Phytopathol. Mediterr. (2005) 44, 00–00

4TH INTERNATIONAL WORKSHOP ON GRAPEVINE TRUNK DISEASES

“ESCA AND GRAPEVINE DECLINES”

jointly organised by

INTERNATIONAL COUNCIL ON GRAPEVINE TRUNK DISEASES (ICGTD) SOUTHERN AFRICAN SOCIETY FOR PLANT PATHOLOGY (SASPP) SOUTH AFRICAN SOCIETY FOR ENOLOGY AND VITICULTURE (SASEV)

20–21 January 2005 University of Stellenbosch, South Africa

ABSTRACTS

83 AA.VV.

Pathogen identification Petri disease or black goo is a serious disease of and detection young and old vines in most areas where grape- vines are cultivated. The disease is typically asso- Phaeoacremonium and Phaeoacremonium- ciated with grapevines exhibiting a slow dieback like fungi in the Calosphaeriales. P.W. C ROUS, as well as stunted growth. The dominant fungus W. G AMS and L. MOSTERT. Centraalbureau voor associated with Petri disease is Phaeomoniella Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, chlamydospora. Several species of Phaeoacremo- The Netherlands. nium (Pm.) are also associated with these disease E-mail: [email protected] symptoms, namely Pm. aleophilum, Pm. angustius, Although it is now commonly accepted that the ge- Pm. inflatipes, Pm. mortoniae, Pm. parasiticum, nus Phaeoacremonium has teleomorphs in Togninia, Pm. rubrigenum and Pm. viticola. Fourteen new hardly anything is known about other fungi within species of Phaeoacremonium have been identified the Calosphaeriales. During a survey of perithecial on the basis of cultural and morphological charac- ascomycetes in New Zealand, two collections of a ters as well as DNA phylogeny of the partial ß-tu- Togninia-like fungus were made on decayed wood. bulin and actin genes. Of these new species the In culture, colonies produced a Phaeoacremonium- following have been isolated from Vitis vinifera: like anamorph. In order to reveal the phylogenetic Pm. alvesii, Pm. australiense, Pm. krajdenii, Pm. relationships of the unknown fungus and its affini- scolyti, Pm. subulatum, Pm. venezuelense and a ty to Togninia and other genera in the Calosphaeri- further two unnamed species. New species isolat- ales, sequences of nuclear LSU and SSU ribosomal ed from hosts other than grapevines include: Pm. DNA were obtained of several members of this or- tardicrescens (human), Pm. griseorubrum (human), der. These data, supported by morphological and Pm. amstelodamense (human) and another three, cultural characteristics, confirm that the New Zea- yet unnamed species (soil, Theobroma grandiflo- land fungus represents a new genus very close to rum, Cupressus macrocarpa, Pinus radiata and Calosphaeria. The genus Togniniella is proposed to Desmoschoenus spiralis). Phaeoacremonium spe- accommodate these collections, while Phaeocrella is cies that were isolated from grapevines as well as established for their anamorphs. Furthermore, Ca- human infections are Pm. alvesii, Pm. krajdenii, losphaeria pulchella was found to form a distinct Pm. parasiticum and Pm. venezuelense. Re-exami- Acremonium-like anamorph in culture, for which the nation of isolates obtained as Pm. inflatipes showed genus Calosphaeriophora is proposed. Similarily the that those isolated from grapevines and humans genus Pleurostomophora is proposed to accommo- had originally been misidentified and belong to ei- date anamorphs of Pleurostoma, which are accom- ther Pm. aleophilum or the new taxa described modated in a new family, Pleurostomataceae. Togni- here. The status of Pm. angustius has been clari- nia with its Phaeoacremonium anamorphs, togeth- fied with the designation of an epitype from the er with Jobellisia, are closer to the Diaporthales, and original type locality. Isolates representing Pm. represent yet another distinct family, for which the angustius proved to be phylogenetically distinct Togniniaceae is proposed. The presence of signifi- from their sister species, Pm. viticola. Furthermore, cantly different anamorphs in the Calosphaeriales, a multiple-entry online electronic key based on as well as obvious differences in teleomorph mor- morphological, cultural and ß-tubulin sequence phology of species accommodated in Calosphaeria, data was developed to facilitate routine identifica- suggest that both the Calosphaeriales and Ca- tion of species. losphaeria, as presently perceived, are polyphyletic. Occurrence of Togninia fraxinopennsylvanica Delimitation of new species in Phaeoacremo- perithecia and Phaeoacremonium species in nium and the development of an identifica- California vineyards. A. ESKALEN, S. ROONEY-LATHAM tion system. L. MOSTERT, J.Z. GROENEWALD, W. and W.D. GUBLER. Department of Plant Pathology, GAMS, R.C. SUMMERBELL, V. ROBERT and P.W. CROUS. University of California, Davis, CA 95616, USA. Centraalbureau voor Schimmelcultures, Uppsala- E-mail: [email protected] laan 8, 3584 CT Utrecht, The Netherlands. Esca and Petri disease are two of the most destruc- E-mail: [email protected] tive diseases of grapevines in California and many

84 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines” other grape production countries. Esca has been a plification of TomRSV. Cuttings were taken from problem in California for over 70 years and is sus- grapevines naturally infected with TomRSV and pected of being caused by multiple species of Phae- grown under greenhouse conditions. After 6 oacremonium and Phaeomoniella chlamydospora. months, cuttings were tested using ELISA and real- In our study, we isolated Togninia fraxinopennsyl- time RT-PCR. Samples that tested positive based vanica multiple times from symptomatic grape- on ELISA were also positive based on real time RT- vines. Furthermore, spore trapping studies suggest PCR. The primers were specific and showed no that spores of this fungus are aerially present in primer dimers. Real-time PCR affords nursery infected vineyards, and perithecia of T. fraxi- owners and growers a non-destructive method for nopennsylvanica were identified in these vine- detecting the presence of pathogens involved in yards. Molecular analyses of the ITS region of the grapevine decline. The methods presented here nuclear ribosomal DNA confirmed that these could be utilized as detection systems in a clean perithecia were T. fraxinopennsylvanica. This work vine certification program. presents the first report of T. fraxinopennsylvani- ca on grapevines. Additionally, Pa. rubrigenum, Pa. White rot symptoms in esca affected grape- parasiticum, and Pa. angustius were also isolated vine: further insights into the biodiversity, from symptomatic grapevines. Pathogenicity tests host range and molecular diagnosis of asso- 1 2 have shown that many of these fungi effectively ciated basidiomycetes. M. FISCHER , F. MELA , L. 2 3 4 4 colonise pruning wounds resulting in vascular dis- MUGNAI , F. HALLEEN , J. EDWARDS and I. PASCOE . coloration. The probability of these fungi being non- 1Staatliches Weinbauinstitut Freiburg, Merzhauser injurious endophytes also exists. Str. 119, D-79100 Freiburg. 2Dipartimento di Bio- tecnologie Agrarie-Patologia vegetale, P.le delle Real-Time PCR-SYBR® Green detection of Cascine 28, I-50144 Firenze. 3Disease Management grapevine decline pathogens. B.E. OVERTON, Division, ARC Infruitec-Nietvoorbij, Private Bag 4 E.L. STEWART, XINSHUN QU, N.G. WENNER, B.J. X5026, Stellenbosch 7599, South Africa. Institute CHRIST and F.E. GILDOW. Department of Plant Pa- for Horticultural Development - Knoxfield, Depart- thology, Pennsylvania State University, University ment of Primary Industries, Private Bag 15, Fern- Park, PA 16802, USA. E-mail: [email protected] tree Gully, Victoria 3156, Australia. Petri disease of grape and Tomato ring spot virus E-mail: [email protected] (TomRSV) have been shown to be associated with While the spectrum of fungal organisms associat- vine decline in commercial vineyards. Petri disease ed with Petri disease seems to be pretty much uni- is caused by Phaeomoniella chlamydospora and form throughout wine-growing regions, available species in the genus Phaeoacremonium. The prim- evidence suggests that the situation is more com- er pairs, Pmo1f + Pmo2r and Pac1f + Pac2r, were plex for esca related white rot basidiomycetes. For designed for genus-specific amplification of Phae- instance, Fomitiporia mediterranea (Fmed) is as- omoniella chlamydospora (Pch) and Phaeoacremo- sociated with esca disease of grapevine mainly in nium spp. respectively, using real-time PCR. The Europe. The geographic distribution of Fmed is primers were specific for each target genus and distinct, covering different climatic zones in Eu- showed no primer dimers in the first 35 cycles. Pch rope and Asia; it is suggested to be closely linked was detected in roots, shoots, and young trunks of with viticulture, and is likely to have spread in drill-inoculated vines. Phaeoacremonium ale- recent years. Besides Vitis, host plants include a ophilum (Pal) was detected in trunk cross sections variety of other hardwood genera. During the last of naturally infected vines from which Pal had been century, increasing amounts of grapevine have been isolated. TomRSV is a polyhedral nematode-trans- cultivated in non-European countries. In recent missible virus (nepovirus) that causes various de- years, plantations throughout the world have been grees of systemic necrosis and stunting of growth found to suffer from white rot symptoms, caused in many hosts. The primer pair Trv2f + Trv2r was by so far unknown basidiomycetes. In general, designed based on sequences in Genbank from sev- fruiting structures of esca-related basidiomycetes eral hosts, including grape, for the one-step, re- are hard to find, or they may mostly occur on non- verse transcription (RT) and real-time PCR am- Vitis hosts, which are not well investigated. The

Vol. 44, No. 1, April 2005 85 AA.VV. existence of these fungi can be demonstrated only sociation among isolates from the localities evalu- by the vegetative mycelia that may be isolated from ated. This study demonstrated morphological and infected wood. In this study, molecular sequences molecular differences between the basidiomycete (nuclear encoded ribosomal ITS region) were gen- species associated with grapevine trunk diseases, erated for basidiomycete isolates from esca affect- Phellinus igniarius and Fomitiporia punctata, and ed grapevine in Europe, Australia and South Afri- the Hymenomycete fungus associated with chlo- ca. In a phylogenetic approach the sequences ob- rotic leaf roll symptoms, which was named Fomi- tained were compared with selected representa- tiporella vitis Auger, Aguilera & Esterio, sp. nov. tives of the Hymenochaetales. With these data, several taxa not easily assignable to already de- Molecular characterisation and identifica- scribed species were detected. In addition, taxon- tion of Eutypa spp. from grapevines in South 1 1 2 specific primers, prFmed1 and prFmed2, were de- Africa. S. SAFODIEN , F. HALLEEN , P.W. CROUS , W.A. 1 3 1 veloped. In a first-hand assay, these may help to SMIT and A. BOTHA . ARC Infruitec-Nietvoorbij, resolve the question to which extent Fmed and/or Private Bag X5026, Stellenbosch 7599, South Afri- closely related taxa are responible for the problem ca. 2Centraalbureau voor Schimmelcultures, Up- of esca-related white rot in grapevine. psalalaan 8, 3584 CT Utrecht, The Netherlands. 3Department of Microbiology, University of Stellen- Identification of basidiomycete species asso- bosch, Private Bag X1, Matieland (Stellenbosch) ciated with wood decay symptoms of grape- 7602, South Africa. vine chlorotic leaf roll in Chile. J. AUGER, N. E-mail: [email protected] AGUILERA and M. ESTERIO. Departamento de Sani- Eutypa dieback of grapevines holds enormous eco- dad Vegetal, Facultad de Ciencias Agronómicas, nomic implications for the vine and wine industry Universidad de Chile, Casilla 1004, Santiago, Chile. of South Africa. The pathogen responsible for the E-mail: [email protected] disease, Eutypa lata, is an ascomycetous fungus The grapevine disease named chlorotic leaf roll in that has a wide host range and has become a sig- Chile is comparable to esca disease in Europe, de- nificant problem in grape growing areas worldwide. spite some differences in the foliar symptoms and Since infected grapevines gradually decline and in the basidiomycete fungi associated with the wood eventually die, infection with this pathogen is caus- decay symptoms. The Phellinus sp. associated with ing significant reductions in yield. Consequently, chlorotic leaf roll symptoms might indicate that this an investigation of local Eutypa populations occur- basidiomycete is involved in the disease. Correct ring on grapevine was undertaken. Isolates were identification of the basidiomycete may also be rel- collected from grapevines and fruit trees with die- evant in determining possible sources of inoculum back symptoms, as well as from fruiting structures in vineyards. Identification was carried out on the on infected wood. Eutypa isolates were also ob- basis of morphological characteristics of the fruit- tained from Australia and France. The isolates ing bodies, such as size and form of the pores, ba- were analysed using sequence data from the inter- sidia and basidiospores, rate of growth and myce- nal transcribed spacer (ITS) region of the nuclear lial biochemical reactions, and sequence of the large ribosomal DNA, the ß-tubulin gene region and the subunit of ribosomal DNA, which was extracted large subunit to infer the phylogenetic relationship from fruit bodies. From the morphological charac- of local Eutypa populations. Analysis of the ITS teristics and phylogenetic analysis of the section sequence data revealed the presence of two groups: sequenced, it was concluded that the hymenomyc- Group I designated for isolates that produced an ete fungi associated with symptoms of chlorotic leaf ~600bp fragment, and Group II designated for roll belong to the genus Fomitiporella. They dif- those that produced an ~560bp fragment. The re- fered from the species Fomitiporella cavicola (Kotl. sults grouped most of the isolates from South Afri- & Pouz) T. Wagner & M. Fisher comb. nov. and F. ca and those obtained from Australia and France caryophylli (Racib) T. Wagner & M. Fisher comb. (Group I) in a well-supported clade with a boot- nov. described for this genus. Genetic variability strap support value of 81% with E. lata, except for of the isolates was determined by means of the STE-U 5581, which was closely related to E. lepto- RAPD technique and revealed a phylogenetic as- placa (bootstrap support value, 100%). The grape-

86 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines” vine isolates STE-U 5561 and 5562 and the fruit tabolite as a diagnostic marker of E. lata. Analysis tree isolates (STE-U 5550–5559) formed a sepa- of leaves, fruits, stems and cordons of grapevines rate cluster (Group II) showing sequence similari- exhibiting symptoms of dieback failed to show the ty to Eutypella vitis. The molecular data derived presence of metabolites. However, when cordon from the amplification of the three markers helped sections were placed in water and cultured for 5 to elucidate the phylogenetic relationship of local days, eutypinol was readily detected in the aque- Eutypa populations, and in particular revealed the ous solution; metabolites were not produced from presence of a second species, E. leptoplaca, which uninfected tissue. This provides a method for de- could be capable of causing disease. tection of infected tissue and indicates that the toxic metabolites react at the point of production, dis- Diagnosis of Eutypa infection by metabolite rupting the vascular structure, rather than being 1 1 analysis. R.J. MOLYNEUX , N. MAHONEY , L.R. translocated to symptomatic tissues. 1 2 3 SMITH , P.E. ROLSHAUSEN , R. LARDNER and E. 3 1 SCOTT . Western Regional Research Center, Agri- Further studies into Botryosphaeria as path- 1 cultural Research Service, U.S. Department of Ag- ogens of grapevine in California. J.R. URBEZ , 2 1 1 riculture, 800 Buchanan Street, Albany, CA 94710, G.M. LEAVITT and W.D. GUBLER . Department of USA. 2Department of Plant Pathology, University Plant Pathology, U.C. Davis, Davis, CA 95616, of California, One Shields Avenue, Davis, CA USA. 2Farm Advisor, UCCE, 328 Madera Ave. 95616, USA. 3Cooperative Research Centre for Viti- Madera, CA 93637, USA. culture, PO Box 154 and School of Agriculture and E-mail: [email protected] Wine, University of Adelaide, PMB 1, Glen Osmond, Botryosphaeria Ces. & De Not (1863) constitutes SA 5064, USA. a complex fungal genus with many taxonomic and E-mail: [email protected] nomenclature problems. Different species of Bo- Dying arm disease in grapevines is responsible for tryosphaeria occur on grapevines causing a wide vineyard production losses around the world, in- variety of diseases and symptoms in various geo- cluding California, and is believed to be due to acet- graphic regions worldwide. In this study, in order ylenic phenol fungal metabolites produced by Eu- to determine the occurrence of Botryosphaeria spp. typa lata. In order to identify specific metabolites in the main grape-producing areas in California, that could potentially be used for diagnosis of in- one hundred vineyards of different ages among fection, eight Eutypa strains isolated from grape- the predominant table and wine grape varieties vines were grown in vitro on hot water extracts, from eleven different counties (Mendocino, Sono- fortified with sucrose, of the susceptible grape va- ma, Napa, San Joaquin, Stanislaus, Madera, Fres- rieties Cabernet Sauvignon, Sauvignon Blanc and no, Monterey, Tulare, Kern and Riverside) were Syrah, and the tolerant varieties Merlot and Sem- sampled throughout the state. Botryosphaeria was illon. Analysis by HPLC showed that eutypinol was found associated with the internal wedge-shaped consistently produced in large amounts, together canker at a different percentage in every grape- with smaller amounts of methyleutypinol and eu- growing area surveyed in California. Preliminary latachromene. Eutypine, the putative toxin, was results, based on morphological colony characters rarely produced, and when present, amounts were and phylogenetic analysis of the internal tran- low. Since differences did not correlate with sus- scribed spacer (ITS) of the rDNA and a partial se- ceptible and tolerant grape varieties, the time- quence of the ß-tubulin gene, showed that at least course and profiles of metabolites produced by E. four different species of Botryosphaeria: B. obtusa, lata strains isolated from Cabernet Sauvignon and B. rhodina, B. dothidea and the unidentified Bo- Merlot grown on identical media were examined tryosphaeria sp1, occur in grapevines in Califor- to determine the effect of fungal source. Although nia. B. obtusa was the most common species iso- the amounts of metabolites produced differed sig- lated in California. Only B. rhodina was found in nificantly between strains, the pattern of metabo- vineyards sampled in southern California (River- lites was quite similar, with eutypinol again pre- side County). This study represents the first re- dominating. The consistent production of eutypi- port describing the population and distribution of nol indicated that this was the most suitable me- Botryosphaeria species on Californian grapevines.

Vol. 44, No. 1, April 2005 87 AA.VV.

Variability of Cylindrocarpon spp. associat- Merzhauser Str. 119, D-79100 Freiburg, Germany. ed with black foot disease of grapevine. C. 2Cooperative Research Centre for Viticulture, PO 1 1 2 2 REGO , T. NASCIMENTO , A. CABRAL , P. TALHINHAS , A. Box 154, Glen Osmond, South Australia 5064, Aus- 3 2 1 3 PHILLIPS and H. OLIVEIRA . Laboratório de Patolo- tralia. Primary Industries Research Victoria – gia Vegetal “Veríssimo de Almeida”, Tapada da Aju- Knoxfield Centre, Department of Primary Indus- da, 1349-017 Lisboa, Portugal. 2Instituto Superior tries, Private Bag 15, Ferntree Gully Delivery Cen- de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, tre, Victoria 3156, Australia. Portugal. 3Universidade Nova de Lisboa, Monte da E-mail: [email protected] Caparica, 2829-516 Caparica, Portugal. Phylogenetic species recognition allows identifica- E-mail: [email protected] tion of a new basidiomycetous species, Fomitipo- Black-foot disease caused by Cylindrocarpon spe- ria australiensis, associated with white heart rot cies is a serious problem of grapevine in many coun- of esca-affected grapevines in Australia. Microscop- tries. Seventy Cylindrocarpon isolates, obtained ic characters of fruit bodies are very similar to those from young grapevines with black foot symptoms of the closely related species, F. punctata and F. and from rootstocks showing decline symptoms in mediterranea. Fomitiporia australiensis is distinct the nursery, were analysed for morphological, cul- in forming both resupinate and pileate fruit bod- tural, pathogenicity characteristics and random ies and in the sequences of its ribosomal ITS re- amplified polymorphic DNA (RAPD). Culture mor- gion. Fruit bodies of this species are rarely found phology on PDA, especially colony colour, was high- in the field; it occurs mostly as vegetative myc- ly variable among the isolates studied. However, no elium. No definite statements are possible regard- meaningful pattern of variation was detected. Anal- ing the exact geographic distribution or host range ysis of morphological structures in SNAY and CLA of the species. media revealed that the majority of isolates formed abundant round slimy conidial heads and abundant Characterisation and identification of the chlamydospores. In contrast, a small group of iso- basidiomycetous fungus associated to ‘hoja lates formed longer and wider conidia and produced de malvón’ grapevine disease in Argentina. 1 1 1 1 few chlamydospores. Of the isolates, only eight grew L. BETTUCCI , S. LUPO , A. PÉREZ , S. MARTÍNEZ , C. 2 2 2 1 at 34°C and the optimum temperature for growth CÉSARI , G. ESCORIAZA and M. GATICA . Facultad was 24°C. Pathogenicity experiments on rooted cut- de Ciencias/Ingeniería, Laboratorio de Micología, tings of grapevine cv. Periquita under greenhouse Universidad de la República, J. Herrera y Reissig conditions revealed that all isolates induced black 565, 11300, Montevideo, Uruguay. 2INTA, EEA foot symptoms and the majority significantly Mendoza, San Martín 3853, Mayor Drummond, (P=0.05) decreased plant height, number of inter- 5507, Luján de Cuyo, Mendoza, Argentina. nodes, number of roots and root elongation. The E-mail: [email protected] percentage of pathogen re-isolations varied from 50 to 96%. RAPD profiles clearly distinguished two The pathogenic fungus Inocutis jamaicensis (Mur- main groups, and a third represented by a single rill) Gottlieb, J.E. Wright & Moncalvo was identi- isolate. These main RAPD groups were not clearly fied as the basidiomycetous species associated with correlated with the morphological characteristics hoja de malvón grapevine disease. Macro and mi- and pathogenicity tests. However, they indicated the cromorphological characteristics of fruiting bodies existence of a high degree of genetic variability (sim- collected from grapevine trunks corresponded to ilarity coefficient 0.29) within the Cylindrocarpon- those of this fungus. Cultural characters of the iso- complex on grapevines. It remains to be clarified if lates analysed also corresponded to isolates from this variability is intra- or inter-specific. Uruguayan specimens of this species from Euca- lyptus globulus and other native plant species. In Fomitiporia australiensis, a new species of order to compare the strains, polymerase chain Fomitiporia from Australian grapevines reaction (PCR) of the internal transcribed spacer found associated with heart rot of grapevines. (ITS) regions was performed. RFLPs from this re- 1 2,3 3 M. FISCHER , J. EDWARDS , J.H. CUNNINGTON and gion generated by restriction digestion with Alu I, 2,3 1 I.G. PASCOE . Weinbauinstitut Freiburg, Hae III, Taq I, Msp I and Hha I were compared

88 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines” with RFLPs of the product amplified by the same and plated onto 2% AMA. Isolates were identified primers from DNA obtained from cultures of I. ja- to genus using morphological and molecular means. maicensis from decayed wood of E. globulus. The The most common fungi isolated from the declin- profile of some enzymes matched those obtained ing vines were Botryosphaeria spp. 7/9, Clonostach- from I. jamaicensis associated with E. globulus. ys 5/9, Fusarium spp. 6/9, Phaeoacremonium spp. Moreover, an amplification product generated with 4/9, Phaeomoniella 5/9, and Phialophora-like iso- specific primers (INO1 and INO7) was observed lates. The necrotic wood and bark was often in- for the isolates of I. jamaicensis used in this study. fected with Clonostachys. Peeling back bark tis- The PCR products revealed a fragment of approx- sue showed necrotic phloem, new callus growth and imately 333 bp, which was expected. In addition, necrotic lesions bordered by a blackish film simi- the aligned partial sequence of LSU rDNA showed lar to symptoms associated with “Diplodia dieback.” a high percentage of identity. Differences found in The climate in Virginia includes summers with some restriction patterns could reflect a certain high humidity, summer rainfall, and heavy morn- degree of variability between strains, probably re- ing dew. This study suggests that graft unions in- lated to a particular lifestyle, host specificity or fected with Botryosphaeria and other fungi repre- geographic origin. A somatic incompatibility test sent a latent threat to vine health. In areas with and other methods used to identify genotypes are cold winters, the external symptoms may be con- necessary to clarify these differences. fused with cold injury-induced crown gall disease.

Fungi isolated from failing graft unions with Fungi associated with grapevine wood de- 1 2 necrotic lesions. L. MORTON , N.G. WENNER , E.L. cay and young vine decline in Chile. J. AU- 2 2 1 1 1 1 2 3 STEWART and B.E. OVERTON . Box 208, Broad Run, GER , I. PÉREZ , M. ESTERIO , V. NAVIA , W.D. GUBLER 2 3 1 VA 20137, USA. Department of Plant Pathology, The and A. ESKALEN . Departamento de Sanidad Vege- Pennsylvania State University, University Park, PA tal, Facultad de Ciencias Agronómicas, Universi- 16802, USA. dad de Chile, Casilla 1004, Santiago, Chile. 2Bayer E-mail: [email protected] CropScience, Santiago, Chile. 3Department of Declining vines in three Virginia vineyards exhib- Plant Pathology, University of California, Davis, ited fissured and failing graft unions with inter- CA, 95616, USA. nal necrotic lesions. External symptoms included E-mail: [email protected] marginal leaf chlorosis, total leaf reddening, lack The incidence of grapevine wood decay and young of fruit, severe cordon dieback, trunk mortality, and vine decline in Chile has increased continously vine death. Graft union deformation was visible over the last few years. Although Chile is one of accompanied by abnormal regenerative callus the world’s largest table grape exporters and the growth beneath the bark resembling crown gall. wine grape growing area has been increasing in Symptomatic vines were randomly scattered in the last few years, no systematic analysis has yet planting blocks of Merlot, Cabernet Sauvignon, and been carried out to evaluate the presence and in- Cabernet Franc on SO4, 5 BB, 3309, and Riparia cidence of fungal pathogens associated with trunk Gloire rootstocks. Vines originated from four dif- diseases of grapevine. In response to grower con- ferent nursery sources and were 3, 4, 5, and 20 cern, a survey of vineyards was initiated to deter- years old. Single and double-trunked vines in this mine the status and causes of grapevine decline study grew to full size (on lyre or VSP, at 2.1 m in the country. A significant number of samples of and 1.2 m between vines respectively). A 6-month rooted cuttings of different varieties grafted on study, from October 2003-March 2004, investigat- different rootstocks, ready for out-planting, or ed the fungi associated with these failing vines. planted and declined a few months or years later, The vineyards sampled were in three different ge- were collected or sent to our laboratory to inves- ographically- and geologically-distinct regions at tigate the presence of phytopatogenic fungi. Two- elevations of 10 m, 150 m, and 300 m. Vine sam- to nine-year-old and older vines were also exam- ples were cross-sectioned into disks on a band saw, ined. Additionally, plant and soil samples were and surface sterilized using a brief ethanol dip and taken to ascertain the presence of fungi and lev- flaming. Discolored wood was aseptically excised els of grapevine viruses, nematodes, and soil fer-

Vol. 44, No. 1, April 2005 89 AA.VV. tility. Cross sections of declining vines revealed infected with at least one of these fungi. With re- brown wood-streaking, wedge-shaped discolour- spect to the distribution of pathogens within the ation and pockets of white rot fungi. Tissue ex- plants, Phomopsis and Botryosphaeria were found plants from symptomatic plants were transferred mainly in the upper part of the rootstock, while to acidified PDA or Malt agar. Phaeomoniella Phaeomoniella chlamydospora was isolated from chlamydospora (Pc) and Phaeoacremonium spp., the lower half. Surprisingly, Cylindrocarpon spp. which included P. aleophilum (Pa), P. rubrigenum were isolated from the graft union in 15% of path- (Pr) and P. parasiticum (Pp), were isolated and ogen isolations. None of the rootstocks sampled identified. These pathogens were associated with (110R, 140Ru, 161-49C, 41B and SO4) were free young vine decline and with brown wood streak- from pathogens. ing in Pinot Noir, Cabernet Sauvignon, Ruby Seed- less, Thompson Seedless, Flame Seedless, Autumn Molecular phylogenetics of grapevine decline Royal, Red Globe and the rootstocks Kober 5BB, fungi from Pennsylvania and New York. B.E. C3309 and Harmony from 27 locations in the V, OVERTON, E.L. STEWART, and N.G. WENNER. Depart- VI, VII and Metropolitan regions of Chile. Pc oc- ment of Plant Pathology, The Pennsylvania State curred more frequently than Pa. Botryosphaeria University, University Park, PA 16802, USA. obtusa was isolated and identified. It was always E-mail: [email protected] associated with wedge-shaped wood discoloura- Twenty-seven vineyard sites comprising more tion or dead-arm symptoms, and mainly from the than 200 ha of vines were examined over three table grape cultivars Red Globe, Ruby Seedless growing seasons (2001–03) to quantify the extent and Thompson Seedless. A basidiomycete identi- of vine decline and to isolate fungi from declining fied as Fomitiporella vitis sp. nov. was always iso- vineyards. Three hundred and sixty-two fungal lated from wood with white soft rot and chlorotic isolates were recovered from vines exhibiting de- leaf roll symptoms. cline symptoms in Pennsylvania (PA) and New York (NY). Twenty-nine percent of the fungi iso- Occurrence of fungi associated with Petri lated were those associated with Petri disease. disease in bench-grafted vines. A. AROCA and Phaeomoniella chlamydospora, Phaeoacremonium R. RAPOSO. INIA, Ctra. Coruña km. 7.5, 28040 Ma- aleophilum, Phaeoacremonium mortoniae, Phae- drid, Spain. oacremonium rubrigenum and Phaeoacremonium E-mail: [email protected] angustius/viticola were identified by morpholog- Bench-grafted vines were sampled at the time of ical and molecular characters. Phaeoacremonium their delivery from Spanish grapevine nurseries inflatipes and P. paristicum were not isolated from to growers (March 2004) to evaluate the presence declining vines in PA or NY. Twenty-four percent of fungi associated with Petri disease and wood of the fungi isolated were Phialophora-like, refer- decay. A total of 34 samples (135 plants) were col- able to the genera Cadophora, Harpophora, and lected. Each sample consisted of 3–5 plants tak- Phialophora s.s. Twenty-four percent were pyc- en at random from a batch of bench-grafted vines. nidial fungi, with 8% identified to the genus Pho- Internal examination was done by cutting the mopsis. To date, the ITS rDNA gene region has rootstocks transversely at different intervals been sequenced for 96 of the 362 isolates. Data (roots, internodes and graft union). Six wood sec- obtained from the ITS rDNA region will be used tions 1–2-mm thick were obtained at each inter- to determine target strains for further phyloge- val to carry out fungal isolations. All plants netic study, including phylogenetic analyses based showed wood browning or blackening around the on other gene regions. Cadophora and Harpopho- pith at the basal end of the rootstock, decreasing ra species were isolated with high frequency from towards the apex. The fungi most frequently iso- declining vines and consequently will be included lated from the rootstocks were: Botryosphaeria in the overall sequencing project. The end result spp. (isolated from 46% of plants), Phaeomoniella will be an established culture collection that can chlamydospora (39%), Phaeoacremonium spp. be used for ecological and plant pathological stud- (26%), Phomopsis spp. (17%), and Cylindrocarpon ies; and the generation of sequence data can be used spp. (16%). Sixty-nine plants out of 135 (51%) were for regional phylogenetic analyses, for identifica-

90 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines” tion of potentially new species, and for developing psis were collected from grapevines in Ohio, Mich- pathogen detection systems (real-time PCR). igan, New York, and Pennsylvania. Isolates were also obtained from California, Canada, France, and New teleomorph findings for species in the Italy. The isolates were grouped on the basis of DNA genus Phaeoacremonium. L. MOSTERT, W. GAMS sequences from intron regions in the translation and P.W. CROUS. Centraalbureau voor Schimmel- elongation factor 1-Α and calmodulin genes. Accord- cultures, Uppsalalaan 8, 3584 CT Utrecht, The ing to DNA sequence comparisons with the type Netherlands. isolate, almost all isolates were found to be P. viti- E-mail: [email protected] cola. One isolate resembled Diaporthe phaseolorum while another isolate was Phomopsis eucommico- Several species of Phaeoacremonium (Pm.) are as- la, a tentative species. Both of these isolates had sociated with Petri disease symptoms, namely Pm. significantly higher mycelial growth rates and short- aleophilum, Pm. angustius, Pm. mortoniae, Pm. er conidia than the P. viticola isolates. Neither of parasiticum, Pm. rubrigenum and Pm. viticola. Tel- these species has been previously reported on Vitis, eomorphs of Phaeoacremonium have been estab- and they were only slightly or not pathogenic to Vi- lished in the genus Togninia (T.). Three teleo- tis. Thirty representative isolates were evaluated morphs have thus far been identified in the genus, for morphological characters and for pathogenicity namely T. minima (Pm. aleophilum), T. novae-zea- on grapevine (Vitis interspecific hybrid ‘Seyval’) landiae and T. fraxinopennsylvanica. To investigate leaves and canes, and 13 of these isolates were also possible teleomorph associations, isolates of vari- evaluated for pathogenicity on fruit and rachises. ous Phaeoacremonium spp. were mated in vitro on All isolates of P. viticola caused disease on grape. sterile grapevine canes. Perithecia were observed However, they differed in virulence on the different for Pm. parasiticum, Pm. viticola, Pm. krajdenii, grape tissues, suggesting some degree of speciali- Pm. rubrigenum and two as yet unnamed species sation with respect to host tissue. Virulence on the of Phaeoacremonium. Of these species Pm. para- fruits and rachises, but not on the leaves and canes, siticum, Pm. viticola, Pm. krajdenii and one un- was positively correlated with the rate of mycelial named species have been isolated from grapevines. growth of the P. viticola isolates. The dominant mating system was heterothallic, though one taxon was homothallic. DNA sequence Phomopsis spp. on grapevines: characterisa- 1 data from the partial ß-tubulin and actin genes tion and pathogenicity. J.M. VAN NIEKERK , J.Z. 2 3 1 4 confirms the link of T. fraxinopennsylvanica with GROENEWALD , D.F. FARR , P.H. FOURIE , F. HALLEEN 2 1 Pm. mortoniae, a species originally described from and P.W. CROUS . Department of Plant Pathology, grapevines in California. University of Stellenbosch, Private Bag X1, Matie- land 7602, South Africa. 2Centraalbureau voor Characterisation of Phomopsis spp. infecting Schimmelcultures, Uppsalalaan 8, 3584 CT grapes in the midwestern and northeastern Utrecht, The Netherlands. 3USDA-ARS, Systemat- 1 2 United States. O. ERINCIK , L. CASTLEBURY , A.M.C. ic Botany and Mycology Lab., Rm 304, Bldg 011A, 3 2 1 1 SCHILDER , A. ROSSMAN , and M.A. ELLIS . Dept. Beltsville, MD 20705, USA. 4ARC Infruitec-Ni- Plant Pathology, Ohio State University, 1680 Mad- etvoorbij, Private Bag X5026, Stellenbosch 7599, ison Ave, Wooster, OH 44691, USA. 2Systemic Bot- South Africa. any and Mycology, USDA-ARS, 10300 Baltimore E-mail: [email protected] Ave, Beltsville, MD 20705, USA. 3Dept. Plant Pa- thology, 104 CIPS Building, Michigan State Uni- The fungi in the genus Phomopsis, with teleo- versity, East Lansing, MI 48824, USA. morphs in Diaporthe, are pathogens of a wide va- E-mail: [email protected] riety of crops. Due to wide host ranges of species, morphological overlap between different species Phomopsis cane and leaf spot is an economically and morphological plasticity, within species, mor- important disease of grapes (Vitis spp.) in the east- phological and cultural characteristics must be ern US. The objective of this study was to deter- combined with DNA sequence data to distinguish mine which Phomopsis spp. are associated with the taxa. Ten species have previously been reported disease in this region. Eighty isolates of Phomo- from grapevines, of which P. viticola (the causal

Vol. 44, No. 1, April 2005 91 AA.VV. organism of Phomopsis cane and leaf spot) and P. rica. Several of these species were new host records, vitimegaspora (causal organism of swelling arm while Diplodia porosum, Fusicoccum viticlavatum of grapevine) have been confirmed as pathogens. and F. vitifusiforme are described as new. From in However, several unidentified Phomopsis species vitro and in vivo pathogenicity trials on grapevine were isolated from diseased grapevines, and from shoots it was concluded that B. australis, B. parva, symptoms atypical of the known Phomopsis dis- B. ribis and B. stevensii were more virulent than eases on this crop. This prompted the need to re- the other species studied. assess Phomopsis species occurring on grapevines. Sixty-one isolates obtained from grapevines were Identification and characterisation of Botryo- subjected to DNA analysis. A phylogenetic analy- sphaeria fungi in New South Wales vineyards. 1 1 2,3 sis of sequence data obtained from the rRNA oper- S. SAVOCCHIA , E.N. LAURENT , B.J. STODART , A. SOM- 2,4 1 2,5 1 on’s internal transcribed spacer regions (ITS-1, ERS , C.C. STEEL and M. PRIEST . National Wine ITS-2) and the 5.8S rRNA gene, combined with and Grape Industry Centre, School of Wine and Food morphological and cultural studies, differentiat- Sciences, Charles Sturt University, Locked Bag 588, ed at least fifteen Phomopsis species, of which 12 Wagga Wagga, NSW 2678, Australia. 22New South occurred on grapevines in South Africa. Patho- Wales Department of Primary Industries, Tocal Ag- genicity tests indicated that of the 12 species from ricultural College, Paterson, NSW, 2421, Australia. South Africa, P. viticola and P. amygdali caused 3Wagga Wagga Agricultural Institute, PMB Pine the most severe lesions on green grapevine shoots. Gully Road, Wagga Wagga, NSW 2650, Australia. P. amygdali is frequently isolated from grape- 4Tocal Agricultural College, Paterson, NSW 2421, vines, but P. amygdali, a known peach pathogen Australia. 5Orange Agricultural Institute, Forest in the USA, has been isolated only twice from Road, Orange, NSW 2800, Australia. grapevines in South Africa. E-mail: [email protected] Botryosphaeria fungi were isolated from declin- Characterisation and pathogenicity of Botryo- ing grapevines in the Hunter Valley and Mudgee sphaeria species occurring on grapevines. J.M. 1 2 2 grape-growing regions of New South Wales, Aus- VAN NIEKERK , P.W. CROUS , J.Z. GROENEWALD , P.H. 1 3 1 tralia, during a survey of 4- to 122-year-old grape- FOURIE and F. HALLEEN . Department of Plant Pa- vines across 21 vineyards. Grapevines infected thology, University of Stellenbosch, Private Bag X1, 2 with Botryosphaeria produced symptoms in the Matieland 7602, South Africa. Centraalbureau voor wood similar to those caused by Eutypa lata. Loss Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, 3 of spur positions, dead arms, cankers and wedge- The Netherlands. ARC Infruitec-Nietvoorbij, Private shaped lesions in the wood were observed in in- Bag X5026, Stellenbosch 7599, South Africa. fected vines. Field observations suggest that Char- E-mail: [email protected] donnay is most susceptible to infection by Botry- Several Botryosphaeria spp. are isolated from grape- osphaeria fungi. We are currently in the process vines, frequently from symptoms traditionally as- of identifying which species are present in these sociated with other diseases such as cane and leaf vineyards, and whether they are responsible for spot (caused by Phomopsis viticola) and Eutypa die- the symptoms observed. To date, two species, B. back (caused by Eutypa lata), making field diagno- obtusa and B. lutea, have been identified amongst sis very difficult. Nevertheless, uncertainty still re- the 30 strains examined. Pathogenicity studies mains as to the species identity and pathogenicity revealed that both these species can infect wound- of the various Botryosphaeria species. The aim of ed and non-wounded grape berries and one-year- this study was to characterise Botryosphaeria spe- old canes in vitro. Symptoms on infected grape cies from grapevines with regard to species identity berries include the presence of black pycnidia, and pathogenicity. A total of 112 Botryosphaeria iso- splitting and collapse of the berry skin. Lesions lates from diseased grapevines, pruning debris and were observed in infected one-year-old canes of symptomless nursery plants were studied. Based Chardonnay and Shiraz. Studies are also in on morphology and DNA sequences (ITS 1, 5.8S, ITS progress to determine the ability of Botryosophae- 2 and EF1-α) 11 Botryosphaeria species were dis- ria fungi to infect glasshouse-grown grapevines tinguished of which 8 species occurred in South Af- (cv. Chardonnay).

92 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

Black dead arm and basal canker of Vitis vini- ing black dead arm and basal canker on Red Globe fera cv. Red Globe caused by Botryosphaeria grapevines in Chile. obtusa in Chile. J. AUGER, M. ESTERIO, G. RICKE and I. PÉREZ. Departamento de Sanidad Vegetal, Cryptovalsa ampelina on grapevine: identi- 1 Facultad de Ciencias Agronómicas, Universidad de fication and pathogenicity. J. LUQUE , F. GAR- 2 2 3 1 Chile. Casilla 1004, Santiago, Chile. CIA , E. TORRES and D. SIERRA . Dep. Protecció Veg- E-mail: [email protected] etal, IRTA - Centre de Cabrils; Ctra. de Cabrils s/ 2 Vitis vinifera cv. Red Globe vines, over 6 years old, n, E-08348 Cabrils, Spain. Laboratori de Sanitat Vegetal; Via Circulació Nord, Tram VI, Carrer 3, started to show disease symptoms about ten weeks 3 after bud break. Symptoms first appeared on the Zona Franca, E-08040 Barcelona, Spain. Dep. Bio- leaves at the base of the shoots and then spread to logia Vegetal (Botànica), Facultat de Biologia, Uni- the other leaves, continuing to appear throughout versitat de Barcelona, Avda. Diagonal 645, E-08028 the growing season. Two forms of the disease were Barcelona, Spain. observed. The mild form led to premature leaf drop, E-mail: [email protected] while the severe form was characterised by die- In recent years, Cryptovalsa ampelina (Diatrypace- back of one or more shoots, accompanied by leaf ae) has been found repeatedly on grapevine canes drop, and the shrivelling and drying of fruit clus- in several countries, including Australia, South ters. The mild form was characterised by wine-red Africa and Spain. Surveys conducted in 2003–2004 spots on the leaf margins or on the leaf blade, which in Catalonia (NE Spain) have shown this fungus coalesced to form large zones of necrosis between to be very abundant on old pruned canes, although the veins and the margins of the leaf. Fruit clus- it has been recorded only rarely from cankered ters may wither. If the bark was scraped off, a wood of living plants. The polysporous asci and brown streak 1–2 cm wide was often seen in the slightly pigmented ascospores of Cryptovalsa am- wood. The streaking began at the base of the af- pelina distinguish the fungus from Eutypa lata, the fected shoot and extended to the ground level, even- causal agent of Eutypa dieback. However, cultures tually resulting in a basal canker. Botryosphaeria of both anamorphic forms are practically indistin- obtusa (Schwein.) Shoemaker (anamorph = Sphae- guishable by their morphology alone. Therefore, ropsis malorum Berk.) was isolated from 86% of our first objective was to design species-specific samples from 6- to 10-year-old vines from 12 loca- primers to be used in a PCR-based diagnostic test tions in the IV, V, VI and Metropolitan regions of for C. ampelina. A primer pair was designed to am- Chile. Isolations were made from the brown plify part of the ITS1-5.8S-ITS2 rDNA region and streaked wood. Isolates were identified based on its specificity was later confirmed on E. lata, sev- previous description and by internal transcribed eral other fungi occurring on Vitis, and the host spacer (ITS1-5.8-ITS2) rDNA sequences identical plant. To test the pathogenicity of C. ampelina, to those of B. obtusa. Pathogenicity tests were done artificial inoculations were conducted on one-year- by inoculating 20 µl of mycelial suspension via in- old potted grapevine plants of Ull de llebre (red) jection into the pith of 16 single-node, rooted cut- and Macabeu (white) cultivars. Wounds made on tings of V. vinifera cv. Red Globe. Sixteen control the bark of canes were inoculated with a mycelial cuttings were injected with an equal volume of ster- plug of the fungus and the plants were then main- ile distilled water. Twenty weeks after inoculation, tained in a greenhouse with regular watering. Vas- all B. obtusa-inoculated cuttings exhibited brown cular lesions were measured twice during the ex- streaks in the wood extending 50 to 60 mm from periment, at six and ten months after the inocula- the point of inoculation. The wood streaking ob- tions. The results of the pathogenicity test showed served in inoculated plants was identical to symp- that C. ampelina has low virulence, confirming toms in naturally infected black dead arm vines in some previous reports on the pathogenicity of this the vineyard. No symptoms were observed in the fungus. controls. B. obtusa was reisolated from the region of brown streaking in all the inoculated cuttings. Biochemical detection of Eutypa lata in 1 2 1 B. obtusa was not isolated from the water-treated grapevines. R. LARDNER , N. MAHONEY , E. SCOTT 2 1 controls. This is the first report of B. obtusa caus- and R. MOLYNEUX . Cooperative Research Centre for

Vol. 44, No. 1, April 2005 93 AA.VV.

Viticulture, PO Box 154 and School of Agriculture tre for Viticulture, PO Box 154 and School of Agri- and Wine, University of Adelaide, PMB 1, Glen culture and Wine, University of Adelaide, PMB 1, Osmond, SA 5064, Australia. 2Western Regional Glen Osmond, SA 5064, Australia. Research Center, Agricultural Research Service, US E-mail: [email protected] Department of Agriculture, 800 Buchanan Street, Eutypa dieback, caused by Eutypa lata, is a major Albany, CA 94710, USA. threat to the sustainability and productivity of E-mail: [email protected] vineyards worldwide. The pathogen is difficult to Eutypa dieback of grapevines, caused by Eutypa identify on the basis of colony morphology and is lata, is a major threat to the sustainability and often out-competed by other fungi when isolated productivity of vineyards worldwide. Foliar symp- from wood. Incubation for 1–2 months may be re- toms, believed to be caused by translocatable tox- quired to confirm the identity of cultures suspect- ins produced by the fungus in the xylem tissue, do ed to be E. lata. To facilitate diagnosis of the dis- not become visible until several years after infec- ease, we designed SCAR primers specific to E. lata tion. Infected vines may not display symptoms eve- and constructed a genomic DNA library from which ry season, or may display symptoms only at the an E. lata-specific probe was identified and se- start of a season. The aim of this research was to quenced. The SCAR primers were used to amplify develop methods based upon the detection of fun- DNA of E. lata directly from mycelium without the gal metabolites in grapevine sap or foliage to facil- need for prolonged incubation or DNA extraction. itate early diagnosis of the disease. High perform- These primers could also detect the pathogen in ance liquid chromatography (HPLC) was used to infected grapevine wood, although only when DNA identify metabolite(s) that were both specific to E. was isolated using the Bio-101 FastDNA® SPIN kit lata and ubiquitous. E. lata isolates and other fungi for soil (Qbiogene). The E. lata-specific DNA probe were grown in media derived from grapevine wood. was used to detect the pathogen in grapevine wood Although E. lata produces a range of secondary using a slot blot assay. The probe was validated metabolites in culture, one compound, eutypinol, for the detection of E. lata in wood using total DNA appeared to be predominant. This compound was isolated from 198 grapevine trunks, in association not detected in any other fungal species. Because with culturing of wood chips to enable morpholog- cultivars of grapevine exhibited varying degrees ical identification of E. lata. Comparison of data of tolerance to eutypa dieback, we examined dif- obtained using molecular and culturing techniques ferences in metabolite production following growth showed that the DNA probe was more sensitive of selected isolates of E. lata on media derived from than culturing, with E. lata detected in 54% of sam- various cultivars of grapevine. Preliminary results ples using the probe and in 38% of the same sam- suggested that cultivar has no influence on sec- ples when wood chips were cultured on agar. The ondary metabolite production in vitro. Grapevine primers and probe can be used to gather informa- tissue-culture plantlets were inoculated with E. tion on epidemiology and to assess the efficacy of lata to assess metabolite production in planta. To potential control agents for eutypa dieback. examine metabolite uptake and to optimise extrac- tion protocols for detecting E. lata metabolites in Detection of Eutypa lata from grapevines by 1 planta, shoots excised from tissue-culture plant- reverse dot blot hybridisation. S. SAFODIEN , F. 1 2 1 3 lets were immersed in purified culture filtrates of HALLEEN , P.W. CROUS , W.A. SMIT and A. BOTHA . 1 E. lata. To date, eutypinol is the only compound ARC Infruitec-Nietvoorbij, Private Bag X5026, 2 which has been detected by HPLC in plantlets in- Stellenbosch 7599, South Africa. Centraalbureau oculated with E. lata, although eulatachromene, voor Schimmelcultures, Uppsalalaan 8, 3584 CT 3 eutypine and 2-isoprenyl-5-formyl-benzofuran Utrecht, The Netherlands. Department of Micro- were also detected in excised shoots treated with biology, University of Stellenbosch, Private Bag X1, culture filtrates of E. lata. Matieland, 7602, South Africa. E-mail: [email protected] Molecular diagnostic tools for the detection Eutypa dieback of grapevines, caused by the fun- of Eutypa lata in grapevines. R. LARDNER, B. gus Eutypa lata, has been recognised as a serious

STUMMER and E. SCOTT. Cooperative Research Cen- disease of grapevines worldwide. The disease is

94 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines” responsible for a slow decline of vineyards by re- ed States, Europe, Australia and South Africa. This ducing growth and yield, and eventually killing the disease is also common in older ‘Concord’ vineyards grapevine. Infection occurs primarily through in Michigan. The objective of this study was to de- pruning wounds. After infection the disease devel- velop a rapid and reliable procedure for the detec- ops slowly and initial symptoms may even appear tion of the causal agent. However, two fungi, E. only after several years. Consequently, it is impor- lata and Eutypella vitis, were consistently isolat- tant to develop detection techniques to identify the ed from ‘Concord’ grapevines with Eutypa dieback presence of the disease as early as possible. Fast, symptoms, sometimes from the same vine. These accurate and reliable detection techniques could fungi are difficult to distinguish morphologically, be used to screen grapevine in an effort to provide but are genetically distinct, as is shown by sequenc- clean and healthy grapevine material to the grape- ing the internal transcribed spacer (ITS) region of vine industry. With the aim of fulfilling these cri- their ribosomal DNA. It is not known whether E. teria, a method using reverse dot blot hybridisa- vitis is pathogenic on grapes. Isolates of both spe- tion was developed. This method makes use of spe- cies were collected from vineyards in southwest- cies-specific oligonucleotides derived from the in- ern Michigan, and the ITS region of 25 E. lata and ternal transcribed spacer (ITS) region, which are 15 Eutypella vitis isolates was sequenced. The se- blotted onto a membrane. In a PCR amplification quences were compared to each other and to exist- with genomic DNA as the template, universal prim- ing sequences in GenBank using the BLAST search ers and digoxigenin-dUTP, the ITS region was si- program. Eutypa lata sequences were more varia- multaneously amplified and labelled. An aliquot ble than those of Eutypella vitis. Specific PCR of this PCR product was used as a probe for hy- probes were designed for both species and evalu- bridisation to a membrane containing the immobi- ated for specificity against a range of fungal iso- lised oligonucleotides. A positive signal was ob- lates from grapes and other fruit crops. The spe- served when hybridisation occurred between the cific primers were selective for each species. A labelled DNA sample and the immobilised specific multiplex PCR protocol was developed that detect- oligonucleotide. In this study, species-specific oli- ed each fungus in culture, in sawdust from can- gonucleotides were designed for E. lata and E. lep- kers, and in young infected canes. toplaca. The oligonucleotides developed to detect E. lata hybridised to pure DNA preparations of First detection of Eutypa lata (Pers.:Fr.) Tul. Eutypa isolates and work is currently being done to with PCR directly out of grapevine trunks in 1 2 1 make the method amenable to the identification of Germany. P. S CHWAPPACH and M. GRIMM . Bavari- Eutypa directly from environmental samples such an State Institute for Viticulture and Horticulture, as woody tissue. The oligonucleotide designed to Section for Grapevine Protection, Herrnstrasse 8, D- identify E. leptoplaca hybridised to E. lata isolates 97080 Veitshoechheim, Germany. 2Julius-Maximil- as well, therefore, more work is needed to improve ians-University Wuerzburg, Institute for Biochem- the specificity of the probe by optimising the hy- istry, Am Hubland, D-97074 Wuerzburg, Germany. bridisation conditions. Once the method has been E-mail: [email protected] fully optimised it would be a rapid, consistent, reli- In 1999, symptoms of Eutypa lata were discovered able and highly reproducible PCR-based assay for in a Frankonian vineyard and have been observed the routine diagnosis of this plant disease. there since then every year in all rootstocks. In 1999, only 16% of all vines showed the typical PCR-based detection of Eutypa lata and Eu- symptoms of eutypa dieback but by 2003 the inci- typella vitis. M. CATAL, S.A. JORDAN, S.C. BUTTER- dence of symptomatic vines had increased to 58% WORTH and A.M.C. SCHILDER. Department of Plant and almost one third of all vines (29%) were dead Pathology, Michigan State University, 104 Center or already removed. Another 11% developed poor- for Integrated Plant Systems, East Lansing, MI ly and 18% showed typical signs of eutypa dieback. 48824, USA. When cutting the grapevine trunk vertically the E-mail: [email protected] extent of the typical dark regions inside of the trunk Eutypa dieback, caused by Eutypa lata, affects was equivalent to the severity of the external symp- grapevines around the world, including the Unit- toms. Up to now, no direct method exists to control

Vol. 44, No. 1, April 2005 95 AA.VV. this disease. Therefore, a non-destructive meth- was added to enhance the subsequent PCR reac- od is needed to detect infected plants prior to ex- tion with species-specific primers (Pch1 and Pch2). ternal symptom expression so that they can be As little as 1 pg of Pa. chlamydospora genomic DNA eradicated from the vineyards. Small pieces of could be detected from spiked wood samples. For wood were taken from the dark region of infected validation of the protocol, different combinations grapevine trunks for molecular analysis. After of grafted grapevines were sampled after uproot- isolating the DNA by means of a purification kit ing, hot water treated (HWT; 50°C for 30 min) or and an extensive denaturing step, species-specif- left untreated. Isolations were made from the ba- ic ITS-primers were added. DNA of any E. lata sal ends of rootstocks on potato dextrose agar that may have been present in the eluate was amended with streptomycin. The same section was amplified by means of a polymerase chain reac- also used for molecular detection. The identity of tion (PCR). Following PCR, fragments of DNA the PCR products was confirmed by digestion with were separated by agarose gel electrophoresis. A the restriction enzymes Aat II and MluN I. Pa. prominent band at 385 bp was typical for positive chlamydospora DNA was detected in 80.9% of sam- detection of E. lata. The DNA of these bands was ples, compared to only 24.1% positive detections excised from the gel, phosphorylised and, after li- by means of isolations. However, Pa. chlamydospo- gation in a pUC19-vector, transformed into E.coli- ra DNA was detected from 100% of samples that DH5α. Thus we obtained sufficient material for tested positive after isolation. Although none of the DNA sequence analysis. Ninety-nine percent of isolations from HWTed plants yielded Pa. chlamy- the tested DNA was identical to the reference-se- dospora, molecular detection was not adversely quence of E. lata as described in the literature. affected. The applicability and robustness of the We furthermore found the same conformity be- molecular detection protocol will be evaluated in tween the tested grapevine DNA and the DNA of subsequent epidemiological studies. an E. lata control isolate. Molecular detection of grapevine trunk dis- Development and validation of a protocol for ease pathogens in nursery and vineyard soils molecular detection of Phaeomoniella chlamy- in South Africa. U. DAMM, E. RETIEF and P.H. dospora in grapevine wood. E. RETIEF, U. DAMM, FOURIE. Department of Plant Pathology, University J.M. VAN NIEKERK and P.H. FOURIE. Department of of Stellenbosch, Private Bag X1, Matieland (Stel- Plant Pathology, University of Stellenbosch, Private lenbosch) 7602, South Africa. Bag X1, Stellenbosch, 7602, South Africa. E-mail: [email protected] E-mail: [email protected] Management of grapevine trunk diseases such as Petri disease, caused mainly by Phaeomoniella Petri and black foot is difficult, especially because chlamydospora, is a serious decline and dieback the disease cycles are currently poorly understood. disease of young grapevines. Pathogen detection Contaminated soil was reported as a source of and accurate diagnosis are currently based on fun- inoculum, especially for black foot in nursery vines gal isolation from artificial growth media. The fun- at susceptible stages after grafting. Use of molec- gus is, however, extremely slow-growing and cul- ular detection of these pathogens in the soil is still tures are often overgrown by saprophytes before it limited because of PCR-inhibiting soil components can be identified: this may lead to false negative and the high costs of commercial DNA extraction results. The aim of this study was to develop a time- kits. The aim of this study was therefore to devel- efficient and cost-effective protocol for molecular op a cost-effective, easy and sensitive method for detection of Pa. chlamydospora in grapevine wood. extraction of fungal DNA from the soil and to use Different published methods for extraction of fun- this method to determine whether South African gal DNA were tested on grapevine wood, some were nursery and vineyard soils are contaminated with modified, and certain steps combined. A protocol grapevine trunk disease pathogens. Soil samples involving the pulverisation of wood samples in liq- were collected from 27 nurseries, 16 rootstock uid nitrogen and DNA extraction in a hexadecylt- motherblocks and 7 vineyards in the Western rimethyl ammonium bromide (CTAB) extraction Cape province. DNA was extracted using a meth- buffer was most effective. Bovine serum albumin od based on sodium dodecyl sulphate buffer, the

96 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

FastPrep‚ instrument (Bio101) and a spin column described Cylindrocarpon-like species. The latter for removal of PCR inhibitors. The extracted DNA two clades formed a paraphyletic group by LSU was tested for Phaeomoniella chlamydospora, analysis, but ITS and ß-tubulin gene analysis made Phaeoacremonium aleophilum and Cylindrocar- them a monophyletic group. Strains of the C. de- pon spp. by means of PCR reactions with specific structans complex isolated from grapevines matched primers. Pm. aleophilum was not detected in any C. destructans in morphology and DNA sequences. of the soils tested. However, Pa. chlamydospora C. macrodidymum formed micro- and macroconid- and Cylindrocarpon spp. were frequently detect- ia, but rarely formed chlamydospores. Its mostly 3- ed in the soils. This is the first time Pa. chlamy- septate macroconidia were more or less straight, dospora was detected in soils in South Africa. This minutely widening towards the tip, and had an api- fungus occurred very often (66%) in homogenous- cal cell slightly bent to one side. Its teleomorph, ly low amounts in soils, independent of the local- Neonectria macrodidyma, was obtained in mating ity and the history of grapevine cultivation. While experiments, and was characterised by smooth to the overall incidence of Cylindrocarpon spp. was finely warted ascospores, smooth to finely warted similar (66%), results suggest these pathogens perithecia, and moderately sized angular to subglo- might be more prevalent in certain areas, but fur- bose cells in the outer region of the perithecial wall. ther research needs to be conducted to substanti- The two undescribed Cylindrocarpon-like species ate these findings. Soils should therefore be re- were characterised by mostly 3–5-septate, curved garded as potential inoculum sources of grapevine macroconidia, and lacked microconidia. For these trunk disease pathogens. Crop rotation, soil fu- species a new genus, Campylocarpon, was estab- migation, composting or the use of antagonistic lished. It comprised the new species Campyl. fas- microorganisms should be considered as means ciculare and Campyl. pseudofasciculare respective- to reduce inoculum levels in soils. ly. Inoculation of 6-month-old potted grapevine root- stocks (cv. Ramsey) with selected isolates of C. de- Fungi associated with black foot disease in structans, C. macrodidymum, Campyl. fasciculare, South African vineyards and nurseries. F. and Campyl. pseudofasciculare resulted in a reduced 1 2, 3 3 HALLEEN , H.-J. SCHROERS , J.Z. GROENEWALD and root and shoot mass of inoculated plants. 3 1 P. W. C ROUS . ARC Infruitec-Nietvoorbij, P. Bag X5026, Stellenbosch, 7599, South Africa. 2Agricul- Surveys of Oklahoma grapevines for Xylella tural Institute of Slovenia, Hacquetova 17, p.p. fastidiosa. S.L. VON BROEMBSEN and B. OLSON. De- 2553, 1001 Ljubljana, Slovenia. 3Centraalbureau partment of Entomology and Plant Pathology, 127 voor Schimmelcultures, Uppsalalaan 8, 3584 CT Noble Research Center, Oklahoma State Universi- Utrecht, The Netherlands. ty, Stillwater, OK 74078, USA. E-mail: [email protected] E-mail: [email protected] Cylindrocarpon and Cylindrocarpon-like taxa iso- Xylella fastidiosa (XF), a xylem-limited bacteri- lated from Vitis vinifera plants in nurseries and um with a wide host range, is indigenous to the vineyards were compared morphologically and Gulf Coast Region of the USA and is vectored by phylogenetically with other Cylindrocarpon taxa. xylem feeding insects. Grape strains of XF cause LSU, ITS and ß-tubulin sequences were used for a vascular wilt, Pierce’s disease of grapevines phylogenetic inferences. Cylindrocarpon species (PD), which often results in the death of suscepti- clustered mainly in three groups: a monophyletic ble cultivars, especially those of Vitis vinifera. group consisting of three subclades comprising (i) Although Oklahoma (OK) grape production is still members of the Cylindrocarpon destructans com- quite limited, it has dramatically increased in the plex; (ii) a Cylindrocarpon species newly described past five years and supports a fledgling regional as C. macrodidymum; and (iii) an assemblage of wine industry. XF has not previously been record- species closely related to strains identified as C. ed on native or cultivated hosts in OK, but has cylindroides, the type species of Cylindrocarpon. been reported from grapevines in Texas counties This monophyletic group excluded two other that border OK to the south. A number of poten- groups, (i) comprising members of the Neonectria tial vectors of XF have been recorded in OK. Sev- mammoidea complex; and (ii) comprising two un- enteen vineyards located primarily in southern

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OK were surveyed in 2003. During 2004, 18 vine- system II. 2. Secondly, the activation of defence yards were surveyed that were more generally mechanisms was monitored in order to confirm distributed throughout OK production areas. Dur- whether grapevines infected with esca are able to ing both surveys, all grapevines in the second leaf react. Here we observed the expression of genes or greater in these vineyards were inspected and encoding for proteins involved in this process. The petiole samples from visually unhealthy vines for results of vineyard experiments carried out in which no apparent cause was evident were taken 2003 and 2004 indicate that esca disease leads to for laboratory analysis. Products resulting from grapevine physiological changes relating to foliar DNA amplification using polymerase chain reac- symptoms. First, carbon nutrition damage was tion technology (PCR) were elucidated on gels in observed resulting in a reduction in net photosyn- 2003, but real time PCR was used for the 2004 thesis and in carbohydrate reserves in the canes analyses. XF was not detected in any samples in during winter dormancy. This reduction could in- either 2003 or 2004. Surveys in 2005 will include duce losses in yield and grapevine vigour, there- native grapevines and other potential hosts. by affecting plant longevity. With regard to de- fence mechanisms, grapevines seemed to detect infection by esca pathogens. The vines react by Host-pathogen interaction inducing several genes involved in defence mech- anisms, such as chitinase and lipoxygenase. Consequences of esca on carbohydrate phys- 1 iology in French vineyards. A.N. PETIT , M. Purification and characterisation of extracel- 2 1 1 1 BOULAY , F. BAILLIEUL , C. CLÉMENT and F. FONTAINE . lular laccase from liquid culture of Fomiti- 1 1 2 Université de Reims Champagne-Ardenne, UFR poria mediterranea. E. ABOU-MANSOUR , R. PEZET 1 1 Sciences, Laboratoire Stress, Defenses et Reproduc- and R. TABACCHI . Laboratory of Analytical Organic tion des Plantes, BP 1039, 51 687 Reims Cedex, Chemistry, University of Neuchâtel, CH-2007 Neu- France. 2Moet et Chandon, 6 rue de Bussy, 51 200 châtel, Switzerland. 2Agroscope-RAC, Swiss Fed- Epernay, France. eral Agriculture Research of Changins, Route de E-mail: [email protected] Duiller, CH-1260 Nyon, Switzerland. Esca is a complex wood disease characterised by E-mail: [email protected] symptoms on the plant organs and in the woody The common internal symptom in chronic esca syn- tissue of grapevines. Several fungi are involved drome is white rot, which gradually changes the in esca and the disease is not yet fully elucidated. hard wood to a soft, friable, spongy mass. The wood In the last few years a dramatic upsurge of esca decay symptoms of esca have been most commonly has occurred in all grape-growing areas, includ- associated with the basidiomycete fungus Fomiti- ing the Champagne. Sodium arsenite, which used poria mediterranea, which produces various extra- to be the only mode of control against esca, was cellular lignin-degrading enzymes, including per- banned in 2001 and other chemical or alternative oxidases and laccases. Laccases are involved in control methods need to be developed rapidly. For several physiological functions, such as lignin bio- this purpose it is necessary to know more about synthesis, plant pathogenesis, insect sclerotisation, this disease. The aim of this work was therefore and degradation of lignocellulosic materials. Lac- to characterise the physiological impact of esca cases are also involved in both the polymerisation on vineyards in the Champagne area. We focused and depolymerisation processes of lignin. In this particularly on the effects of foliar symptoms on study we report for the first time on the purifica- two parameters: 1. carbon nutrition, which is the tion and characterisation of a 60-kDa extracellu- key factor for grapevine yield and vigour, was de- lar laccase enzyme from a liquid culture of F. medi- termined. In this context gas exchanges were terranea. Using gel filtration and ion exchange measured, and the biochemical analysis of photo- chromatography, a 2006-fold purification and a synthetic pigments and carbohydrate reserves yield of 9.6% of the laccase was obtained. Using was undertaken. Moreover, we followed the ex- 2.6-DMP as the substrate, the enzyme exhibited pression of a gene encoding an oxygen-evolving an optimum pH of 3 and an optimum temperature enhancer, which is a protein involved in photo- of 40°C against ABTS, and 50°C for DMP, and its

98 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

Km was 25 mM. Substrate specificity and inhibitor ment of Pomology, University of California, Davis, studies indicated the enzyme to be a typical fun- CA, 95616, USA. gal laccase. E-mail: [email protected] Eutypa lata is a vascular pathogen of grapevines. Chemical characterisation of stilbenic The fungus spreads by ascospores, infects open wood polyphenols from esca diseased wood and vessels and slowly decays the wood. The goal of this their role in defence mechanisms. C. AMALFIT- 1 2 2 1 study was to identify the fungal population inhab- ANO , F. PEDUTO , L. MUGNAI , A. EVIDENTE and G. 2 1 iting wood cankers and to determine the polymer SURICO . Dipartimento di Scienze del Suolo della targets and products of degradation of the cell wall Pianta e dell’Ambiente, Università di Napoli Fede- degraded by E. lata. The cordons of naturally in- rico II, Via Università 100, 80055 Portici, Italy. 2 fected grapevine cv. Merlot and Cabernet Sauvignon, Dipartimento di Biotecnologie Agrarie, Sezione di which are reported to be respectively tolerant and Patologia Vegetale, Università degli Studi, Piazzale susceptible to the disease, were sampled for two delle Cascine 28, 50144 Firenze, Italy. consecutive years in California. The sapwood was E-mail: [email protected] separated into three zones, healthy, cankered, and It has been demonstrated that stress metabolites canker margin. Isolations were conduced from all such as resveratrol (3,5,4’-trihydroxystilbene) and three wood zones on PDA medium and fungi were its oligomers (viniferins) accumulate in grapevine identified based on their mitosporic stage, or by tissues in response to fungal infection. In this work BLAST search in the GenBank database when mor- we report on the purification of three resveratrol phological characters were missing. Wood from all tetramers extracted from the brown-red wood of esca three zones parasitised by E. lata only was ground diseased Vitis vinifera cv. Sangiovese by extensive to powder and the hemicellulose fraction of the cell chromatographic methods (CC and HPLC). Resver- wall was analysed by gas chromatography, while atrol, cis and trans ε-viniferin and ampelopsin B lignin, pectin, and cellulose levels were determined were detected in these grapevines in previous stud- by colorimetric assays. The microbial population of ies. The tetramers were identified mainly by 1H and cankered tissue showed frequent association of E. 13C mono and bidimensional NMR spectroscopy, ESI- lata with one or more additional parasitic fungi (Bot- MS spectrometry and optical activity, and were found ryosphaeria obtusa, Phomopsis viticola, Phaeoacre- to be hopeaphenol, isohopeaphenol and ampelopsin monium aleophilum, Phaeomoniella chlamydospo- H. Resveratrol and its oligomers were also found in ra, Verticillium sp.) in a complex of fungi. Diatrypella higher concentrations in diseased, discoloured wood sp., a fungus closely related to E. lata, was also found than in healthy wood. This suggests that some of in some wood cankers, indicating that other dia- these metabolites could be involved in the plant de- trypaceous fungi may be associated with grapevine fence response. Some of the isolated polyphenols dieback. Biochemical analysis of the wood revealed were assayed in vitro and compared with resvera- no degradation of cellulose and pectin. However, trol in germination tests against the fungi involved glucose in the hemicellulose fraction of the cell wall in esca (Phaeomoniella chlamydospora and Phae- was targeted by fungal attacks. Moreover, a higher oacremonium aleophilum). No or low antifungal ac- lignin content was measured for cv. Merlot as com- tivity was found at the assayed concentrations. How- pared with Cabernet Sauvignon in all three wood ever, to fully elucidate the biological activity of such zones, suggesting that phenolic compounds may be compounds, more in vitro tests (including activity involved in disease resistance. on hyphal growth, sporulation and spore vitality) are under way to elucidate the role of stress metab- Isolation of esca-associated fungi, chemical olites in the plant/pathogen interaction. composition of xylem exudate from bleeding spurs of infected grapevines and annual Fungal identification and biochemical anal- trend of sap flux. G. BRUNO and L. SPARAPANO. ysis of wood canker caused by Eutypa lata. Dipartimento di Biologia e Patologia vegetale, 1 2 P.E. ROLSHAUSEN , J.M. LABAVITCH and W.D. Università di Bari, Italy. 1 1 GUBLER . Department of Plant Pathology, Univer- E-mail: [email protected] sity of California, Davis, CA, 95616, USA. 2Depart- Phaeomoniella chlamydospora (Pch), Phaeoacremo-

Vol. 44, No. 1, April 2005 99 AA.VV.

nium aleophilum (Pal) and Fomitiporia mediterra- RIO, E. APARICIO, G. HERNAIZ and M.L. TELLO. Insti- nea (Fme) cause at least a part of the external symp- tuto Madrileño de Investigación Agraria. Autovía toms of esca by phytotoxic metabolites produced in A-2, Km. 38,200. Alcalá de Henares E-28800- Ma- the discoloured or decayed woody tissue. Both Pch drid, Spain. and Pal produced in vitro several phytotoxic com- E-mail: [email protected] pounds: exopolysaccharides (EPS) (pullulans) and Petri disease is a serious problem in newly plant- two pentaketides (scytalone and isosclerone). The ed vineyards, causing decline and dieback of young aim of this study was to: 1. isolate fungal patho- grapevines. Being a complex syndrome, its aetiol- gens; 2. detect phytotoxic pullulans and pen- ogy is still partly unknown. The aim of this work taketides; 3. detect proteins, glycoproteins and was to acquire a better understanding of the aeti- polyphenols of the host response; 4. assess the an- ology of this disease through analysis of the rela- nual trend of sap flux and its chemical composition. tionships between the main causal agents. Two During vine bleeding (early spring: 2000–2004), the year old asymptomatic grapevines of var. Tempra- xylem sap was collected from ‘Sangiovese’ vines nillo clone RJ-78/140-Ruggeri clone 101 were in- showing severe symptoms of brown wood-streaking, fected with the fungi Phaeomoniella chlamydospo- with or without white rot. Aliquots of sap were ra, Phaeoacremonium aleophilum and Pm. in- poured on malt-agar dishes or filtered and assayed flatipes. Inoculations were carried out by insert- on leaves of grapevine cv. Italia and Sangiovese. ing plugs, removed from actively growing colonies, Numerous phialidic conidia were found in the xy- into shoot wounds (5 mm diam. ϫ 2–4 mm depth). lem sap from esca-affected grapevines; isolated in The three strains were inoculated separately and pure culture, these conidia gave origin to colonies in dual combinations, introducing pairs of fungi in of Pch and Pal. In addition, strains of Sphaeropsis the same wound, with an interval of fifteen days. malorum, Alternaria sp., Diplodia sp., Fusarium sp. Lesion lengths were measured weekly for three and bacteria were also isolated. Symptoms on the months and the differences between wounds inoc- leaves after absorption of the xylem sap solutions ulated with each fungus or combination of fungi were: irregular, pale green areas located among the were compared. Pa. chlamydospora was the most main veins or at the leaf margins, yellow-brown or virulent fungus causing the largest lesions, both red-brown necrotic spots. Significant differences in in single inoculations and in combinations, when- the trend of the parameters examined each year ever it was inoculated first. However, it had the (flux, pH, and organic compounds) were recorded. lowest percent isolation due to its less competitive The main components of the EPS were pullulans, behaviour compared with other saprophytic fungi. galactomannans, glucogalactomannans, and arabi- Pm. aleophilum showed a synergistic effect with nogalactans. Proteins and glycoproteins were detect- Pa. chlamydospora when it was inoculated last. ed ranging from 14 to 60 kDa. Benzoic acid deriva- Pm. inflatipes did not seem to have a predominant tives, stilbenes and flavonol-O-glycosides were the role in the rot. These results point to Pa. chlamy- dominant polyphenols of the xylem sap. The results dospora and Pm. aleophilum as the causal organ- of this study provide new information on the pro- isms of Petri disease. duction of toxic metabolites in planta by Pch and Pal associated with esca disease. Conidia of Pch and The influence of Phaeomoniella chlamydospo- Pal, their metabolites and host response compounds ra and other fungi on the strength of the graft were found to occur in the xylem sap of naturally union and biomass of young grapevines. A.B. 1 1 1 2 infected vines, indicating that the pathogens, their REED , L.D. MELTON , B.G. SMITH and P. JOHNSTON . by-products and defense substances were translo- 1Food Science Programme, Department of Chemis- cated from the infected woody tissue of the trunk to try, University of Auckland, Private Bag 92019, the aerial part of the affected vines. Auckland, New Zealand. 2Mycology Department, Landcare Research, University of Auckland (Tam- Differential virulence effects of single and aki Campus), Auckland, New Zealand. dual inoculations of Phaeomoniella chlamy- E-mail: [email protected] dospora, Phaeoacremonium aleophilum and During nursery propagation, some grafted vines Pm. inflatipes on young grapevines. L. GAFO- die and weak grafts were observed in otherwise

100 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines” healthy looking 8-month-old vines. Petri disease, In this study, we demonstrated the involvement of caused by Phaeomoniella chlamydospora and Phae- ethylene in the first phase of the development of oacremonium spp., is associated with decline and Petri disease in grapevine plants growing in vitro, dieback of young grapevines that are subjected to which had been infected with Pa. chlamydospora stress. The effects of inoculating spore suspensions and Pm. aleophilum. The presence of Brotomax in of P. chlamydospora into the graft wound were ex- the culture media decreased the biosynthesis of amined. Root and shoot dry weights were meas- ethylene and reduced the progress made by these ured as well as the tensile strength of the graft fungi. The results suggest that the increase of nat- assessed. Field trials were conducted in conjunc- ural defences brought about by Brotomax can re- tion with a commercial viticulture nursery, using duce the incidence of these pathogens in grapevine Sauvignon Blanc and Pinot Noir grafted onto 101- plants. 14 Mgt. Vines were inoculated with P. chlamydospo- ra spore suspensions (102, 103, 104, 106 and 108 cfu Bioactive secondary metabolites from Pho- ml-1) or water into the omega graft wound at the mopsis viticola, a fungus responsible for time of grafting. Stock-takes of young vines grow- grapevine excoriosis. R. TABACCHI, M.-L. GOD- ing in the nursery were performed at 10 and 24 DARD, N. MOTTIER and E. ABOU-MANSOUR. Laborato- weeks. The average root and shoot biomass was ry of Analytical Organic Chemistry, University of recorded (n=10). After 8 months, the graft tensile Neuchâtel, CH-2007 Neuchâtel, Switzerland. strength was assessed as well as the development E-mail: [email protected] of black streaking in the xylem tissue. Results in- Phomopsis cane and leaf spot, also known as exco- 4 -1 dicated that at least 10 cfu ml of P. chlamydospo- riosis, is widely distributed throughout the viticul- ra was necessary to significantly reduce root and tural world. The causal agent, Phomopsis viticola shoot biomass and weaken grafts. Some inoculat- (Sacc.), infects all green parts of vines such as the 6 -1 ed (up to 10 cfu ml ) vines, did not show obvious canes, leaves and fruits. The symptoms are black symptoms of vine decline, but all inoculated vines necrotic lesions on the lowest internodes of the developed black streaking in the xylem of both root- shoots and small black spots on the leaves and ber- stock and scion. The extent of black streaking var- ries. Crop losses are due to weakening canes and ied with the inoculation dosage. poor fruit development or fruit loss. In a continu- ing effort to identify phytotoxins from pathogens Ethylene metabolism in grapevine plants in- implicated in grapevine trunk diseases, we fected by Phaeomoniella chlamydospora and screened several strains of P. viticola for cytotoxic- 1 Phaeoacremonium aleophilum. P. G ÓMEZ , A. G. ity, antifungal, and antimicrobial activities. From 1 1 1 2 BÁIDEZ , A. ORTUÑO , J.A. DEL RÍO , A. CARBALLO and three different strains, a variety of bioactive com- 3 1 V. F RÍAS . Department of Plant Biology, Faculty of pounds, including Phomopsides B, four new Biology, Campus Espinardo, University of Murcia, pyranones and a new xanthenone, were character- 2 30100 Murcia, Spain. Department of Genetic, Uni- ised. We describe here the isolation of the fungi, 3 versity of Sevilla, 41080 Sevilla, Spain. Depart- the culture conditions, the structure elucidation ment of I+D of Agrométodos S.A., Alamos 1, Urb. and the biological activity of the isolated com- Monteclaro, 28223 Pozuelo de Alarcón, Madrid, pounds. Spain. E-mail: [email protected] Antagonistic behaviour of Phaeomoniella Petri disease is caused by different fungi such as chlamydospora and Phaeocremonium spp. vs. Phaeomoniella chlamydospora and Phaeoacremo- Fomitiporia mediterranea: isolation, purifi- nium aleophilum. The disease causes decline and cation, chemical and biological characterisa- dieback of young grapevines because the xylem tion of active compounds. G. BRUNO and L. becomes blocked by tyloses, which form when the SPARAPANO. Dipartimento di Biologia e Patologia xylem lumen is invaded by the protoplasm of the vegetale, Università di Bari, Italy. adjacent parenchyma cells. This invasion is possi- E-mail: [email protected] ble because of the softening of the xylem walls, Recent advances in research on esca of grapevine which is caused by various wall hydrolitic enzymes. shed further light upon the aetiology and patho-

Vol. 44, No. 1, April 2005 101 AA.VV. genesis of this disease complex. The pathogenicity azzale delle Cascine 28, 50144 Firenze, Italy. of the three fungi most commonly associated with E-mail: [email protected] the disease, the thracheiphilous hyphomycetes Changes in the host metabolism or in metabolic Phaeomoniella chlamydospora (Pch) and Phaeoa- and catabolic processes following pathogen attack cremonium aleophilum (Pal), and the wood-rotting can result in localised variations in the micro and basidiomycete Fomitiporia mediterranea (Fme) has macro elements content. Such changes are often been clearly demonstrated. The non-synergistic, accompanied by an increase in the resistance or competitive association of the two hyphomycetes tolerance to a given pathogen. In a disease such as and the inhibition of Fme growth have been de- esca, whose foliar symptoms are similar to symp- picted. The objectives of this study were to: 1. as- toms of nutrient deficiency, an investigation was sess the toxigenicity of P. angustius (Pan), P. in- started on the mineral content of leaves from flatipes (Pin), P. parasiticum (Ppa), P. rubrigenum healthy and esca diseased vines, and on the main (Pru), P. viticola (Pvi) and their inhibition of Fme biochemical and physiological pathways of the de- growth; 2. study how Pch, Pal and Fme interact fence response in which those minerals were in- with each other and with host tissue; 3. isolate and volved. Leaves from healthy vines and vines show- characterise chemically and biologically the sub- ing chronic symptoms (leaves showing only in- stances involved, showing how the mycelium of terveinal chlorotic spots, and leaves showing chlo- each fungus functions and how the fungi co-ordi- rotic and necrotic interveinal areas) of the table nate their activities. The culture filtrates of all grape cv. Cardinal were collected at the end of flow- strains, assayed on detached leaves of ‘Italia’ or ering (middle of May), at the end of colour change ‘Sangiovese’ grapevines, were phytotoxic. Isoscle- (beginning of July) and just after harvest (middle rone was produced by Pal, Pch, Pin, Pan, Pru, Pvi, of September). The content of Ca, Cu, K, Fe, Mg, whereas scytalone was produced by Pal, Pch, Pin, Mn, Mo, Na, P and Zn were determined by spec- Pan and Ppa. Pullulan was also produced by Pan, troscopic methods (A.A. and UV-Vis spectrometers), Pin, Ppa and Pvi. In dual cultures, the antagonism and C, H, N and S by elemental analysis (HCNS against Fme was clearly shown on malt-agar plates: elemental analyser). Significant differences were the margin of the Fme colonies turned brown, be- found in the mineral content of leaves between dis- came thicker, and aerial hyphae formed a ridge- eased and healthy vines. While such differences like barrier between the two fungi. This experiment could be simply the result of physiological events showed that the antagonistic effect against Fme was linked to the early senescence of leaves in diseased probably caused by strains of Phaeoacremonium spp. vines, triggered by toxic compounds produced by and Pch producing substances that spread through the esca pathogens, the possibility cannot be ex- the medium and affected the Fme colony. All Phae- cluded that the imbalance in nutrients in some way oacremonium spp. and Pch strains were used for caused these leaf symptoms. the concentration and desalting of culture filtrates through an anisotropic membrane. Most of the Effect of fungi associated with decline symp- growth inhibitor activity of the crude culture filtrate toms on growth curves and berry develop- 1 was located in the 3–10 kDa or up to 3 kDa frac- ment of Vitis vinifera cv. Red Globe. J. AUGER , 1 1 2 tions. These fractions were found to contain pep- M. ESTERIO , C. CARRERAS , P. NAULIN and M. PIN- 3 1 tides and proteins that suppressed Fme growth. TO . Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas, Universidad de Chile. Preliminary studies on micro and macro ele- 2Departamento de Silvicultura, Facultad de Cien- ments in esca diseased leaves of Vitis vinif- cias Forestales, Universidad de Chile 3Departamen- 1 1 era cv. Cardinal. C. AMALFITANO , V. COZZOLINO , to de Producción Agrícola, Facultad de Ciencias 1 1 2 1 D. AGRELLI , V. DI MEO , L. MUGNAI , A. EVIDENTE Agronómicas, Universidad de Chile, Casilla 1004, 2 1 and G. SURICO . Dipartimento di Scienze del Suolo Santiago-Chile. della Pianta e dell’Ambiente, Università di Napoli E-mail: [email protected] Federico II, Via Università 100, 80055 Portici, Ita- Table grape is the main exported Chilean fruit- ly. 2Dipartimento di Biotecnologie Agrarie, Sezione bearing species and the table grape cultivar Red di Patologia Vegetale, Università degli Studi, Pi- Globe is the second most important cultivar grown.

102 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

This cultivar is subject to a decline syndrome which strictum are considered to be non-pathogens or has been associated with Botryosphaeria obtusa endophytes due to their small lesions and low re- and Phaeoacremonium sp. The symptoms are: de- isolation percentages. Field trials after 14 months layed budding, poor shoot and berry growth and confirmed Pa. chlamydospora as the most aggres- uneven ripeness; other symptoms are: short inter- sive pathogen, since it produced the largest lesions nodes, wine-red spots on the leaf margins or on in the trunks, as well as on inoculated pruning the leaf blade, and dark wood streaking of the xy- wounds. It was also re-isolated more frequently lem. To study the effect of this pathology on berry than any other fungus, especially from the inocu- development, healthy and diseased plants were lated pruning wounds. The Massarina sp. was the compared by evaluating grape berries for size, col- only fungus that could not be re-isolated from any our, soluble solid content, fresh and dry weight, of the inoculated trunks or pruning wounds and calcium and potassium, and number of functional the wounding reaction did not differ from the con- vascular bundles. Diseased plants had a 2-week trol treatment. The Massarina sp. should there- delay in growth, ripeness and xylem closing com- fore be regarded as a non-pathogen or endophyte. pared with healthy plants. After xylem closing, All the other fungi infected and colonised the vines there was a decrease in the calcium content of both and produced lesions statistically different from plants, but the potassium content decreased only those caused by the water control and the non-path- in diseased plants, which suggested a phloem de- ogen in the field trial. terioration. At harvest time, berries from diseased plants had a much lower sugar content. The influence of Phaeomoniella chlamydospo- ra on the production of polyphenols and Pathogenicity testing of Phialophora, Phia- polyphenol oxidase (PPO) in nursery grown lophora–like, Phaeoacremonium and Acremo- grapevines. A.B. REED, L.D. MELTON and B.G. nium species isolated from vascular tissues SMITH. Food Science Programme, Department of 1 2 of grapevines. F. H ALLEEN , L. MOSTERT and P.W. Chemistry, University of Auckland, Private Bag 2 1 CROUS . ARC Infruitec-Nietvoorbij, Private Bag 92019, Auckland, New Zealand. X5026, Stellenbosch, 7599, South Africa. 2Centraal- E-mail: [email protected] bureau voor Schimmelcultures, Uppsalalaan 8, Petri disease, caused by Phaeomoniella chlamy- 3584 CT Utrecht, The Netherlands. dospora and Phaeoacremonium spp., is associated E-mail: [email protected] with vine decline and with the development of black Several Phialophora, Phialophora-like, Phaeoacre- streaking in the xylem of young grapevines sub- monium and Acremonium-like species were recent- ject to stress. Plants produce some polyphenols ly isolated from vascular tissues of grapevines in such as resveratrol, trans-piceid and the oligomer South Africa. In most cases their status as patho- viniferin, in response to fungal invasion. Polyphe- gens was unknown and pathogenicity studies were nols are oxidised by polyphenol oxidases (PPO), therefore conducted to determine their potential resulting in the formation of brown/black pigments. as decline pathogens. The fungi included Acremo- Both grapevines and fungi are known to produce nium cf. charticola, A. cf. strictum, Cadophora PPO enzymes. It is not known whether the brown/ luteo-olivacea, Massarina sp., Phialemonium cf. black streaking is as a result of PPO activity on curvatum, Phialophora richardsiae, four new Phae- the endogenous polyphenols. Field trials conduct- oacremonium species (Pm. krajdenii, Pm. venezue- ed in conjunction with a commercial viticulture lense, Pm. subulatum and Pm. Sp. 3). Phaeomo- nursery were carried out using Sauvignon blanc niella chlamydospora, Pm. aleophilum, Pm. para- grafted onto 101-14 Mgt. Vines were inoculated at siticum, and Pm. viticola were also included. Data the time of grafting on the omega graft wound with obtained from the glasshouse trial three months P. chlamydosporum spore suspensions (106 cfu ml-1) post inoculation were difficult to interpret due to or water. After 10 weeks, polyphenols were extract- the small lesions, and the similarity of lesions from ed from the xylem of rootstocks (n=10) and ana- different species. However, Pa. chlamydospora pro- lysed by the HPLC and Folin-Ciocalteu methods. duced the largest lesions and proved to be the most PPO was also extracted and the activity of the aggressive pathogen. A. cf. charticola and A. cf. crude extract of both inoculated vines and controls

Vol. 44, No. 1, April 2005 103 AA.VV. was measured using several polyphenol substrates. gus was also isolated from the substrate. Results The amount of resveratrol, trans-piceid and sever- confirmed that P. chlamydospora infected un-root- al other oligomeric polyphenols was greater in the ed cuttings of grapevine from the substrate and inoculated vines than in the controls. PPO extract- kept its viability throughout the assay. The tissue ed from both inoculated and control grapevines colonisation rate was moderate but sufficiently produced brown/black products when catechol, res- severe to render future use of infected propagation veratrol and chlorogenic acid were used as sub- material unadvisable. strates. Little initial activity was measured by fun- gal PPO alone, with no formation of brown/black pigments. Epidemiology Phaeomoniella chlamydospora: infection abil- Esca – a disease or a case of commensalism? 1 2 3 1 ity and survival in soil. L. GAFORIO, S. PASTOR, T. GRÄFENHAN , R. ZARE and W. GAMS . Federal C. REDONDO and M.L. TELLO. Instituto Madrileño Biological Research Centre for Agriculture and de Investigación Agraria (IMIA). Autovia del Nor- Forestry (BBA), Institute for Plant Virology, Micro- deste A-2, km. 38.200, E-28800 Alcala de Henares, biology and Biological Safety, Königin-Luise-Str. Madrid, Spain. 19, 14195 Berlin, Germany. 2Plant Pests and Dis- E-mail: [email protected] eases Research Institute (PPDRI), Department of Botany, PO Box 1454, Tehran 19395, Iran. 3Cen- Petri disease affects young grapevines. The main traalbureau voor Schimmelcultures, Uppsalalaan causal agent is the fungus Phaeomoniella chlamy- 8, 3584 CT Utrecht, The Netherlands. dospora, which causes decline and dieback. Infec- E-mail: [email protected] tion, as proposed by several researches, occurs by penetration through pruning wounds, by the use Esca is a complex disease of increasing importance of infected propagation material, and by the infec- around the globe. Its causal agents are well known tion of rooted cuttings through the soil. This last and now have a cosmopolitan distribution. Reasons way of transmission could well be occurring in prop- for their spread are found in modern viticultural agation plots in nurseries. The aim of this work practices: in the worldwide trade of scions and root- was to test the ability of P. chlamydospora to in- stocks without certification, and in current tech- fect un-rooted cuttings from the soil; the ability of niques of grafting and high-density planting. When the fungus to survive in the soil; and to determine looking for unspoiled situations of viticulture with its infection rate inside plant tissues. Healthy cut- an ancient history, we expected that Iran would of- tings of Vitis vinifera var. Tempranillo, with no fer insights into a traditional, apparently primitive roots, were planted in pots that were artificially- style of wine growing. A high diversity of indige- inoculated with a spore suspension of 1.3ϫ107 sp nous cultivars has been preserved and own-rooted ml-1 and incubated under semi-controlled condi- grapevine plants are grown in a traditional way with tions (22°C, 30% RH, 14-hr light). The inoculum limited commercial incentive. Therefore we were was applied to the substrate (peat/vermiculite 1:1) particularly interested to study the distribution of before planting the cuttings. Samples from sub- esca and its causal agents under these conditions. strate and cuttings (wood) were collected periodi- We assumed that the most natural growing condi- cally over 3 months, and the pathogen was reiso- tions would be found in the foothills of the Minor lated from them. Even though the cuttings re- Caucasus, on the north-western border of Iran, mained asymptomatic, the fungus was isolated where notable populations of wild grapevine (Vitis from plant tissue throughout the assay, penetrat- sylvestris) still occur. During a summer visit, few ing the plant tissue up to 10 cm from the cutting diseased plants with clear-cut external and inter- base at 90 days after inoculation. The progression nal esca symptoms were found in various vineyards rate was also confirmed by microscopic observa- in the provinces of Ardabil and West Azarbaijan and tion of tissue slides and estimated as 16.7 mm/day the pathogens of these plants were isolated. During in the first month, 3.3 mm/day in the second month a spring visit, symptoms mainly consisting of yel- and 13.3 mm/day in the third month. Tylose for- lowing were seen in some vines in the provinces of mation started during the second month. The fun- Fars, Qom, Qazvin and Semnan, and the fungi con-

104 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

cerned were isolated. Apoplexy has neither been Petri disease. S.A. WHITEMAN, M.V. JASPERS, A. seen nor reported in Iran. Wild grapevine hardly STEWART and H.J. RIDGWAY. National Centre for showed any symptoms, but some apparently endo- Advanced Bio-Protection Technologies, PO Box 84, phytic fungi were isolated. Obviously grapevine Lincoln University, Canterbury, New Zealand. trunks are a suitable biotope for a diversity of fun- E-mail: [email protected] gi, including species that are pathogenic in intensi- Phaeomoniella chlamydospora is believed to be the fied viticulture, but they cause little harm in more primary causal agent of Petri disease of grapevines natural situations. in New Zealand. A number of researchers have hy- pothesised that planting young vines in soil infest- First report of naturally occurring Togninia ed with the fungus can result in infection. DNA minima perithecia in California vineyards. S. was extracted from soil samples collected from be- ROONEY-LATHAM, A. ESKALEN and W.D. GUBLER. De- neath rootstock mother-vines known to be infect- partment of Plant Pathology, University of Califor- ed with P. chlamydospora, using a SDS/phenol/chlo- nia, Davis 95616, USA. roform method and then tested using a nested spe- E-mail: [email protected] cies-specific PCR assay. The detection level of the Esca can be a severe disease of grapevines in Cal- assay was determined to be 103 conidia g-1 when a ifornia, capable of causing serious economic dam- conidial suspension was added to sterilised soil age to table, raisin and wine grape cultivars. One samples, and 5 fg when P. chlamydospora genomic of the most commonly isolated fungal pathogens DNA was added directly to the PCR reaction. Of associated with esca-diseased vines in California the 20 bulked samples tested, 20% were positive. is Togninia minima (P. aleophilum). Although the In order to determine whether infested soil was a perfect stage was recently identified in vitro by source of inoculum, young vines were planted in successful crossings of compatible isolates, the soil that was artificially infested with a conidial natural occurrence of perithecia in vineyards was suspension of P. chlamydospora for which an iso- hitherto unknown. The existence of perithecia in late-specific genetic marker had been developed. vineyards was recently suggested in California and Use of this isolate allowed differentiation between Australia, after T. minima perithecia developed on natural infection and infection due to the intro- naturally infected, moist-incubated grapevine wood duced inoculum. Three months after planting, sam- after extended periods of time. Vineyards through- ples of woody tissue from all vines were plated on out California, shown to contain aerial propagules agar. DNA was extracted from all recovered P. of T. minima by spore trapping, were surveyed for chlamydospora isolates using a commercially avail- the source of these spores. Perithecia morphologi- able kit and amplified using an isolate-specific cally identical to those of published T. minima de- primer pair. A restriction digest was performed on scriptions and to those produced in culture, were the resulting products. Identification of the inocu- identified on grapevines in multiple vineyards in lated isolate was possible by the observation of a California. After thorough moistening, perithecia specific banding pattern following digestion. Of the forcibly discharged their ascospores, as previously 40 vines planted in infested soil, 5% were found to documented for T. minima perithecia produced in be infected with the inoculated isolate. P. chlamy- vitro. Furthermore, molecular analyses of the ITS dospora was not reisolated from any of the control region of the nuclear ribosomal DNA confirmed vines. The results of this work confirm that P. these perithecia to be T. minima. Additional de- chlamydospora is present in rootstock mother-vine tails regarding ascospore release and where these block soil and has limited potential to infect young perithecia reside on the grapevines will be dis- vines. Options for further research to determine cussed. This is the first report of the occurrence of the importance of this inoculum source will be dis- T. minima perithecia on standing grapevines and cussed. suggests the importance of ascospores as a source of inoculum for new grapevine infections. Susceptibility of grapevine spring wounds to Eutypa lata: further results and present Infested soil as a source of inoculum for Phae- epidemiological status. P. L ECOMTE, S. CARDON, omoniella chlamydospora, causal agent of N. BASTIEN, S. GIRY LATERRIERE and D. BAILEY. In-

Vol. 44, No. 1, April 2005 105 AA.VV. stitut National de la Recherche Agronomique, thology, Michigan State University, 104 CIPS UMR Santé Végétale (INRA-ENITAB), CR Bor- Building, East Lansing, MI 48824, USA. deaux-Aquitaine, Avenue Edouard Bourleaux, BP E-mail: [email protected] 81, 33883 - Villenave d’Ornon cedex, France. The objective of this study was to monitor ‘Con- E-mail: [email protected] cord’ vines with Eutypa dieback over several years The susceptibility of grapevine to Eutypa dieback and assess losses in yield and quality due to the by the contamination of wounds caused when ex- disease. A ‘Concord’ vineyard in southwest Michi- cess shoots are pruned and removed in spring may gan was monitored from 1999 to 2002. The inci- play a role in the epidemiology of this wood decline dence of vines with typical symptoms (cupped disease. Preliminary findings, following inoculation leaves, chlorosis, and stunted shoots) was 1% in of young Cabernet Sauvignon plants grown in shel- 1999, 0.6% in 2000, 4% in 2001, and 17% in 2002. tered conditions and a 2-week incubation period, led None of the vines showed foliar symptoms every us to hypothesise that this kind of wound provides year and a few vines died. To determine the effect an infection court for ascospores of E. lata. To fur- on yield on a per-shoot basis, individual shoots on ther understand the conditions that affect the colo- infected vines were categorized as having no, mild, nisation of spring wounds, a number of natural or intermediate, or severe symptoms in 1999–2001. artificial infection studies were undertaken in Bor- Compared to nonsymptomatic shoots on the same deaux area between 1999 and 2004. The infection vine, severe infection resulted in significant reduc- efficiency (IE, percentage recovery of E. lata) was tions in fruit weight per shoot, fruit weight per clus- examined for different growth conditions (control- ter, number of berries per cluster, number of clus- led or natural), contamination conditions (artificial ters per shoot, and weight per berry. Even in mild- or natural), wound type (desuckering or disbudding), ly infected shoots, significant reductions were ob- inoculum density (0, 100 or 1000 spores per wound) served in fruit weight per shoot, fruit weight per and incubation period (two weeks or one year). All cluster, and number of berries per cluster. Non- inoculations were performed on Cabernet Sauvi- symptomatic shoots on infected vines had higher gnon. The presence of E. lata was diagnosed by cluster weights and a greater number of berries morphological assessment assisted by PCR. Results per cluster than similar shoots on apparently were highly variable. For plantlets grown under healthy vines, suggesting compensation by infect- controlled conditions, the IE after one year incuba- ed vines. The disease had only a slight effect on tion was lower than had been reported previously. pH, and the Brix, and tartaric acid content of the Moreover, the overall IE recorded in this study was juice of infected vines. On a whole-vine basis, vines lower than that reported generally for winter prun- with mild symptoms showed an average yield re- ing analysed under the same conditions. In this duction of 21% (2003) and 15% (2004) compared to study, we found no consistent effect of inoculum apparently healthy vines. Vines with intermedi- density or wound type on IE. However, the IE was ate symptoms showed an average yield reduction significantly higher with artificial than with natu- of 27% (2003) and 30% (2004). Severely sympto- ral contamination. With artificial contamination, the matic vines had an average yield reduction of 59% IE was higher for wounds assessed after 1 year than (2003) and 83% (2004). These results show that after two weeks. Variability between results ob- Eutypa reduces yield considerably, even in mildly tained from artificially infected wounds was most symptomatic vines. Variability in symptom expres- likely due to assay limitations such as the buildup sion from year to year will make it more difficult of other micro-organisms. On the whole, these re- for growers to monitor their vines for infection sults, combined with relatively low levels of as- based on foliar symptoms. cospore release during spring, suggest that the epi- demiological role of spring wound contamination is Investigation into the occurrence of Botry- less important than that caused by winter pruning. osphaeria dothidea in grape propagative material and pathogenicity studies on differ- Eutypa dieback: disease progress and losses ent woody plants. I.C. RUMBOS. NAGREF, Plant in ‘Concord’ grapes. S.C. BUTTERWORTH, S.A. JOR- Protection Institute of Volos, Volos 380 01, Greece. DAN and A.M.C. SCHILDER. Department of Plant Pa- E-mail: [email protected]

106 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

4 5 1 The fungus Botryosphaeria dothidea (Moug.:Fr.) OSTI and G. SURICO . A.R.S.I.A. Regione Toscana, Ces. & De Not. is the causal agent of black dead Via Pietrapiana 30, 50100 Firenze, Italy. 2Diparti- arm of grapevine and is associated with branch and mento di Scienze degli Alimenti, Università di Ter- trunk dieback, wood necrosis, brown wood streak- amo, Via Spagna 1, 64023 Mosciano S.A., Teramo, ing and incomplete grafts. An extensive study was Italy. 3Dipartimento di Scienze Agronomiche e Ges- carried out during 2002 and 2003 covering the en- tione del Territorio Agro-forestale, Università di tire grape-growing area of Greece in order to in- Firenze, Piazzale delle Cascine 18, 50144 Firenze, vestigate the incidence of B. dothidea and other Italy. 4Istituto di Biometeorologia, Sezione di Bolo- wood destroying fungi in the grape propagative gna, CNR, via Gobetti 101, 40129, Bologna, Italy. material that was produced in Greece or other 5Dipartimento di Biotecnologie Agrarie, Sezione di European countries. Four groups of vine material Patologia vegetale, Piazzale delle Cascine 28, 50144 were examined: a) 12,000 unrooted rootstock cut- Firenze, Italy. tings from governmental or private mother nurs- E-mail: [email protected] eries, b) 3000 rooted rootstock cuttings from Greek Outbreaks of esca were monitored in Tuscany, nurseries or from other European countries, c) 5000 Emilia Romagna and Marche, Italy. Data and anal- scion cuttings, d) 5000 bench-grafted rooted vines yses were consistent with previous observations. from Greek nurseries or from other European coun- Random or near-random distribution of diseased tries. The sampling strategy was carried out by 33 vines was observed in vineyards with low disease agronomists according to our instructions. In to- incidence (<10%). In vineyards with a disease in- tal, over 20,000 cuttings or grafted rooted vines cidence over 10–15%, infected vines were nearly were examined and over 80,000 isolations were uniformly distributed or tended to aggregate in made. B. dothidea was not isolated from any un- clusters. None of the analyses detected a preferen- rooted rootstock or scion cutting. The fungus was tial spread of the disease from plant to plant along isolated in a low percentage (0.3%) from rooted columns or in any other direction. But though dis- rootstock cuttings and in a higher percentage from eased vines did not often influence immediately grafted rooted vines (3.5%). In some grafted root- adjacent plants, infected vines did appear to be ed cultivars from other European countries the aggregated within 10–12 plant-spacings of each percentage was significantly higher (12.5%). It was other in all directions. A single-point source of out- concluded that planting material colonised by Bo- breaks could not be identified: several foci were tryosphaeria dothidea at the nursery was one rea- present in the vineyards surveyed. From the time son for the decline of young grapevines. Patho- the outbreaks started (in some vineyards two or genicity studies with isolates from kiwifruit (B. three years after establishment) disease incidence parva), olive (Botryosphaeria sp.), walnut (B. increased in almost linear progression. Weather dothidea), Ilex aquifolium (B. parva), grapevine (B. data were compared with esca incidence over 4-to- dothidea) and pistachio (B. dothidea) on grapevine 10-year periods in all vineyards. to look for a rela- (different cultivars and rootstocks) and other woody tion between annual esca incidence and the number hosts (kiwifruit, olive, Ilex aquifolium, walnut, of days with rain, total rainfall during the entire quince, pear, almond, plum, cherry), showed that growth period, and rainfall in single months. The all isolates from any host would infect other hosts results seem to confirm what was previously re- although considerable variation in aggressiveness ported, that cool, rainy growing seasons are more was noticed. No evidence for host specificity was favourable to the emergence of foliar esca symp- observed. The growth rate of Botryosphaeria iso- toms. lates from different hosts at different temperatures (5, 10, 15, 20, 25, 30, 33, 37°C) on PDA was also Enviro-spatial distribution of grapevine studied. trunk pathogens in South Africa. J.M. VAN 1 1 1 2 NIEKERK , W. BESTER , U. DAMM , F. HALLEEN , P.W. 3 1 1 Relation of esca foliar symptoms to rainfall CROUS and P.H. FOURIE . Department of Plant Pa- 1 and rainfall-related parameters. P. B RACCINI , thology, University of Stellenbosch, Private Bag 2 3 4 2 F. C ALZARANO , A. DALLA MARTA , S. DI MARCO , G. X1, Matieland 7602, South Africa. ARC Infruit- 5 5 5 3 MARCHI , L. MUGNAI , F. PEDUTO , S. ORLANDINI , F. ec-Nietvoorbij, Private Bag X5026, Stellenbosch

Vol. 44, No. 1, April 2005 107 AA.VV.

7599, South Africa. 3Centraalbureau voor Schim- Black foot disease in South African vineyards 1 melcultures, Uppsalalaan 8, 3584 CT Utrecht, The and grapevine nurseries. F. H ALLEEN , P.H. FOU- 2 3 1 Netherlands. RIE and P.W. CROUS . ARC Infruitec-Nietvoorbij, E-mail: [email protected] Private Bag X5026, Stellenbosch, 7599, South Af- 2 Recent studies have identified several trunk path- rica. Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Matieland 7602, ogen complexes from grapevines in South Africa. 3 These include Eutypa lata, Phaeomoniella chlamy- South Africa. Centraalbureau voor Schimmelcul- dospora, Phaeoacremonium, Botryosphaeria and tures, Uppsalalaan 8, 3584 CT Utrecht, The Neth- Phomopsis spp. Climatic differences between erlands. grape growing regions are likely to affect the de- E-mail: [email protected] mographics of regional pathogen populations. In The survival rate of grapevine cuttings in nurs- order to develop management strategies for spe- eries, and young grapevine plants in newly estab- cific regions, it was therefore necessary to deter- lished vineyards in South Africa is being increas- mine the distribution of grapevine trunk patho- ingly compromised by root and trunk pathogens. gens in the different grape-growing regions of Cylindrocarpon spp., which cause black foot dis- South Africa. During 2003 and 2004, a survey of ease of grapevine, were associated with 52, 22 and visually healthy grapevines was conducted in 15 29% of diseased grapevines during 1999–2002, wine (Cabernet Sauvignon) and 15 table grape 2002–2003 and 2003–2004 respectively. Howev- (Dan-ben-Hannah) vineyards that were 10 years er, very little information is available regarding and older. These vineyards were situated in the the aetiology and epidemiology of the pathogens summer-, intermediate- or winter-rainfall regions believed to be involved in black foot disease. The of South Africa. The occurrence of trunk patho- primary aims of research have been (1) to con- gens was determined by making isolations from duct nursery surveys in order to determine which areas with wood decay symptoms, observed in fungi are involved in the decline phenomenon, (2) cross sections of the samples taken (distal portions to identify the organisms believed to be the caus- of arms). Symptoms observed were brown inter- al organisms of black foot disease, and (3) to de- nal necrosis (BIN), black (Bl-st) and brown (Br- velop management strategies to prevent or erad- st) streaking, esca-like yellow (E-Y) and brown (E- icate these infections. The diversity of species as- B) soft rot, V-shaped necrosis (V-N) and watery sociated with black foot disease was confirmed by necrosis (W-N). Results indicate that Pa. chlamy- this study and four Cylindrocarpon-like species dospora was isolated significantly more often in were identified: Cylindrocarpon destructans, C. the winter-rainfall region (19%) than in the inter- macrodidymum, Campylocarpon fasciculare and mediate-rainfall (8.9%) and summer-rainfall Campyl. pseudofasciculare. The observation that (3.1%) regions. In the winter-rainfall region, the these species infect grapevine cuttings in nurs- fungus had a broad symptom profile, whereas in ery soils has clearly placed the emphasis on the the other regions it was isolated mainly from BIN importance of suitable measures to prevent or and Bl-st. Phaeoacremonium spp. had similar dis- eradicate these infections. However, none of the tribution patterns and symptom profiles, but were chemical and biological treatments evaluated in isolated at slightly higher levels in the summer this study prevented infection of nursery grape- rainfall region. Botryosphaeria spp. were isolated vines. On the other hand, the reduction of infec- most frequently from the intermediate-rainfall tion in uprooted dormant nursery grapevines (12.4%) and summer-rainfall (5.9%) regions and caused by hot water treatment clearly demon- mostly from BIN, V-N and Bl-st. Phomopsis spp. strated the potential of this control measure. were isolated mostly from the winter- (3.4%) and Apart from these pro-active management strate- intermediate-rainfall (2.8%) regions, with a vary- gies in grapevine nurseries, no curative strategy ing symptom profile between regions. E. lata was is known for declining grapevines in vineyards. infrequently isolated and only from the winter- Producers are therefore urged to prevent or cor- rainfall region. Once all isolates have been iden- rect predisposing stress situations, such as un- tified to species level, regional management strat- balanced root development, soil compaction and egies will be formulated accordingly. poor drainage.

108 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

Petri disease: potential inoculum sources in more than those with esca (18%). In addition, Phae- South African grapevine nurseries. E. RETIEF, omoniella chlamydospora was isolated from all but U. DAMM, A. MCLEOD and P.H. FOURIE. Department two of the samples, whereas Phaeoacremonium of Plant Pathology, University of Stellenbosch, Pri- aleophilum was isolated from only nineteen sam- vate Bag X1, Matieland (Stellenbosch) 7602, South ples. Other fungi associated with grapevine decline Africa. diseases, such as species of Phomopsis, Cylindro- E-mail: [email protected] carpon, Botryosphaeria and heart-rotting basidio- Petri disease of grapevine is primarily caused by mycetes, were also isolated but not consistently. Phaeomoniella chlamydospora. This pathogen af- Thirty-two different grapevine cultivars were rep- fects mostly young grapevines, but is also impli- resented, and samples were received from most cated in esca disease of older grapevines. Little is major grape-growing regions of New South Wales, known about the disease cycle of this fungus. In- South Australia, Victoria and Western Australia. fected propagation material was identified as a The majority (62%) were ungrafted grapevines. Pa. major means of dissemination of the pathogen. chlamydospora was also found to be present in Recently, the pathogen was also detected from soil grapevines with no external symptoms of disease, in South Africa, and airborne conidia have been including mother grapevines used for propagation. trapped in vineyards. The aim of this study was to use a molecular detection technique to test differ- Stone fruit trees as alternative hosts of grape- 1 ent samples (water, soil, rootstock and scion cut- vine trunk disease pathogens. U. DAMM , P.W. 2 1 1 tings and callusing medium) collected from nurs- CROUS and P.H. FOURIE . Department of Plant Pa- eries in South Africa at different nursery stages thology, University of Stellenbosch, Private Bag X1, 2 for the presence of Pa. chlamydospora. All sam- Matieland 7602, South Africa. Centraalbureau ples were tested using a DNA extraction and spe- voor Schimmelcultures, Uppsalalaan 8, 3584 CT cies-specific PCR protocol that was previously de- Utrecht, The Netherlands. veloped and validated. Pa. chlamydospora was E-mail: [email protected] detected from the rootstock cuttings, hydration Grapevine (Vitis vinifera) and stone fruits (several water and soil. These media can therefore be con- Prunus species) are often grown in close proximity sidered possible inoculum sources of the pathogen. to each other. Both species of woody plants are at- Management strategies should include pruning- tacked by fungal trunk disease pathogens, for ex- wound protection of rootstock mother plants, path- ample Botryosphaeria, Phomopsis and Eutypa spe- ogen eradication from rootstock cuttings (e.g. by cies. The aim of this study was to determine wheth- hot water treatment), biological or chemical amend- er stone fruits are inhabited by known grapevine ments in hydration water and prevention of wound- trunk disease pathogens as either pathogens or infection of grafted cuttings in nursery soils. saprobes, and whether stone fruits could act as al- ternative hosts of grapevine trunk disease patho- The occurrence of Petri disease and esca and gens. A survey of wood-inhabiting fungi of plum and their causal organisms in Australia. J. ED- peach trees from the Western Cape province in 1,2 2 1 WARDS and I.G. PASCOE . Cooperative Research South Africa was conducted by sampling living wood Centre for Viticulture, PO Box 154, Glen Osmond, with dieback, canker or necrotic symptoms as well 2 South Australia 5064, Australia. Primary Indus- as pruning debris. Fungi occurring in/on the sam- tries Research Victoria – Knoxfield Centre, Depart- ples were isolated and morphologically and phylo- ment of Primary Industries, Private Bag 15, Fern- genetically characterised. DNA sequences of the 5.8S tree Gully Delivery Centre, Victoria 3156, Australia. ribosomal gene with the two flanking internal tran- E-mail: [email protected] scribed spacers (ITS-1, ITS-2) were compared to Over a five year period (1998–2002), 124 samples those of known taxa currently available in of diseased grapevines sent to the Department of GeneBank. The genus Botryosphaeria dominated Primary Industries, Knoxfield, in Victoria were among the fungi isolated from necrotic plum wood, diagnosed with the grapevine decline diseases, and its fruiting structures were often observed on Petri disease and esca. The proportion of grape- dead peach wood. Ribosomal ITS sequences of the vines with Petri disease (82%) proved to be much isolates were the same as or very similar to those

Vol. 44, No. 1, April 2005 109 AA.VV. isolated from grapevine and belonging to B. obtusa, 2.4% in 2002, 3.55% newly infected vines in 2003 B. australis, B. rhodina, B. stevensii and Fusicoc- and 5.25% in 2004 (until September). The fungi cum vitifusiforme. Based on sequence comparison most frequently isolated were Phaeoacremonium analysis we also identified Togninia minima (an- spp., Phaeomoniella chlamydospora, Fomitiporia amorph: Phaeoacremonium aleophilum) and Pho- punctata and Fusicoccum sp. Annual and cumula- mopsis amygdali from necrotic plum wood. All these tive field maps will be presented as well as an in- species are reported to be pathogenic on grapevine. dex of dispersion of diseased plants. According to these results, stone fruit orchards should be considered a potential inoculum source of Relationship between incidence of esca and grapevine trunk disease pathogens. Further exam- black dead arm foliar symptom expression in inations will include similar surveys in climatically the vineyard, ecophysiological indicators and 1 different areas of South Africa and pathogenicity cultural practices. L. GUÉRIN-DUBRANA , A. 1 2 2 trails on grapevine and stone fruit wood. DESTRAC-IRVINE , J.P. GOUTOULY , A. LETOUZE and 2 1 J.P. GAUDILLÈRE . U.M.R Santé végétale INRA/ Spatial distribution of esca symptomatic ENITAB, CR Bordeaux, BP 81 33 883 Villenave plants in Dão vineyards (Centre Portugal) d’Ornon, France. 2U.M.R Oeno-Ampélologie INRA/ 1 and isolation of associated fungi. J. SOFIA , M.T. Faculté d’œnologie, Université Bordeaux II, CR 2 3 1 GONÇALVES and H. OLIVEIRA . DRABL, CEVDão, Bordeaux BP 81 33 883 Villenave d’Ornon, France. Quinta da Cal, 3520-090 Nelas, Portugal. 2Dep. E-mail: [email protected] Botânica, FCTUC, Universidade de Coimbra, 3000 3 Esca or black dead arm (BDA) foliar symptoms Coimbra, Portugal. Laboratório de Patologia Veg- were recorded at different frequencies in vineyards etal “Veríssimo de Almeida”, Tapada da Ajuda, of the same variety and similar age in the same 1399 Lisboa Codex, Portugal. area of production. This may be explained as due E-mail: [email protected] to variation in (i) inoculum dispersal of the fungi In recent years there has been a noticeable increase associated with these syndromes, (ii) contamina- in the incidence of esca disease in the Portuguese tion-related factors, (iii) the physiological status wine region of Dão. Since wine grape production is of the vine before and after infection and (iv) cul- the most important agricultural activity of this tural practices. In order to evaluate variations in region the occurrence of esca is of great concern symptom expression of esca and BDA and to study and deserves our attention. The objectives of this its relationship to host and environmental factors, study were to evaluate the spatial distribution of a 5-year field survey was set up around Bordeaux symptomatic plants and to investigate the fungi in 2004. Twenty-four vineyards of about 15-year- associated with diseased wood in these grapevines. old Cabernet Sauvignon vines trained to a Guyot The plot studied, with an area of ca. 4000 m2, com- Double trellis system were selected. The frequen- prised 1296 grapevines cv. Touriga Nacional graft- cy and severity of BDA and esca symptoms on 2000 ed on SO4 rootstock and was located in a charac- vines in each vineyard were recorded at two dates teristic 11 ha vineyard. Vines were planted in 1989. in summer. Pedological and ecophysiological indi- During three years (2002–2004) the presence/ab- cators were measured to estimate the water, ni- sence of foliar symptoms of esca in each vine was trogen and carbon nutrition status of the vines and recorded as well as any dead vines. Surveys took their vegetative vigour. Cultural practices were also place after the first symptoms on the leaves were recorded. Assessed disease results show a large noticed, especially between June and September, range of vines with BDA and esca foliar symptoms: with an average of one visit per month. Occurrence from 0 to 12.9% and from 0 to 30.8% respectively. maps were drawn each year, and cumulative maps To investigate the relationship between the inci- in the last two years. After the harvest, dead and dence of diseases with physiological and site traits, strongly symptomatic grapevines were uprooted; collected data were analysed by means of princi- transversal slices of the trunk were cut off and pal component analysis (PCA). small portions of deteriorated wood tissues used for mycological analysis. Annual disease incidence Long term vineyard monoculture influences increased over the three-year sampling period: the rhizosphere microbial communities. M.

110 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

1 2 2 1 SVERCEL , J. HAMELIN , M. ARAGNO and G. DÉFAGO . sition of grapevines to these diseases, internal 1Plant pathology group, Institute of Plant Scienc- wood symptoms are usually far advanced by the es, Swiss Federal Institute of Technology Zürich, time they are noticed. No curative control meas- Switzerland. 2Laboratoire de Microbiologie, Uni- ures for these diseases are known and remedial versity of Neuchâtel, Switzerland. management strategies in established and mature E-mail: [email protected] grapevines are expensive and in most cases only Root colonising Pseudomonas fluorescens strains, marginally effective. Research into the manage- which produce 2,4-diacetylphloroglucinol (Phl), are ment of these diseases has therefore focused on biocontrol agents against fungal pathogens in dif- pro-active management during the nursery stag- ferent pathosystems. The secondary metabolite Phl es in order to prevent and eradicate contamina- is toxic to certain bacteria, fungi and nematodes tion and infection of propagation material and and plays a key role in plant protection. Four vine- nursery grapevines, and consequently to ensure yards were identified where cultures of up to 1653 the production of healthy, pathogen-free grape- years old were growing next to others of 9–70 years. vines. Several semi-commercial nursery trials Long-term cultivation increases (i) the diversity of were conducted to study the effect of various treat- the soil bacteria in the deep (60–120 cm) soil lay- ments on the occurrence of natural infections of ers, (ii) the content of calcite in these deep layers grapevine trunk disease pathogens in graft un- and (iii) the frequency of the grape root associated ions, rootstocks and/or roots of nursery grape- pseudomonads carrying the key biocontrol gene vines. Results from these projects culminated in phlD. The copper added during cultivation accu- an extensive management strategy that integrat- mulated at the top of the soil profile following the ed pruning wound protection in rootstock mother soil organic matter. This project is funded by the blocks, chemical or biological amendments in hy- Swiss National Competence Centre for Research dration water, hot water treatment of rootstock “Plant survival”. cuttings and/or dormant nursery plants, hygienic grafting, wound protection during callusing and in field nursery soils and soil amendments with Disease management Trichoderma formulations. Integrated strategies for pro-active manage- The effects of hot water treatment on grape- ment of grapevine trunk diseases in nurser- vine cutting physiology and cell ultrastruc- 1 2 1 1,2 3 4 ies. P.H. FOURIE and F. HALLEEN . Department of ture. H. WAITE , J. FARAGHER and G. JAUDZEMS . Plant Pathology, University of Stellenbosch, Private 1Cooperative Research Centre for Viticulture, PO Bag X1, Matieland (Stellenbosch) 7602, South Af- Box 154, Glen Osmond, South Australia,506, Aus- rica. 2Disease Management Division, ARC Infruit- tralia. 2Institute of Land and Food Resources, Uni- ec-Nietvoorbij, Private Bag X5026, Stellenbosch versity of Melbourne, Dookie Campus, Dookie Col- 7599, South Africa. lege, Victoria, 3647, Australia. 3Department of Pri- E-mail: [email protected] mary Industries, Knoxfield Centre, Private Bag 15, Ferntree Gully Delivery Centre, Victoria 3156, Aus- Phaeomoniella chlamydospora and Phaeoacremo- tralia. 4Monash Microimaging, School of Biologi- nium spp. (causal organisms of Petri disease), Cy- cal Sciences, Monash University 3800, Victoria, lindrocarpon and Campylocarpon spp. (causal or- Australia. ganisms of black foot rot), Botryosphaeria spp. E-mail: [email protected] (causal organisms of black dead arm) and Pho- mopsis spp. (causal organisms of Phomopsis cane Hot water treatment (HWT; 50°C/30min) of dor- and leaf blight and dead arm) are economically mant grapevine cuttings is a useful measure to important grapevine trunk disease causing path- control endogenous pathogens including Phaeo- ogens, which can infect grapevines through moniella chlamydospora. However, there are re- wounds made during nursery processes or through ports of sporadic losses of HWT cuttings. To de- unprotected pruning wounds at later vineyard termine the underlying cause of cutting failure, stages. Given long periods prior to external symp- the effects of HWT on respiration and cell ul- tom expression in plants and/or stress predispo- trastructure were investigated. Emission of CO2

Vol. 44, No. 1, April 2005 111 AA.VV. in Cabernet Sauvignon, Chardonnay and Pinot ease over time. The evidence suggests that stress Noir cuttings collected in early, mid- and late win- is a major factor in development of the disease, ter was measured before, and 1 and 24 hours af- particularly stress due to inadequate irrigation ter HWT and after 14 weeks cold storage. In ear- and overcropping before the vine has established ly and mid season the pre-HWT respiration rate a decent root system, and if the stress is relieved of Chardonnay was higher (P<0.05) than that of the vines will recover. It is still unknown, howev- Pinot Noir and Cabernet Sauvignon, but by late er, whether recovery is permanent or whether dis- season the respiration rate of Pinot Noir was high- ease expression will re-occur some time in the est and Cabernet Sauvignon lowest (P<0.05). One future. hour after HWT, there was a rise (P<0.05) in all respiration rates except that of early-season Char- Degradation of trunk disease toxins by fun- donnay. After 24 hours the respiration rates of all gal antagonistic strains as a new prospect in cuttings had fallen (P<0.05). Respiration rates biocontrol strategies against Eutypa dieback 1 after cold storage were variable. Ethanol emis- and esca disease on grapevine. D. CHRISTEN , 2 2 2 sion was measured in Cabernet Sauvignon and M. THARIN , S. PERRIN-CHERIOUX , E. ABOU-MANSOUR , 2 1 1 Pinot Noir cuttings 24 hours and 1 month post R. TABACCHI and G. DÉFAGO . Phytopathology HWT and detected in HWT cuttings 24 hours post Group, Institute of Plant Sciences, Swiss Federal HWT. High post-HWT respiration rates and emis- Institute of Technology, Zürich, Switzerland. 2An- sion of ethanol by HWT cuttings indicated that alytical and Structural Organic Chemistry, Uni- HWT tissue became anoxic in the immediate post- versity of Neuchâtel, Switzerland. HWT period. Preliminary transmission electron E-mail: [email protected] microscopy examination of ray tissue in Caber- Eutypine, 4-hydroxybenzaldehyde and 3-phenyl- net Sauvignon 24 hours post HWT showed rup- lactic acid are some of the phytotoxins produced tured amyloplasts, fractured cell walls and in- by Eutypa lata causing Eutypa dieback, and by creased ribosomal activity in HWT cuttings +/- Phaeomoniella chlamydospora, Phaeoacremonium hydration and hydration only. We conclude that spp. and Fomitiporia mediterranea the causal HWT is a significant stress for grapevine cuttings agents causing esca. In order to develop a biocon- and that therefore post-HWT handling is likely trol strategy for disease prevention, known bio- to be critical to cutting viability. control agents such as Fusarium lateritium and Trichoderma sp. were tested for their ability to Experiences with amelioration treatments degrade these toxins. Detoxification processes trialed on Petri disease in Australian vine- 1,2 2 1 were investigated by incubating these potential yards. J. EDWARDS and I.G. PASCOE . Coopera- biocontrol agents in a liquid medium containing tive Research Centre for Viticulture, PO Box 154, the individual pure toxins. An HPLC-based meth- Glen Osmond, South Australia 5064, Australia. 3 od using UV- and MS-detection was developed to Department of Primary Industries, Knoxfield Cen- analyse toxin metabolisation, and a quantifica- tre, Private Bag 15, Ferntree Gully Delivery Cen- tion of degradation products was performed. The tre, Victoria 3156, Australia.. results show that the aldehyde function was re- E-mail: [email protected] duced to the corresponding alcohol and oxidised This paper reports on the results of four field tri- to the acid form simultaneously for eutypine and als investigating amelioration treatments for for 4-hydroxybenzaldehyde. While the Trichoder- Petri disease of grapevines. Treatments included ma strains degraded completely every toxin test- applications of compost, nutrient fertilisers, ex- ed, the F. lateritium strains degraded eutypine tra water, phosphonates and Brotomax. Vine and 4-hydroxybenzaldehyde but not 3-phenyllac- health was monitored for two to five years. To tic acid. In biological assays on cells of Vitis vini- summarise our experience with field trials to date, fera cv. Chasselas, the degradation products ex- development of management strategies to deal hibited a lower toxicity than the toxins. The pos- with infected vineyards has been very difficult sibility of developing new control strategies because no single treatment has proven effective. against trunk diseases of grapevine based on mi- Despite this, the vines recovered from Petri dis- crobial detoxification is discussed.

112 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

Wood-inhabiting fungi isolated from New wood-infecting pathogens vines intended for vine- Zealand grapevines and the potential for pro- yard planting stock. 1 tective control with Trichoderma. I.C. HARVEY 2 1 and J.S. HUNT . Plantwise Services Ltd, PO Box Biological control of Eutypa dieback in Aus- 181, Lincoln, New Zealand. 2Agrimm Technologies tralia: field evaluation of fungal antagonists. 1 1 1 2 Ltd, PO Box 13-245, Christchurch, New Zealand. S. JOHN , J. MALONE , E.S. SCOTT , T.J. WICKS and 3 1 E-mail: [email protected] J.S.HUNT . The University of Adelaide, Waite Cam- 2 The incidence of disease-causing fungi in samples pus, Glen Osmond, SA 5064, Australia. South Australian Research and Development Institute, received from the major winegrowing regions of 3 New Zealand was assessed from August 2000 un- GPO Box 397, Adelaide SA 5001, Australia. til December 2003. The pathogens were isolated Agrimm Technologies Ltd, PO Box 13-245, from sections taken from the roots, rootstocks, graft Christchurch, New Zealand. unions, bud wood and stems of grapevine wood in E-mail: [email protected] specimens showing vineyard symptoms and sent Eutypa dieback, caused by Eutypa lata, reduces to the Lincoln laboratory for diagnosis. The four the longevity of premium vineyards in Austral- most common pathogens isolated from all 37 sam- ia. Since ascospores of the pathogen enter the ples were Botryosphaeria spp. (68%), Phomopsis vine via wounds, wound protection offers a means (40%), Cylindrocarpon destructans (27%) and Phae- of control. Preliminary research on the biologi- omoniella chlamydosphora (19%). The in vitro bio- cal control of E. lata using Trichoderma har- control potential of a cohort of 8 Trichoderma har- zianum and Fusarium lateritium was presented zianum and T. atroviride antagonist strains were at the 3rd International Workshop on Grapevine assessed in a dual inoculum culture system against Trunk Diseases (February 2003, Christchurch, five species of Botryosphaeria isolated from these New Zealand). Five trials were established over samples (B. dothideae, B lutea, B. obtusa, B. parva 3 years using mature vines of cv. Cabernet Sau- and B. stevensii). Morphological changes observed vignon, Shiraz, Rondella and Palomino at vari- in the mycelium of these Botryosphareria spp. at ous sites in South Australia. One-year-old canes the interface with the Trichoderma included abut- were pruned in winter (July–August), treated ment and coiling associated with myco-parasitism with T. harzianum or F. lateritium within an hour by the Trichoderma as well as evidence of cell lysis. of pruning, and inoculated with spores of E. lata There was no evidence of antibiosis in any of the 1 or 14 days later. T. harzianum was applied as pathogen/antagonist combinations. Sauvignon a spore suspension or in commercial formulations blanc vines on SO4 rootstock were inoculated at (Trichoseal Spray™ or Vinevax™), F. lateritium the graft union with Trichoderma at the time of as a spore suspension only. Colonisation of the grafting (14 reps), rooted and compared with the spurs by E. lata was assessed by isolation after untreated controls after growing under glasshouse 10 weeks to 11 months. Also, the colonisation of conditions. Vines were destructively analysed (2 vines of cv. Nyora by T. harzianum, applied as reps of each group) by sampling each plant in 10 Trichodowels inserted into the trunk, was as- locations from 5 cm below the graft union to 5 cm sessed over 20 months. Interactions of T. har- above. Trichoderma was found to have grown 5 cm zianum and F. lateritium in vitro were examined into the rootstock wood and for varying distances to assess the potential for dual application of the into the scion wood. Trichoderma was not isolated antagonists. Recovery of E. lata from inoculated from the new cordon growth or from any of the con- controls varied and was greatest (77% of spurs) trol samples. B. parva, Phomopsis spp. and Alter- when wounds were inoculated 1 day after wound- naria alternata were isolated from the graft union ing in July (mid-winter). Pruning wounds treated and scion wood of both control plants. These re- with T. harzianum or F. lateritium showed reduced sults suggest that a) Botryosphaeria and Phomo- incidence of infection by E. lata in most experi- psis spp. and C. destructans may be more preva- ments when the wounds were challenged 1 and 14 lent in New Zealand vineyards than previously days after treatment with the antagonist. T. har- thought; b) Trichoderma inoculation of grafted zianum spread as far as the crown of the Nyora material may have potential in protecting against vines and persisted for at least 20 months in some

Vol. 44, No. 1, April 2005 113 AA.VV. vines. T. harzianum inhibited growth of F. lateri- al of benomyl products from the market, alterna- tium in vitro. T. harzianum and F. lateritium, ap- tives such as carbendazim fungicides or acrylic plied separately, have potential to protect wounds paint are suitable for preventing infection of prun- from infection by E. lata. The spread and persist- ing wounds by E. lata. Furthermore, our study ence of T. harzianum in the vine suggest that it identified several other fungicides which have po- may also offer systemic protection. tential as pruning wound protectants and further testing is under way. Field evaluation of pruning wound treat- ments for control of Eutypa dieback. M.R. Effect of fosetyl Al foliar applications towards 1,2 1,2 2 1 SOSNOWSKI , M.L. CREASER and T.J. WICKS . Co- esca fungi in grapevine. S. DI MARCO and F. OSTI. operative Research Centre for Viticulture, PO Box Istituto di Biometeorologia, Sezione di Bologna, 154, Glen Osmond SA 5064, Australia. 2South Aus- CNR, via Gobetti 101, 40129, Bologna, Italy. tralian Research and Development Institute, GPO E-mail: [email protected] Box 397, Adelaide SA 5001, Australia. This study reports on the activity and effect of fo- E-mail: [email protected] setyl Al applications on esca and associated patho- Eutypa dieback causes a gradual decline of grape- gens. Different trials were conducted treating pot- vine production in many wine regions around the ted grafted vines with fosetyl Al formulations. world. Ascospores of the fungus Eutypa lata enter Phaeoacremonium aleophilum and Phaeomoniel- via pruning wounds and colonise the wood tissue la chlamydospora were then inoculated into the in the cordons and trunks. Pruning wound protect- rootstock. Assessments were made by measuring ants can be used to reduce infection, especially on the average length of wood discolouration and in- larger wounds. In Australia, the only product cur- vestigating in vitro the viability of the fungi. Field rently registered for this purpose is Vinevax™ trials were carried out either before or soon after pruning wound dressing (Trichoderma sp., formerly the appearance of the disease in properly managed Trichoseal®) and this study may lead to the regis- vineyards with vigorous vines and abundant foli- tration of additional products. Trials were conduct- age. About 4500 vines in three vineyards were as- ed on Cabernet Sauvignon vines in the Barossa sessed annually by visual inspection of foliar symp- Valley, South Australia, between 2000 and 2003 to toms, vine mortality and esca incidence. Six fosetyl evaluate the efficacy of various fungicides, a bio- Al treatments were given annually for 4 to 6 years control agent and paint as wound protectants with the routine plan for downy mildew control and against E. lata infection. Pruning wounds were were compared to vines that were never treated made in winter (August) on 1-year-old canes, and with the fungicide. Commercial formulations of the treatments applied with a paintbrush within 1 fosetyl Al in combination with mancozeb and cy- hour. Wounds were inoculated with 500 ascospores moxanil, and/or copper oxychloride were used. The of E. lata either 1 day before or 1, 7 or 14 days effects of fungicide application on plant physiolo- after treatment. Canes were removed in the fol- gy were continuously assessed by a gas exchange lowing winter and the presence or absence of E. measurement system. Most of the greenhouse tri- lata was determined by isolation on potato dextrose als showed a reduction in the average length of agar with streptomycin. Benlate® (benomyl), Bavis- internal necrosis, especially P. aleophilum necro- tin® (carbendazim), Solucuivre® (copper and sis, in fosetyl Al treated plants compared with the carbendazim), Garrison® (cyproconazole and iodo- control. Both fungi were re-isolated from the treat- carb in paste) and ATCS Tree Wound Dressing ed vines. Field trials generally showed that mor- (acrylic paint) reduced infection by E. lata at all tality of grapevines was not influenced by fosetyl inoculation times. Fungaflor® (imazalil sulphate), Al treatment. Fungicide application generally led Scala® (pyrimethanil), Cabrio® (pyraclostrobin), to a decrease in the number of vines showing esca Bayfidan® (triadimenol), Teldor® (fenhexamide) and symptoms in the year compared to the control. The Topas® (penconazole) reduced infection, but not at incidence of esca in the fosetyl Al treated vineyards all inoculation times. Trichoseal® (Trichoderma sp.) at the end of the trials was lower than that in the reduced infection when the pathogen was applied control. The net photosynthetic rates of treated 14 days after treatment. With the recent withdraw- plants were depressed, especially when fosetyl Al

114 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines”

+ mancozeb + cymoxanil was applied. This decrease treated plot and 63% in the control plot. The an- in photosynthesis probably affected parameters nual percentage of symptomatic plants followed a associated with transport inside the plants. Al- different pattern between the two treatments: in though the production of foliar symptoms is not the pruned sprayed plots it was represented by a yet well understood, a relationship between phys- quadratic model (y= -0.6463x2 + 9.3639x – 6.6939; iological effects caused by fosetyl Al and foliar R2=0.9916), while the control plot showed a power symptom expression cannot be excluded. Our find- pattern (y=2.7507x1,593; R2=0.9992). This difference ings indicate that fosetyl Al decreased the severity suggests that spraying the cutter and the pruning of esca. The activity seemed to be linked to the fa- cuts significantly reduced the number of sympto- vourable combination between the fungicide mode matic esca plants. This technique deserves to be of action and the particular condition of esca-af- better evaluated as a means to reduce esca spread fected vines. in the vineyard.

Agronomical approach to reduce esca disease Survival of fungi associated with grapevine 1 spread in the vineyard. G. SCALABRELLI , G. FER- wood declines in pruned material following 1 1 2 1 1 1 1 RONI , C. D’ONOFRIO and A. FELICIOLI . Dipartimen- composting. P. L ECOMTE , G. LOUVET , B. VACHER 2 1 to di Coltivazione e Difesa delle Specie Legnose, and P. GUILBAULT . Institut National de la Recher- Università di Pisa, Via del Borghetto 80, 56124 che Agronomique, UMR Santé Végétale (INRA-EN- Pisa, Italy. 2Dipartimento di Anatomia Biochimi- ITAB), CR Bordeaux-Aquitaine, Avenue Edouard ca e Fisiologia Veterinaria, Università di Pisa, Viale Bourleaux, BP 81, 33883 - Villenave d’Ornon cedex, delle Piagge, 56124 Pisa, Italy. France. 2Chambre d’Agriculture de la Gironde, 39 E-mail: [email protected] rue Michel Montaigne, BP 115, 33294 - Blanquefort, Pruning wounds are one of the main infection France. courts for the fungal agents of esca. A trial was set E-mail: [email protected] up to evaluate the outcome of suitable cultural In viticulture, a range of management practices practices aimed at limiting or avoiding contami- exist to deal with canes or undesirable debris re- nation during pruning. A 22-year-old cv. Sangio- moved during the pruning period in winter. Among vese vineyard (2 ha) was divided in two plots of 1 these, grinding, composting and reapplication in ha in order to set up two treatments. In the first the vineyard are the most efficient methods for plot, pruning was carried out using pneumatic scis- recycling this waste. However, such practices may sors mounted on a tractor equipped with a pressu- be thought to pose a risk of recontamination with rised container that sprayed a water solution (1/5 wood decline fungi. To examine the survival of four by volume) of sodium hypochlorite (8%) on the cut- ascomycete fungi (Eutypa lata, Botryosphaeria ter to keep the cut surface clean. The second plot, obtusa, Phaeomoniella chlamydospora and Phae- taken as the control, was pruned in the same way oacremonium aleophilum) in composted cane ma- but no spray was applied. The treatment was re- terial, a 3-year-study was initiated in 2002 in a peated for 7 years, and every year the vines show- commercial vineyard located at Pauillac (Médoc). ing leaf and shoot symptoms of esca were record- In each of the three years, about 140 m3 of pruned ed. After seven years the vines showing clear symp- shoot material was ground and mixed with 125 m3 toms of esca (leaf discoloration, necrosis and shoot of sheep dung and 60 m3 of stalks and garden res- drying) were removed and replaced the following idues (leaves, grass). The internal temperature of year with new vines. In the control plot sympto- the compost increased to 75°C a few hours after matic vines were not removed. Disease incidence mixing and thereafter fluctuated between 50°C to at the beginning of the trial was around 3% in both 75°C during the six-month period of composting. plots, and after three years it reached about 16%, The compost was regularly wetted. To assess the with no difference between treatments. Starting effect of composting on the survival of fungi, natu- from the fourth year, however, the control plot rally or artificially infested material (labelled pieces showed a rapid increase of symptomatic plants, of wood) were incorporated into the compost prior while in the sprayed plot the incidence increased to composting in 2003 and 2004. Samples were more slowly, so that after 7 years it was 27% in the examined by classical isolation methods with or

Vol. 44, No. 1, April 2005 115 AA.VV.

without disinfection with calcium hypochlorite. E.A. BARBOPOULOU. NAGREF, Institute of Olive Tree Results were consistent over the years. Before com- and Subtropical Plants of Chania, Laboratory of posting, only B. obtusa was isolated from ground Plant Pathology and Ecotoxicology of Plant Pro- canes and all introduced infested samples were tective Products, Agrokipio, 73100, Chania, Greece. viable. However, after composting, no mycelial de- E-mail: [email protected] velopment was observed from ground cane mate- In Crete (Greece) during the last few years, seri- rial or introduced samples for any of the fungi (ex- ous damage has been caused by the wood fungus cept some colonies of B. obtusa in 2003 from one Eutypa lata (Pers. Fr.) Tul. (=E. armeniacae) to piece of wood). These results clearly showed that table grape as well as wine grape varieties. Apart composting eradicates the target fungi. We con- from precautionary and cultural practices, the only clude that composting not only reduces inoculum effective chemical method after the ban on sodium pressure and thereby the incidence of some grape- arsenite is the utilisation of benomyl in the form vine declines, but can also be safely recommended of a paste. However, this drastic substance is diffi- to growers as not furnishing a potential source of cult to apply and is also prohibited. This trial ex- inoculum for wood declines. amined whether the pathogen responsible for Eu- typa dieback could be controlled by carbendazim Growth control of Inocutis jamaicensis by -1 1 (100 gr hl of the commercial product Bavistin 50 antagonistic fungi in vitro. G. ESCORIAZA , C. -1 1 2 1 1 WP), fluazinam (150 ml hl of the commercial prod- CÉSARI , M. CÉSARI and M. GATICA . Instituto Na- uct Ohayo 50 SC) or the biological product Promot, cional de Tecnología Agropecuaria, EEA Mendoza, containing Trichoderma harzianum Rifai and T. San Martín 3853, Mayor Drummond, 5507, Luján koningii Oudem (100 g hl-1). These products were de Cuyo, Mendoza, Argentina. 2Instituto Tecnológ- used either alone or in combination with the bio- ico de Buenos Aires, Av. E. Madero 399, 1106, Cap- logical product in two applications at a 12-day in- ital Federal, Argentina. terval. Pruning wounds were artificially contami- E-mail: [email protected] nated with a solution of 1.2ϫ104 ascospores one The effectiveness of Trichoderma harzianum, Clon- hour after cutting and were sprayed with the prod- ostachys rosea and Epicoccum purpurascens against ucts one day later. Products were judged effective Inocutis jamaicensis (Murril) Gottlieb, J.E. Wright if they prevented the appearance of circular sec- & Moncalvo, the basidiomycetous species associat- tors of infected wood. Best results were observed ed with hoja de malvón grapevine disease in Argen- when fluazinam was used alone at one or two ap- tina, was investigated. In vitro assays to determine plications and carbendazim at two applications the forms of antagonism were carried out. The fun- (100%). The biological product alone controlled the gi were grown on malt extract agar (MEA). Colony pathogen with an effectiveness of 40–54.6% after growth was measured and results were expressed one application and 80–81.8% after two applica- as colony diameters and percentage inhibition of tions. The combination of the biological product radial growth. All fungi were found to have an an- with the fungicides achieved an effectiveness rang- tagonistic effect by competition and produced solu- ing from 81.8 to 100%. ble antifungal metabolites against the target fun- gus at varying distances. Statistical analysis indi- Managing grapevines infected with Eutypa cated that T. harzianum strain II produced the dieback using remedial surgery. M.R. SOS- greatest inhibition in dual culture. It also produced 1,2 1,2 2 1 NOWSKI , M.L. CREASER and T.J. WICKS . Cooper- volatile and non-volatile compounds inhibiting myc- ative Research Centre for Viticulture, PO Box 154, elial growth. Microscopic examination by phase con- Glen Osmond SA 5064, Australia. 2South Austral- trast revealed lysis of the phytopathogenic fungal ian Research and Development Institute, GPO Box mycelium only by T. harzianum strain II. Further 397, Adelaide SA 5001, Australia. experiments to improve the biocontrol of this strain E-mail: [email protected] in mature field-grown grapevines are under way. Eutypa dieback, caused by the wood colonising fun- Study of the possibility to control Eutypa lata gus Eutypa lata, is a major disease of grapevines (Pers. Fr.) Tul. in grapevine. V.A. BOURBOS and worldwide. Characteristic symptoms include stunt-

116 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines” ed shoots and a wedge of discoloured tissue in the ing infected vines are limited to removal of infect- wood. The disease reduces productivity and lon- ed tissue. The ability of Brotomax™, a liquid ferti- gevity in established vineyards, particularly of pre- liser that stimulates the synthesis of phenolic com- mium red cultivars such as Shiraz, Cabernet Sau- pounds in fruit and vegetable crops, to alleviate vignon and Grenache. Two methods of remedial foliar symptoms and increase yield was evaluated. surgery were evaluated in 11 trials established in Brotomax™ was applied to own-rooted Shiraz commercial vineyards across South Australia; i) the (planted 1971) and Cabernet Sauvignon (1960) “cut and train” method, which involves cutting off vines on two commercial properties in Eden Valley the trunk 30–40 cm above the ground and train- and McLaren Vale, South Australia over three ing a watershoot to replace the lost canopy and ii) growing seasons between 2001 and 2004. At both the “train and cut” method, where cordons are re- sites, vines naturally infected with E. lata were moved and the lowest healthy shoot on the trunk sprayed i) when shoot length reached 20–30 cm, ii) is trained to form a new canopy, followed by re- 20–25 days later, iii) at bunch closure, and iv) 15– moval of the infected trunk. Watershoot produc- 20 days after harvest. All sprays were applied at a tion and recurrence of foliar symptoms of eutypa rate of 10 ml l-1 to leaves and trunks with a back- dieback were monitored over 1–5 years. Watershoot pack-spraying unit that dispensed approximately production was 40% greater for Pinot Noir and 1.5 l per vine. Disease severity was assessed on a Malbec than for Shiraz and Cabernet Sauvignon rating scale from 0 (no foliar symptoms) – 4 (severe when the cut and train method was used. Water- stunting). Since the number of vines for each level shoot production was 30% less on vines subjected of severity was not equal, the data were subjected to the cut and train method compared to vines that to Restricted Maximum Likelihood analysis. Eu- were trained and then cut. Foliar symptoms first typa dieback was well established in both vine- appeared 4 years after surgery in vines cut and yards. In the first year of the study, 68% of the trained (0.1–1.4% of vines). In one trial, foliar vines in Eden Valley and 53% of the vines in symptoms were observed on 3% of vines 1 year af- McLaren Vale produced symptoms of the disease. ter train and cut surgery. The presence of infected In the first 2 years, yield from Brotomax™-treat- wood remaining below the watershoot may influ- ed vines was marginally higher than that in un- ence the reappearance of foliar symptoms. Hence, treated vines, but the difference was not signifi- regardless of the remedial surgery method, if the cant. In the third year of treatment, there was a pathogen is not completely excised, the longevity significant yield increase of 20% for vines treated of the reworked vines may be limited. Cultivar and with Brotomax™ compared to untreated vines. environment may also be important, however. Long However, the severity of foliar symptoms of eu- term monitoring and more trials are necessary to typa dieback was not reduced following 3 years of test this hypothesis. treatment. Application of Brotomax™ and moni- toring of vines is continuing. Effect of Brotomax™ applied to Eutypa die- back-affected grapevines in South Australia. Protection of grapevine pruning wounds 1,2 1,2 2 1 M.R. SOSNOWSKI , M.L. CREASER and T.J. WICKS . against fungal infections. F. HALLEEN and P.H. 1 2 1 Cooperative Research Centre for Viticulture, PO FOURIE . ARC Infruitec-Nietvoorbij, Private Bag Box 154, Glen Osmond SA 5064, Australia. 2South X5026, Stellenbosch, 7599, South Africa. 2Depart- Australian Research and Development Institute, ment of Plant Pathology, University of Stellenbosch, GPO Box 397, Adelaide SA 5001, Australia. Matieland, 7602, South Africa. E-mail: [email protected] E-mail: [email protected] Eutypa dieback, caused by the wood infecting fun- Eutypa dieback, caused by Eutypa lata, is one of gus Eutypa lata, is a major disease of grapevines the most destructive diseases of grapevines world- worldwide causing a gradual decline in vineyard wide. Infection occurs through unprotected wounds production. Characteristic symptoms include stunt- and protection of wounds directly after pruning is ed shoots and a wedge of discoloured tissue in wood. recommended to prevent the disease. However no Disease management strategies are generally fungicide is registered for control of this disease in aimed at preventing infection. Options for restor- South African vineyards. The aim of this study was

Vol. 44, No. 1, April 2005 117 AA.VV. therefore to evaluate the effectiveness of fungicides unknown disease that affects mainly young grape- and biological control agents against E. lata infec- vines. The causal fungi infect grapevine cuttings tions under field conditions. The effect of the treat- planted in infested nursery soil. Control methods ments on other prominent wood-invading patho- should therefore focus on preventing or eradicat- gens was also recorded. The study was conducted ing infection at the basal ends of these cuttings. in two Cabernet Sauvignon vineyards. Treatments No fungicides are registered for control of BFD. In were sprayed onto pruning wounds directly after vitro studies revealed that benomyl, flusilazole and pruning in August 2001 and 2002. Treated wounds prochloraz manganese chloride were the most ef- were inoculated after 24 h with a 1ϫ103 suspen- fective fungicides. Nursery trials were conducted sion of E. lata ascospores. Treatments were beno- to evaluate the effectiveness of various physical, myl, flusilazole, Bacillus subtilis, Trichoderma for- chemical and biological treatments protecting the mulations A, B and C, an inoculated control and basal ends of rootstocks against infection. After an uninoculated control. The incidence of trunk callusing, the basal ends of grafted cuttings were disease pathogens in treated wounds was deter- dipped in various treatments prior to planting. mined 11 months after inoculation by making iso- Additional treatments involved soil amendments lations from dissected pruning wounds. Flusilazole with Trichoderma formulations and hot water (5.5%) and benomyl (5%) were the most effective treatment of dormant nursery grapevines. Nurs- treatments against E. lata compared to the inocu- ery plants were uprooted after eight months. None lated control plants (48.5%). The B. subtilis strain of the treatments affected root or shoot mass neg- did not inhibit E. lata. The Trichoderma treatments atively, although some of the treatments caused a were less effective than the fungicides, especially reduction (7.63% to 15.92%) in the number of cer- during the 2001–2002 season, but results indicat- tifiable nursery grapevines produced compared to ed that Trichoderma survived in pruning wounds the control plants. Isolations from the roots of up- and colonised them. To what extent this colonisa- rooted plants revealed very low incidences (4.1%) tion might prevent or inhibit later infections is of BFD pathogens and no significant differences unclear at this stage. Isolations from the untreat- were observed between treatments. Infection lev- ed, uninoculated control plants also revealed the els at the basal ends were substantially higher. presence of several other trunk disease pathogens BFD pathogens were isolated from 45.33% (2002- including Phomopsis (37.5%), Botryosphaeria 2003) and 16.83% (2003-2004) of the basal ends of (18.5%) and Phaeomoniella chlamydospora control plants. The incidence of BFD pathogens at (19.5%). Flusilazole and benomyl reduced infection the basal ends was not significantly or consistent- of P. chlamydospora by 82 and 77%, respectively, ly reduced by the majority of chemical and biolog- whilst flusilazole reduced Phomopsis infection by ical treatments. However, no BFD pathogens were 53%. The results clearly showed that pruning isolated from the plants that were subjected to hot wound protection could contribute to lower levels water treatment. It is therefore recommended that of infections from trunk disease pathogens under hot water treatment of dormant nursery grapevines vineyard conditions. be included in an integrated strategy for the proac- tive management of BFD in grapevine nurseries. Proactive management of black foot disease in South African grapevine nurseries. F. H AL- Black foot of grapevine: sensitivity of Cylin- 1 2 3 1 1 LEEN , P.H. FOURIE and P.W. CROUS . ARC Infruit- drocarpon destructans to fungicides. C. REGO , 2 1 2 ec-Nietvoorbij, Private Bag X5026, Stellenbosch L. FARROPAS , T. NASCIMENTO , A. CABRAL and H. 2 2 1 7599, South Africa Department of Plant Patholo- OLIVEIRA . Laboratório de Patologia Vegetal “Verís- gy, University of Stellenbosch, Private Bag X1, simo de Almeida”, Tapada da Ajuda, 1349-017 Lis- Matieland 7602, South Africa. 3Centraalbureau boa, Portugal. 2Instituto Superior de Agronomia, voor Schimmelcultures, Uppsalalaan 8, 3584 CT Tapada da Ajuda, 1349-017 Lisboa, Portugal. Utrecht, The Netherlands. E-mail: [email protected] E-mail: [email protected] Black foot disease of grapevine is caused by Cylin- Black foot disease (BFD), caused by species of Cy- drocarpon spp., with Cylindrocarpon destructans lindrocarpon and Campylocarpon, is a relatively being the main pathogen isolated from vine cut-

118 Phytopathologia Mediterranea 4th International Workshop on Grapevine Trunk Diseases “Esca and Grapevine declines” tings and young vineyards in Portugal. Few rec- va and B. rhodina. Iprodione, pyrimethanil, cop- ommendations for black foot disease control are per ammonium acetate, kresoxim-methyl and bos- presently available, and they are not easy to im- calid were ineffective in inhibiting mycelial growth plement in commercial nurseries. In this study, 14 at the highest concentration tested (5 µg ml-1; 20 fungicides were evaluated for their effects on the µg ml-1 for copper ammonium acetate). Benomyl, radial growth and spore germination of four field tebuconazole, prochloraz manganese chloride, isolates of C. destructans. Mycelial growth of the flusilazole and fenarimol were the most effective, pathogen was inhibited by DMI fungicides, prochlo- with EC50 values for the different species ranging -1 raz (EC50 <0.09 mg l ),to a lesser extent by tebuco- from 0.36–0.55, 0.07–0.17, 0.07–1.15, 0.04–0.36 -1 -1 nazole and difenoconazole (EC50 <5.66 mg l ), by and 0.45–3.01 µg ml respectively. All these fungi- the benzimidazole fungicide benomyl (EC50 <0.35 cides, except prochloraz manganese chloride, are mg l-1), and by the mixtures cyprodinil + fludiox- registered for use on grapes in South Africa and onil and carbendazim + flusilazol, which gave EC50 are also reported to be effective against Pa. chlamy- <0.75 mg l-1. Among these, only cyprodinil + fludi- dospora, P. viticola and E. lata. -1 oxonil (EC50 values < 0.15 mg l ), the strobilurin fungicides, azoxystrobin and tryfloxistrobin (EC50 The effects of different post hot water treat- <2.27 mg l-1) and the sulphamide fungicide tolyflu- ment cool down protocols on dormant cut- -1 1,2 anide (EC50 <0.54 mg l ) were effective in reduc- tings of Vitis vinifera cultivars. H. WAITE , S. 2 2 1 1 1 ing spore germination. Results from in vivo stud- BEGGS , H. DARK , P. KWAK and G. MURRELLS . Co- ies carried out on potted grapevine plants (culti- operative research Centre for Viticulture, PO Box var Periquita) showed that benomyl, tebuconazole 154, Glen Osmond, South Australia, 5064, Austral- and the mixtures carbendazim + flusilazol and cy- ia, 2Faculty of Land and Food Resources, Univer- prodinil + fludioxonil significantly (P=0.05) im- sity of Melbourne, Dookie College, 3647 Victoria, proved plant growth (plant height, number of roots Australia. and root elongation) and decreased disease inci- E-mail: [email protected] dence compared with non-treated plants. Hot water treatment (HWT) of dormant grapevine cuttings controls a number of pathogens including Fungicide sensitivity of selected Botry- Phaeomoniella chlamydospora. Dormant cuttings osphaeria species from grapevine. W. B ESTER are plunged into cold water immediately following and P.H. FOURIE. Department of Plant Pathology, hot water treatment (50°C/30 min) to quickly low- University of Stellenbosch, Private Bag X1, Matie- er the temperature and minimise heat damage to land (Stellenbosch) 7602, South Africa. the tissue. Water used in commercial cool down E-mail: [email protected] tanks is chlorinated, but it is not sterile and it is a Protection of wounds against infection by trunk potential source of microbial contaminants includ- disease pathogens is the most efficient and cost- ing Phaeomoniella chlamydospora. It would be effective means to prevent grapevine trunk diseas- desirable to eliminate this stage of the HWT proc- es. Studies done to determine the effectiveness of ess allowing cuttings to air-cool provided that the chemical pruning wound protectants have mostly slower cooling did not affect cutting viability. The focused on the control of Eutypa dieback. Howev- effects of post HWT air-cooling versus water-cool- er, other important wound pathogens, such as ing on dormant cuttings of 4 V. vinifera cultivars, Phaeomoniella chlamydospora, Phomopsis and Pinot Noir, Chardonnay, Semillon and Cabernet Botryosphaeria species, pose just as significant a Sauvignon were examined. Treatments applied threat to sustainable grape production. Fungicide were: HWT plus water-cooling (30 min immersed sensitivity studies have been conducted for Pa. in cold water at 12°C), HWT plus air-cooling (30 chlamydospora, P. viticola and Eutypa lata. How- min cooling on laboratory bench at 17°C), and un- ever, no such studies have been conducted on the treated control. Cuttings were incubated at 28°C pathogenic Botryosphaeria species from grapevine. for 21 days and assessed for percentage callus, Ten fungicides were therefore tested in vitro for root number and length and bud development. All their efficacy on mycelial inhibition of four Botry- cuttings of all varieties, regardless of treatment, osphaeria species, B. australis, B. obtusa, B. par- were alive and viable at callusing. In all varieties

Vol. 44, No. 1, April 2005 119 AA.VV. both HWT treatments tended to stimulate callus of HWT cuttings and reduce contamination by and bud development. Although variable, root de- water borne micro-organisms. However these re- velopment in all treatments of all varieties was sults should be tested in a commercial setting adequate for cutting establishment, indicating before changes to HWT protocols can be recom- that it may be possible to eliminate water-cooling mended.

120 Phytopathologia Mediterranea