 arereported for the first time in Greece: Phaeomarasmiuserinaceus (first record of the genus in Greece); Gleo-cystidiellum luridum and Hypoxylon fuscum on C. ori-entalis; Hemimycena crispula; Tomentella badia; T. fi-brosa and Tubulicrinis sororius on C. sativa; Hyphode-rma argillaceum; Panaeolus guttulatus and Pluteusplautus on Junglans regia; and Tubulicrinis calothrixon A. cephalonica. Furthermore, Cheilymenia sterco-rea, Conocybe coprophila, and Coprinus schroeteri werefound on cow manure, and Entoloma nitens, Omphali-na grisella and Russula alutacea in mixed stands of A.cephalonica and C. sativa. The mycorrhizal taxa Bo-letus calopus, B. queletii, B. reticulatus, Lactariusazonites, L. piperatus, Russula acrifolia and R. nigri-cans are reported for the first time in association withC. sativa, while 17 wood-rot fungi are first host-recordsin Greece.

Nitrate critical concentrations in lettuce plants.Y. TROYANOS and A. ASSIMAKOPOULOU. Benaki Phytopatho-logical Institute, 8 St. Delta str., 14561 Kifissia, Athens,Greece.

In lettuce growing, it is important to apply nitrogen fer-tilizer according to the nitrogen requirements of the crop,so as to produce plants with a nitrate content low enoughfor safe human consumption. Current nitrogen require-ments are assessed by means of plant-tissue analysis.This technique is a useful guide to achieve high nitro-gen use efficiency and minimize environmental pollu-tion. However, the optimization of nitrogen nutrition inthe field by applying diagnostic tissue analysis is time-consuming and sometimes not easy to apply in fast grow-ing plants like lettuce. For this reason a semi-quantita-tive method of nitrate-nitrogen determination, knownas Merckoquant strips, was used in this study, whichwas carried out on butterhead lettuce grown in sandculture with different concentrations of nitrate. The ni-trate-nitrogen content of petioles of young, fully expand-ed leaves, as determined by Merckoquant strips, corre-lated well with the growth rate of the plants. These re-sults along with the additional information obtainedfrom field trials showed that the Merckoquant stripscould be used reliably to check the nitrogen nutrition oflettuce plants.


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Molecular diagnosis of the cucumber pathogensFusarium oxysporum f. sp. cucumerinum and F.oxysporum f. sp. radicis-cucumerinum. G.A. FRAG-KIADAKIS and D.J. VAKALOUNAKIS. National AgriculturalResearch Foundation (N.AG.RE.F), Plant ProtectionInstitute, P.O. Box 2228, 71003 Heraklion, Crete, Greece.

In a previous study (Vakalounakis and. Fragkiadakis,1999. Phytopathology 89, 161–168) we used randomamplification of polymorphic DNA (RAPD) to assess thegenetic variability of 106 pathogenic isolates of Fusari-um oxysporum obtained from cucumber and classifiedby pathogenicity tests as formae speciales of either cu-cumerinum (Fusarium wilt) or radicis-cucumerinum(root and stem rot). For diagnostic purposes, twelve pol-ymorphic RAPD-bands that distinguished the isolatesof the two formae speciales were purified from 2% agar-ose gels, cloned and sequenced in both DNA strands.Sequences were used to design specific primer pairs of18–20 nucleotides in length. The specificity and sensi-tivity of the primer pairs in the detection/identificationof each pathogen was tested using the polymerase chainreaction. Five pairs of primers that distinguished thetwo formae speciales using the polymerase chain reac-tion with an annealing temperature of 54oC were de-tected.

Viruses infecting pea (Pisum sativum L.) crops inGreece. E.K. CHATZIVASSILIOU1, H. BOUBOURAKAS1, S. WIN-TER2, D.E. LESEMANN2, A.D. AVGELIS3 and N.I. KATIS1. 1Ar-istotle University of Thessaloniki, Faculty of Agriculture,Plant Pathology Laboratory, P.O. Box 269, 54124 Thes-saloniki, Greece. 2Department of Plant Virology, Micro-biology and Biosafety, Messeweg 11-12, D-38104 Braun-schweig, Germany. 3National Agricultural ResearchFoundation (N.AG.RE.F), Plant Protection Institute,Plant Virology Laboratory, 71110 Heraklion, Crete,Greece.

During the spring of 2000, pea crops in the Thessalyand Thessaloniki prefectures showed severe virus-likesymptoms consisting of vein-clearing, leaf and pod dis-tortion, stem necrosis, and stunting. Samples from symp-tomatic plants were collected and tested with electronmicroscopy (EM), as well as serologically with DAS andTAS ELISA. In the EM preparations, filamentous virusparticles were observed in most cases, while serologicalmethods identified seven viruses either in single ormixed infections. Pea enation mosaic virus (PEMV)(44%), Bean leaf roll virus (BLRV) (40%) and Pea seed-borne mosaic virus (PSbMV) (12%) were found in themajority of the samples. A lower incidence was recordedfor Cucumber mosaic virus (CMV) (4%), Beet mosaic vi-rus (BtMV) (4%) and Beet western yellows virus (BWYV)(4%). A limited number of samples collected in Thessal-oniki were infected with Tomato spotted wilt virus(TSWV). None of the samples were infected with Potato

virus Y (PVY), Broad bean wilt virus (BBWV), Turnipmosaic virus (TuMV), Watermelon mosaic virus 2 (WMV-2), Lettuce mosaic virus (LMV), Bean common mosaicvirus (BCMV), Alfalfa mosaic virus (AMV) or Tobaccomosaic virus (TMV). Additionally, a primer set was de-signed for the detection of RNA-1 of PEMV, and polymer-ase chain reaction (PCR) tests showed the presence ofboth RNAs of the PEMV complex. The occurrence of oth-er viruses as well as the incidence of seed-borne virusesin the seed lots that were used for the establishment ofthe affected crops are under study.

CHEMICAL CONTROL


The control of late blight of potato and tomato andgrape downy mildew in southern Europe with aFamoxate® based mixture. C. THEOCHARIS1, Y. STAMA-TAS1, P. CAGNIEUL2. 1Du Pont Agro Hellas S.A., 12 Solo-mou str. and Vas. Georgiou 15232 Athens, Greece. 2DuPont de Nemours France S.A., C.P.P., 137 rue del’Université, F-75334 Paris Cédex 07, France.

Famoxadone (Famoxate®) is a new active ingredientfrom DuPont with outstanding fungicidal properties.On grape downy mildew (Plasmopara viticola) and lateblight of potato and tomato (Phytophthora infestans)it primarily exerts a potent preventive activity by in-hibiting spore survival and germination even at verylow concentrations. Mixtures of famoxadone (Famox-ate®) and cymoxanil (Curzate‚) were tested on potatoes,tomatoes and grapes for combined protectant and cur-ative activity. The results of field experiments indicatethat in southern Europe this combination can providebetter control of P. infestans than the standard mix-tures with penetrant properties, resulting in parallelin a reduction of the total fungicide rate of more than80%.

Control of Cercospora leaf-spot disease of sugar-beets (Cercospora beticola) with specific sprayingprograms using fewer fungicide applications. C.DOULIAS1, A. GKIZELIS2, P.M. IOANNIDIS3. K. PASHALIDIS4 andX. NERANTZIS5. 1H.S.I. Orestias Sugar Factory, Plant Pro-tection Service, 68200 Orestiada. 2H.S.I. ThesalonikiHead Quarter, Plant Protection Service, 54000 Thesalo-niki. 3H.S.I. Platy Sugar Factory, Plant Protection Serv-ice, 59032 Platy Hemathias. 4H.S.I. Seres Sugar Facto-ry, Plant Protection Service, 62100 Seres. 5H.S.I. XanthiSugar Factory, Plant Protection Service, 67100 Xanthi,Greece.

The leaf-spot disease Cercospora beticola is the most im-portant disease of sugar beet, and under Greek climaticconditions can cause yield losses of up to 30–35%. Con-trol of this disease with chemical fungicides (6–8 treat-

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ments) is very expensive and has a high risk of develop-ment of resistance to fungicides and of environmentalconcerns. Proper resistant sugar beet varieties for defi-nite solutions have not yet been developed. A study ofthe application of specific fungicide programs givespromising results. An integrated pest management sys-tem for the control of C. beticola is being developed step-by-step based on the use of resistant varieties and spe-cific fungicide programs. This system has already im-proved control of leaf spot and reduced the cost. A widerange of spraying programs was evaluated in field tri-als in the framework of a project lasting several years.Climatic conditions (e.g. temperature, relative humidi-ty) and agronomic parameters (sowing date, row closure,irrigation), which are very important factors in deter-mining the timing and intervals of fungicide spraying,have been determined. The succession of fungicides ap-plied as stand-alone treatments, in mixtures or in al-ternation, has been defined. A warning system for spraysbased on prevailing temperatures was also studied. Highdaily temperatures (more than 32–35°C), which are verycommon during July, inhibit the germination and growthof C. beticola conidia, leading to suppression of the dis-ease. As long as these conditions are met the intervalbetween applications can be prolonged to as much as 30days, according to the susceptibility of the cv., resultingin reduced cost of control to farmers and a lower impacton the environment.

Effect of phenylpyrrole-resistant mutations on thephytopathogenic fitness of Botrytis cinerea. B.N.ZIOGAS, A.N. MARKOGLOU and V. SPYROPOULOU. Agricul-tural University of Athens, Laboratory of Pesticide Sci-ence, 75 Iera Odos str., 11855 Athens, Greece.

Mutants of Botrytis cinerea highly resistant to fludi-oxonil (Rf: 5.000, based on EC50 values) were isolatedfrom a wild-type strain, with a high mutation frequen-cy of 10-3, after chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG). Approximately 95% offludioxonil-resistant mutants were found to be moresensitive to high osmotic pressure than the wild-typestrain of B. cinerea. Cross-resistance studies with otherfungicides showed that the mutations for resistanceto phenylpyrroles affect the sensitivity of mutantstrains to the aromatic hydrocarbons dicloran (Rf: 100),chloroneb (Rf: 130) tecnazene (Rf: 70) and tolclofos-methyl (Rf: 750) and to the dicarboximides iprodione(Rf: 100) and chlozolinate (Rf: 40), but not to benzim-idazole benomyl, the mixture of benzimidazole-phenyl-carbamate (carbendazim+diethofencarb), to the ani-linopyrimidine cyprodinil, to the phenylpyridinaminefluazinam, to the hydroxyanilidine fenhexamid, tochlorothalonil and to the sterol biosynthesis inhibitorstriadimenol and fenpropimorph. A study of fitness pa-rameters in the wild-type and representative fludiox-

onil-resistant mutants of B. cinerea showed that theosmotic-sensitive isolates showed a significant reduc-tion in characteristics determining phytopathogenicfitness, such as mycelial growth, sporulation, conidialgermination, sclerotia production and pathogenicityon cucumber seedlings. By contrast, in the case of theosmotic-resistant isolates the mutations for phenylpyr-role-resistance may or may not affect phytopathogen-ic fitness. In planta experiments on cucumber seed-lings under greenhouse conditions showed that, withthe exception of the osmotic-resistant mutants, all theisolates of the osmotic-sensitive phenotypic class hada decreased infection ability compared with the wild-type. Preventive applications of the commercial for-mulation fludioxonil (Saphire 50 WP) were effectiveagainst lesion development on cotyledons by the wild-type, but were ineffective even in high concentrationsagainst disease caused by both phenotypic classes offludioxonil-resistant isolates. Experiments on the sta-bility of the fludioxonil-resistant phenotype showed asignificant reduction of resistance when the mutantisolates were grown on an inhibitor-free medium. Arecovery of the high resistance level was observed af-ter they were returned to the selection medium. Stud-ies on the competitive ability of resistant isolatesagainst the wild-type strain of B. cinerea by applica-tions of a mixed population showed a significant re-duction of resistant isolates with an increase in thepopulation of the wild-type strain.

Fungicidal action and the resistance risk of flu-azinam. B.N. ZIOGAS, A.G. VITORATOS, A. KYRIAKOU andS. GEORGOUDELI. Laboratory of Pesticide Science, Agri-cultural University of Athens, 75 Iera Odos str., 18855Athens, Greece.

Fluazinam is a new protective fungicide against Bo-trytis cinerea. It belongs to the chemical group of phe-nylpyridinamines with a broad spectrum of activityagainst phycomycetes, ascomycetes, basidiomycetesand adelomycetes. Ustilago maydis strains, with a low-to-moderate resistance to fluazinam (Rf: 5-50), wereisolated with a mutation frequency of 7�10-6 afterchemical mutagenesis with N-methyl-N-nitrosoguani-dine (MNNG). Genetic analysis resulted in the identi-fication of two chromosomal genes. A study of the ef-fect of mutant genes on the phytopathogenic fitness ofUstilago maydis, showed that the resistance mutationshad no apparent effect on the mycelial growth rate andon U. maydis pathogenicity on young corn plants.Cross-resistance studies showed that the mutations forresistance to fluazinam also caused resistance to oli-gomycin but not to dinitrophenol. A dose-dependentinhibition of glycose oxidation in whole cells was ob-served by both fluazinam and oligomycin, and a com-plete inhibition was found at 40 µg ml-1. In contrast,


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substrate oxidation was uninhibited by dinitrophenolat concentration up to 50 µg ml-1. These results sug-gest that fluazinam has a fungicidal action on ATP-synthetase than that it is an uncoupler of the oxida-tive phosphorylation from mitochondrial electron trans-port. Study of resistant risk to fluazinam in Botrytiscinerea showed the possibility of appearance of onlylow-resistant mutants (Rf: 5). Furthermore, study ofphytopathogenic fitness parameters of both wild andmutant strains showed that the mutations for resist-ance to fluazinam may or may not affect growth rate,conidial production and germination, and pathogenic-ity. Preventive applications of commercial formulationof fluazinam IKF-1216 SC (500 g l-1 fluazinam) wereeffective against lesion development on cotyledons byboth wild and mutant strains.

Study of the inherent resistance risk to fenhexa-mid in Botrytis cinerea. B.N. ZIOGAS, A.N. MARKOGLOU

and A.A. MALANDRAKIS. Laboratory of Pesticide Science,Agricultural University of Athens, 75 Iera Odos str.,18855 Athens, Greece.

After chemical mutagenesis of a wild-type strain of Bo-trytis cinerea with N-methyl-N-nitrosoguanidine(MNNG), two phenotypes that are respectively highlyand moderately resistant to fenhexamid were isolatedwith a high mutation frequency of 0.9�10-5. Resistancefactors, based on EC50 values, were 460–570 and 10–15 respectively. The mutation(s) for resistance to fen-hexamid did not affect the sensitivity of mutant strainsto the benzimidazole benomyl, the phenylpyridinaminefluazinam, the anilinopyrimidine cyprodinil, the gua-nidine iminoctadine or the sterol-biosynthesis-inhibit-ing fungicides fenarimol, fenpropimorph and tride-morph, but there was an increased sensitivity (EC50

ratios 0.2 to 0.6) of fenhexamid-resistant strains to thephenylpyrrole fludioxonil and the dicarboximide ipro-dione. Fitness parameters of fenhexamid-resistant iso-lates of both phenotypic classes showed that thesemutation(s) had no apparent effect on mycelial growthand on sensitivity to high osmolarity, but did affect oneor more other characteristics, such as sporulation, co-nidial germination and sclerotium production. In testson cucumber seedlings under greenhouse conditions,all highly fenhexamid-resistant isolates tested had low-er infection ability than the wild-type. Preventive ap-plications of a commercial formulation of fenhexamid,Teldor 50 WP (fenhexamid 500 g kg-1), were effectiveagainst lesion development on cotyledons by the wild-type, but ineffective, even in concentrations higher thanthe manufacturers’ recommended dose, against diseasecaused by the fenhexamid-resistant isolates. The riskof resistance to fenhexamid arising during commercialuse of fenhexamid is discussed in the light of these re-sults.

Protective and curative activity of the fungicideazoxystrobin against Cercospora beticola. T. ANE-SIADIS, G.S. KARAOGLANIDIS and K. TZAVELLA-KLONARI. Fac-ulty of Agriculture, Plant Pathology Laboratory, Aristo-telian University of Thessaloniki, P.O. Box 269, 54006,Thessaloniki, Greece.

Strobilurin fungicides constitute a new group of chemi-cal compounds with a novel mode of action and showingfungicidal activity against several fungal species. Inorder to evaluate the fungicidal activity of azoxystrobinagainst Cercospora beticola, a set of in planta experi-ments was carried out. Fungicides used in this studywere azoxystrobin (Quadris 25 SC) at 8 and 16 µg a.i.ml-1, difenoconazole (Score 25EC) at 8 µg a.i. ml-1 andchlorothalonil (Daconil 75WP) at 118.75 µg a.i. ml-1. Forthe purposes of the study, 4-5-week-old sugarbeet plants(cv. Rizor) were artificially inoculated with a conidialsuspension (11–20�104 spores ml-1) of a fungal isolateobtained from the field. After inoculation plants weremaintained in a growth-chamber at 25°C, RH 95% anda 16-h photoperiod. Fungicides were applied prior (pre-ventive treatments) and after (curative treatments) in-oculation at 24-, 48- and 96-hour-intervals respectively.Disease incidence was measured 20 days after inocula-tion using the 9-scale disease index of KWS. Resultsshowed that azoxystrobin provided mainly a preventiveaction, particularly when applied 24 h before artificialinoculation of the plants, with an efficacy level of 90%.Significantly lower, at 70%, was the control effective-ness provided by chlorothalonil when applied 24 h be-fore inoculation. Azoxystrobin also showed curative ac-tivity, particularly when applied 24 h after inoculationof the plants, with a control effectiveness of 85%; thiswas similar to that obtained by difenoconazole (82%) 24h after inoculation. The control effectiveness of cura-tive applications of chlorothalonil, at 60%, was signifi-cantly lower .

Effectiveness of the fungicide ORTIVA 25 SC(azoxystrobin 25%) on asparagus rust (Pucciniaasparagi). A. KAZANTZIDOU, I. PAPAGEORGIOU andA.TSINGAS. Syngenta Hellas A.E.B.E., Leoforos Anthou-sas, 15349 Anthousa, Attiki, Greece.

Ortiva is a broad-spectrum fungicide belonging to thegroup of strobilurins and it is used on a great number ofcrops. In 1999–2001 in 5 field trials carried out in thearea of Giannitsa, the effectiveness of ORTIVA againstasparagus rust was evaluated. Ortiva was applied atthe rates of 75, 80, 100 cc hl-1, and the reference productwas hexaconazole. Applications started preventively inJune, since ORTIVA is mainly a protective product, dueto its mode of action. Sprays were applied at 10–16-dayintervals. After the beginning of infection, 4–5 assess-ments were made in each trial. The infection appearedin July, and incidence became high (up to 74%) in the

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control plots. Statistical analysis showed that treatmentdifferences were highly significant, and that ORTIVAgave excellent (in some cases 100%) control. Phytotox-icity symptoms did not appear in any of the trials.

Cross-resistance patterns among Ergosterol bio-synthesis inhibiting fungicides (EBIs) in Cercospo-ra beticola. G.S. KARAOGLANIDIS and C.C. THANASSOULO-POULOS. Aristotelian University of Thessaloniki, Facultyof agriculture, Plant Pathology Laboratory P.O. Box 269,54006 Thessaloniki, Greece.

Thirty single-spore isolates of Cercospora beticola, col-lected from several fields in northern Greece and repre-senting a broad-spectrum sensitivity to the demethyla-tion-inhibiting fungicide flutriafol, were tested for sen-sitivity to eleven other ergosterol biuosynthesis inhibit-ing fungicides (EBIs) and to the guanidine fungicidedodine. Sensitivity was measured in terms of EC50 val-ues for each fungicide, log-transformed EC50 values werepairwise correlated and the correlation coefficient wasestimated. These pairwise comparisons showed high cor-relation coefficients between DMIs, indicating the ex-istence of cross-resistance relationships among them.However, the degree of cross-resistance between DMIsvaried greatly. Non-significant correlation coefficientswere measured for morpholine fungicide fenpropimor-ph and each DMI, suggesting the lack of cross-resist-ance between morpholines and DMIs in C. beticola. Sim-ilarly, low and statistically not different from 0 were thecorrelation coefficients measured with the pairs of theguanidine fungicide dodine with all the other fungicidestested, indicating that there is no negative cross-resist-ance between dodine and EBIs in C. beticola.

Sterol analysis in DMIs-resistant and -sensitivestrains of Cercospora beticola. G. S. KARAOGLANIDIS1,O. MENKISSOGLOU-SPIROUDI2 and C.C. THANASSOULOPOU-LOS1. 1Plant Pathology Laboratory. 2Pesticide Laboratoy,Faculty of Agriculture, Aristotelian University of Thes-saloniki, P.O. Box 269, 54006 Thessaloniki, Greece.

The effect of the sterol-demethylation-inhibiting fungi-cide flutriafol on the sterol composition of DMI-sensi-tive and resistant strains of C. beticola, was studied. Inthe absence of flutriafol these strains had the same sterolcomposition regardless of their sensitivity to DMIs. Er-gosterol was the major sterol of fungal membranes. Fol-lowing treatment with flutriafol, 4-desmethyl sterol lev-els were lower and eburicol, obtusifoliol and 14α-meth-ylergosta-8,14,24(28)-trienol were higher, indicating aninhibition of 14α-demethylase in all the C. beticolastrains tested. The accumulation of methyl-sterols inthe resistant strains occurred after their exposure toconcentrations of flutriafol higher than those in the caseof the sensitive strains. Therefore, DMI-resistance can-not be associated with a deficiency of the C-14 demeth-

ylation enzyme in the ergosterol biosynthetic pathway.The reason for the different effects of flutriafol on thesterol composition of the various C. beticola isolates maybe due to lower levels of the fungicide in the myceliumand to other, not yet identified mechanisms of resist-ance.

Pyrenophorin and pyrenophorol production byDrechslera avenae f. sp. sterilis. M.A. KASTANIAS andM. CHRYSAYI. Agricultural University of Athens, Pesti-cide Science Laboratory, 75 Iera Odos str., 11855 Ath-ens, Greece.

Drechslera avenae f. sp. sterilis is a forma specialis witha host-specificity for Avena sterilis. The fungus produc-es the phytotoxic macrodiolides pyrenophorin and pyren-ophorol. In the present study, the production of thesesecondary metabolites in culture medium was studiedover time. Quantitative determinations were made bygas chromatography-mass spectrometry (GC-MS).Pyrenophorin was at detectable levels in cultures incu-bated for three days (5 µg g-1 of culture medium), reacheda maximum yield in 10 days (364 µg g-1 of culture medi-um) and dropped to concentrations below the limit ofdetection after 25–27 days. Pyrenophorol was at detect-able levels in cultures incubated for five days (2 µg g-1 ofculture medium), reached a maximum production in 35days (240 µg g-1 of culture medium) and then the yielddeclined, but remained at detectable quantities (87 µgg-1 of culture medium) even after 90 days. The produc-tion pattern of the two macrodiolides indicates thatpyrenophorin may be a precursor in the pathway ofpyrenophorol biosynthesis.

Development and implementation of a new dy-namic and electrodynamic technique within theframework of electrostatic spraying. S. KOTSOPOU-LOS1, R. CRAMARIUC2, L. PANAGIOTOPOULOS3, N. YIOLDASIS1,S. FOTOPOULOS4, D. ZEVGOLIS4 and A. KOTSOPOULOS3. 1Wire-less Telecommunications Laboratory, Department of Elec-trical and Computer Engineering, School of Engineer-ing, University of Patras, Patra, Greece. 2National In-stitute for Scientific Research in Electrostatics and Elec-tro technologies, Bucharest, Romania. 3Department ofAgricultural Machinery and Irrigation, TechnologicalEducational Institute of Mesologgi, Greece. 4Departmentof Physics, University of Patras, Greece.

Within the framework of the upgrading of the Agri-cultural Sector, advanced machinery plays an impor-tant role, giving new directions to both the performedcultivation procedures and the existing antagonisticmarket environment. In the present work, the tech-nical parameters involved are investigated, in orderto apply new pesticide techniques. Moreover, a newmethod is proposed that is based on a technique ofspraying in an electrostatic field. Using the proposed


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technique, the minimum quantity of pesticide liquidis required. Moreover, for the improvement of the pro-posed technique, the following are examined: (a) Theprocess theoretical and experimental research for theimprovement of the dynamic systems of electrostaticspraying, (b) the process theoretical and experimen-tal research for the improvement of the electrodynam-ic systems of electrostatic spraying; and (c) the proc-ess theoretical and experimental research in the fieldsof scanning and image processing by means of a com-puter, for the analysis of the jet of spraying globules,via: (i) the design and the development of algorithmsfor 3-D image-scanning, (ii) the design and the devel-opment of algorithms for image-analysis and the rec-ognition of specific patterns, (iii) the scanning exper-iments and the image processing of the electrostaticfield of the dynamic and electrodynamic systems forelectrostatic spraying. The present work is part of theResearch Project “DEMETRA”, sponsored by the Hel-lenic General Secretariat of Research and Technolo-gy of the Ministry of Development. This project iswithin the framework of Bilateral Cooperation be-tween Greece and Romania.

Evaluation of the fungicide MAXIM 035 FS appliedas seed treatment for the control of soil-borne dis-eases in Maize. I. PAPAGEORGIOU, A. TSINGAS and A. KA-ZANTZIDOU. Syngenta Hellas A.E.B.E., Leoforos Anthou-sas, 15349 Anthousa Attiki, Greece.

In field trials the effectiveness of the fungicide MAXIM035 FS on soil-borne diseases caused by Pythium spp.and Fusarium spp. and affecting seeds and young plantsof maize was evaluated. MAXIM 035 FS contains 2.5 gl-1 fludioxonil + 1 g l-1 metalaxyl-m. Fludioxonil is a non-systemic active substance belonging to the group of phe-nylpyrroles and is effective against a number of fungisuch as Botrytis cinerea, Sclerotinia spp., Monilinia spp.,Rhizoctonia solani and Fusarium spp. Metalaxyl-m,which belongs to the group of phenylamides, is a sys-temic fungicide and is biologically active on fungi suchas Phytopthora, Pythium, Plasmopara, Peronospora,Pseudoperonospora. In 2000–2001, 4 field trials werecarried out in Greece. MAXIM was applied at two rates(70 and 100 cc 100 kg-1 seed) and was compared to thereference product APRON 35 WS (35% metalaxyl). Af-ter treatment, maize seeds were drilled in the field andemerged plants were counted 21 and 45 days after drill-ing. In plots where MAXIM was applied, seed-emergence21 days after drilling was 93%, while in the control plotsit was 83% (mean from 4 trials). In addition, 45 daysafter drilling the percentages were 89% in the treatedplots and 80% in the control plots. Statistical analysisdetected that differences between treated and untreat-ed plots were significant. Phytotoxicity symptoms werenot observed.

In vitro evaluation of antifungal activity of sapon-ins from the annual medic Medicago arabica. E.J.PAPLOMATAS1, A. TZIMA1, Z. BIALY2 and M. JURZYSTA2. 1Ag-ricultural University of Athens, Laboratory of Plant Pa-thology, 75 Iera Odos, 11855 Athens, Greece. 2Depart-ment of Biochemistry and Plant Quality, Institute of SoilScience and Plant Cultivation, Czartoryskich 8, 24 – 100Pulawy, Poland.

Saponins, glycosylated triterpenoid or steroid com-pounds, are widely found in higher plants. In many cas-es, the antifungal activity of saponins has been shownin vitro and in vivo. Saponins that have been isolatedfrom cultivated medic (alfalfa) Medicago sativa (L.) areusually a composite blend of triterpenoid glycosides.Depending on the structure of the aglycones, they canbe separated into several groups: derivatives of medica-genic acid, oleanolic acid, zanhic acid, hederagenin andsoyasapogenols. In contrast with alfalfa, in the annualherb medic Medicago arabica (L.), Huds. medicagenicor zanhic acid glycosides have never been found. Theaim of the present study was to evaluate in vitro theantifungal activity of eight saponin samples from M.arabica that had never been reported from a Medicagospecies before. The chemical structure of these saponishad been defined by FAB-MS and NMR to be glycosidesof hederagenin, bayogenin and 2-hydroxyoleanolic acid.The biological activity of the samples in the study wastested against the plant pathogenic fungi Pythium ulti-mum and Rhizoctonia solani (AG4). The saponins wereadded to the culture medium (PDA) at concentrationsof 10, 25, 50 and 100 µg ml-1. Their antifungal activitywas estimated as percent inhibition of the linear growthof the fungal mycelium compared with the untreatedcontrol. It was found that inhibition of growth with P.ultimum was 25.5%, while with R. solani it was evengreater: 38.8%. The biological activity of the most effec-tive in vitro saponins samples will be evaluated in plantaon host plants of the above phytopathogenic fungi.

Comparative toxicity of fungicides on Verticilliumfungicola and Agaricus bisporus. M. CHRYSAYI1, M.A.KASTANIAS1, P. DIAMANTOPOULOU2 and A. FILIPPOUSSIS2. 1Ag-ricultural University of Athens, Pesticide Science Labo-ratory, 75 Iera Odos str., 11855 Athens, Greece. 2Nation-al Agricultural Research Foundation (N.AG.RE.F), In-stitute of Agricultural Engineering, Edible Fungi Labo-ratory, 61 Democracies str., 13561 Athens, Greece.

Production of cultivated mushrooms can be very great-ly affected by fungi. The chemical control of fungi thatinfect edible mushrooms requires highly selective fun-gicides. The aim of this work was to evaluate in vitrothe effectiveness of the new fungicides famoxadone, tri-floxystrobin and tebuconazole against Verticillium fun-gicola, which causes dry bubble disease on the whitemushroom Agaricus bisporus. Prochloraz and carben-

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dazim were used as reference fungicides. The ED50 val-ues of famoxadone, trifloxystrobin, tebuconazole,prochloraz and carbendazim were 0.5, 0.12, 0.01, 0.002and 0.07 µg ml-1 respectively. The effect of these fungi-cides was also evaluated on the in vitro growth of A.bisporus on agar culture and, in an attempt to simulatecommercial mushroom cultivation, on a casing layer inglass tubes. All fungicides at concentrations which gavesatisfactory mycelial inhibition of V. fungicola were with-out effect on the mycelial growth of A. bisporus. Basedon the observed in vitro selectivity of the above fungi-cides, further evaluation of their formulated productsat commercial production scale seems of interest.

Effectiveness of BION (50% benzothiadiazole) inthe control of fire blight. J. TSIANTOS1 and P.G. PSAL-LIDAS2. 1National Agricultural Research Foundation(N.AG.RE.F), Plant Protection Institute of Volos, 38001Volos, Greece. 2Benaki Phytopathological Institute, 8 St.Delta str., 14561 Kifissia, Greece.

The effectiveness of BION (50% benzothiadiazole) atdoses of 20 g and 10 g 100 l-1 H2O, which induces sys-temic acquired resistance for controlling fire blight (Er-winia amylovora), was tested in the years 2000 and 2001.BION was compared with Agrept (20% streptomycinsulphate) at a dose of 50 g 100 l-1 H2O in 2000 and 2001,and Aliette (80% phosetyl-Al) at a dose of 250 g 100 l-1

H2O in 2001, and Kocide (50% copper-hydroxide) at adose of 90 g 100 l-1 H2O also in 2001. Whole pear trees(cultivar Krystalli) were sprayed with the chemicals twoand four days before artificial inoculation of preselect-ed flowers with a water suspension of the pathogen atconcentrations of 105 and 107 cfu ml-1. Kocide was ap-plied once two days before inoculation. The evaluationof the infection (percentage of infected flowers) was made15 days after the treatment. The results indicated thatBION at a dose of 20 g, and Aliette provided significantprotection, but were not as effective as Agrept. The chem-ical BION at 10 g and Kocide did not show any signifi-cant protection.

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