ANNUAL MEETING OF WORKING GROUPS 1, 2, 3 AND

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ANNUAL MEETING OF WORKING GROUPS 1, 2, 3 AND Powered By Docstoc
					ANNUAL MEETING OF WORKING
        GROUPS 1, 2, 3 AND 4
   Management Committee Meeting

                 26-29, October, 2009
                     Cetara (SA), Italy




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Scientific Committee:

      Charles MANCEAU           WG 1 leaders
      Marco SCORTICHINI



      Concepció MORAGREGA       WG 2 leaders
      John ELPHINSTONE



      Anita SOLAR
      Diego FRUTOS              WG 3 leaders



      Emilio STEFANI
                                 WG 4 leaders
      Michel GIRAUD



      Brion DUFFY           Chairman of the COST 873 Action




Local Organizer Committee:

      Giuseppe CAPRIOLO
      Alfredo MARANO
      Assunta NESCI
      Marco OLIVA
      Milena PETRICCIONE
      Luigi SCARPATO
      Marco SCORTICHINI




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                               SCHEDULE



Monday October 26th, 2009


9 h 00 – 11 h 00        WG 1

11 h 00 – 11 h 15       Coffee Break

11 h 15 – 12 h 50       WG 1

12 h 50 – 14 h 00       Lunch and Posters

14 h 10 – 16 h 30       WG 3

16 h 30                 Evening




Tuesday October 27th, 2009


9 h 00 - 10 h 00        Poster

10 h 00 – 11 h 00       WG 2

11 h 00 – 11 h 15       Coffee Break

11 h 15 – 12 h 35       WG 2

12 h 40 – 14 h 00       Lunch and Posters

14 h 00 – 16 h 30        WG 4 + Cop - STSM Activities - Emerging disease

16 h 30                 Evening




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                              SCHEDULE



Wednesday October 28th, 2009

9 h 30 - 10 h 30        STF Groups Strategy

10 h 30 - 11 h 15       STF Groups (Specific Research Plans)

11 h 15 - 11 h 30       Coffee break

11 h 30 - 12 h 30       STG Groups (Research Implementation Plans)

12 h 30 - 14 h 00       Lunch - Posters




Thursday October 29th, 2009

8 h 00 - 13 h 00       Management Committee Meeting




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                    PROGRAM OF EVENTS

WG 1 - Monday October 26th, 2009


9 h 00               Registration

9 h 20               Welcome to the participants

9 h 30 - 10 h 00      Keynote Lecture: J. Young - Pseudomonas syringae in
                      stone-fruit in New Zealand

10 h 00 - 10 h 20     S. Gironde, J. Guillaumes, C. Manceau - Specific detection
                      of Xanthomonas arboricola pv. juglandis pathogen on
                      walnut

10 h 20 - 10 h 40     M. Kałużna, P. Ferrante, P. Sobiczewski, M. Scortichini -
                      Characterization and genetic diversity of Pseudomonas
                      syringae isolates originating from stone fruits and hazelnut
                      using repetitive-PCR and MLST methods

10 h 40 - 11 h 00     P. Ferrante, D. De Luca, M. Scortichini – Different
                      evolutionary dynamics of Pseudomonas avellanae inferred by
                      MLST analysis

11 h 00 - 11 h 20     Coffee break

11 h 20 - 11 h 40     A. Palacio-Bielsa, J. Cubero, M.M. López - Sensitive real-
                      time PCR detection of Xanthomonas arboricola pv. pruni in
                      symptomatic and asymptomatic Prunus spp.

11 h 40 - 12 h 00     N. Parkinson - Phylogenetic discrimination of Pseudomonas
                      syringae-related pathogens using the     rpoD locus

12 h 00 - 12 h 20     J. F. Pothier, V. Pflüger, M. Tonolla, G. Vogel, B. Duffy -
                      MALDI-TOF mass spectrometry subspecies-level rapid
                      resolution of the complete genus of phytopathogenic
                      Xanthomonas

12 h 20 - 12 h 50     Keynote Lecture: G. Preston - Integrated use of
                      metabolomic and bioinformatic technology to provide insight
                      into the adaptation of Pseudomonas syringae for growth in
                      plants

12 h 50 - 14 h 00     Lunch – Posters



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                    PROGRAM OF EVENTS


WG 3 - Monday October 26th, 2009



14 h 10 - 14 h 30    M. Petriccione - Ethnobotany of peach, cherry and walnut

14 h 30 - 14 h 50     H. Halbwirth, K. Stich - Transferring expertise with apple
                      defense biochemistry to walnut

14 h 50 - 15 h 10     M.C. Holeva, P.E. Glynos, C. Karafla, D.C. Stylianides, A.E.
                      Voloudakis - Studies on resistance of almond varieties to
                      hyperplastic canker caused by Pseudomonas amygdali and
                      identification of gene homologues of hrpZ and hrpW,
                      potential molecular targets for inhibiting infection process

15 h 10 - 15 h 30     D.    Burokiene      -   Identification and    molecular
                      characterization of bacterial agents of stone fruits and
                      walnuts in Lithuania

15 h 30 - 15 h 50     J. Matias, N. Aletà, M. Rovira - Mesocarp and leaf phenolic
                      composition in Juglans regia commercial cultivars

15 h 50 - 16 h 10     A. Solar, T. Dreo, M. Mikulič-Petkovšek, A. Likozar, M.
                      Šuštaršič, R. Veberič, L. Matičič, M. Ravnikar, F. Štampar -
                      Phenolic compounds as potential markers for walnut blight
                      resistance - present results of Slovene research

16 h 10 - 16 h 30     L. Ruiz, D. Frutos, G. Lopez, A. Fuentes - Walnut molecular
                      characterization and Xanthomona arboricola pv. juglandis
                      damages evaluation in Murcia, Spain




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                    PROGRAM OF EVENTS


WG 2 - Tuesday October 27th, 2009




9 h 00 - 9 h 20       J. Janse - Xylella fastidiosa - an emerging threat?

9 h 20 - 9 h 40       J.L. Peroys - Factors influencing walnut blight symptoms
                      emergence and development

9 h 40 - 10 h 00      C. Moragrega - Epidemiology and causal agents of brown
                      apical necrosis of walnut

10 h 00 - 10 h 20     Coffee break

10 h 20 - 10 h 40     M. Giraud - Incidence of fungi associated with walnut blight

10 h 40 - 11 h 00     H. Ozaktan - Etiological approach to brown apical necrosis
                      on walnut fruits in Turkey

11 h 00 - 11 h 20     A. Garcin, J. Vibert, A. Leclerc - Epidemiology of
                      Xanthomonas arboricola pv. pruni on peach in France

11 h 20 - 11 h 40     M.D. Lang, K.J. Evans - Epidemiology of walnut blight in
                      Tasmania, Australia

11 h 40 - 13 h 20     Lunch - Posters




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                    PROGRAM OF EVENTS

WG 4 +CoP - Tuesday October 27th, 2009


13 h 20 - 13 h 40     Keynote Lecture:     C. Wend – Pantoea agglomerans
                      commercial biocontrol product

13 h 40 - 14 h 00     B. Duffy, F. Rezzonico, T.H.M. Smits, E. Montesinos, J.E.
                      Frey - Genotypic characterisation of the Jekyll-Hyde
                      antagonist Pantoea agglomerans

14 h 00 - 14 h 20     T. Merckling - Development of Serenade as a bacterial
                      biopesticide against bacterial plant diseases

14 h 20 - 14 h 40     B.J. Blum - Gaps, problems and opportunities for the EU
                      registration of microbial biopesticides

14 h 40 - 15 h 00      D. Giovanardi, D. Dallai, E. Cozzolino, E. Stefani - Recent
                      advances on the epidemiology and control of the bacterial
                      blight of walnut, incited by Xanthomonas arboricola pv.
                      juglandis

STSM Activity

15 h 00 - 15 h 10     E. Stefani, A. Obradoviš, D. Dallai, K. Gasiš - Surveys for
                      Xanthomonas arboricola pv. pruni and implementation of
                      detection procedures and certification schemes in Serbia

15 h 10 – 15 h 20     A. Ćalić, K. Gašiš, M. Ivanoviš, A. Obradoviš, P. Ferrante,
                      M. Scortichini – New occurrence of Xanthomonas arboricola
                      pv. corylina on European hazelnut in Serbia

15 h 20 – 15 h 30     S. Akat – Field survey and microorganisms associated with
                      apical necorsis of walnut

15 h 30 – 15 h 40     K. Gasic – Monitoring surveys in Italy for Xanthomonas
                      arboricola pv. pruni in peach and plum orchards


Emerging disease

15 h 40 – 15 h 50     P. Ferrante, M. Scortichini – A severe epidemic of bacterial
                      canker on yellow kiwifruit in central Italy caused by
                      Pseudomonas syringae pv. actinidiae


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                    PROGRAM OF EVENTS



Wednesday October 28th, 2009


9 h 30 - 10 h 30      STF Groups Strategy

10 h 30 - 11 h 15     STF Groups (Specific Research Plans)

11 h 15 - 11 h 30     Coffee break

11 h 30 - 12 h 30     STG Groups (Research Implementation Plans)

12 h 30 - 14 h 00     Lunch - Posters




Thursday October 29th, 2009


8 h 00 - 13 h 00     Management Committee Meeting




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WORKING GROUP 1




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PSEUDOMONAS SYRINGAE IN STONE-FRUIT IN NEW ZEALAND


J.M. Young

Landcare Research, Auckland, New Zealand; youngj@clear.net.nz


The pathovars, Pseudomonas syringae pv. syringae, P. syringae pv. persicae, and P. syringae pv. morsprunorum, have
all been isolated and identified in New Zealand: P.s. syringae from apricot, cherry, nectarine and peach; P.s. persicae
from nectarine, peach and Japanese plum, and P.s. morsprunorum from cherry. All pathovars cause bud death, stem die-
back, and occasional die-back of leaders in spring. For P.s. syringae, this is usually associated with „gummosis‟. These
symptoms are the result of autumn infections of leaf scars, with development of disease in the following spring. Disease
development does not occur in winter and is associated with late frosts. The condition can be controlled by applying 2 –
3 autumn copper sprays. These pathogens all cause fruit blemishes in spring, P.s. persicae being the most severe. The
pathogens cannot be isolated from tissue after late spring. With the exception of P.s. persicae, the pathogens appear not
to infect roots.
These pathogens also cause other very serious conditions in New Zealand stone-fruit that have well-described in other
countries; P.s. syringae is associated with „apoplexy‟ in apricot (described in Hungary) and with short life of peach („PTSL‟
– in the United Sates), and P.s. persicae with dépérissement in peach (in France). The condition is marked by the death
of main leaders and entire trees, first indicated in spring and into mid-summer by the sudden withering of leaves in
affected parts. Dissection invariably reveals extensive necrotic cankers that completely girdle affected limbs.
Observations over several years and experimental inoculations support the conclusion drawn overseas that the condition
is initiated by infection at unidentified sites and is most aggressive in mid-winter, with progressive and extensive
development resulting in killing of the cortical parenchyma. The major conclusion from these observations is that
bacterial activity is dependent on heavy frosts when trees are dormant, and on an imbalance between root and branch
structures caused by root weakening from a combination of possible causes; the presence of impervious clay pans,
nematode injury, and elevated soil acidity caused by the application artificial fertilizers and inadequate liming. In New
Zealand, the condition is clearly associated with plantings on sandy, shallow, alluvial soils, when an anaerobic layer forms
at the sand-gravel interface, killing substantial proportions of root systems. In New Zealand, the disease may have been
exacerbated by a change in the method of frost-fighting, from the use of fire-pots to overhead sprinkling.
An uncertainty was the extent of the delay between infection and expression. Generally it has been assumed that canker
formation in winter leads directly to disease expression (leaf withering) in the following growing season. However, the
desiccated condition of cankers and their distinct smell („sour sap‟) suggested a longer period. A crude experiment, in
which branches were girdled either in autumn or spring by physically removing the entire parenchyma and cambium of a
short section of branch, showed that treated limbs did not usually express symptoms in the season following girdling,
and could appear unaffected through the second season. Apparently, limbs continue to draw adequate water through
their wood for extended periods and may only collapse when their growth creates demands for water that cannot be
satisfied through the constriction caused by a canker.




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SPECIFIC DETECTION OF XANTHOMONAS ARBORICOLA PV. JUGLANDIS PATHOGEN ON WALNUT.


Sophie Gironde, Jacky Guillaumes, Charles Manceau.

INRA, UMR PaVé – 42 rue Georges Morel, BP 57, 49071 Beaucouzé, France



Xanthomonas arboricola pv. juglandis is the causal agent of walnut blight which causes severe damages to leaves, twigs
and fruits, and lead to an important loss of nut‟s harvest. This bacterium has also been reported to be responsible of an
emerging disease on walnut : the vertical oozing canker that has been observed in France for a decade. Moreover, the
development of copper resistant populations makes inefficient the authorized chemical treatment. In order to fight
against this bacterium and to clean up the nurseries, a specific and sensitive detection method become necessary.
We developped a Bio-PCR method to detect specifically X. a. pv. juglandis. The Bio-PCR test combining the biological
amplification on the elective growth medium with the specific PCR detection makes rapid and reliable the detection of X.
a. pv. juglandis in plant samples. Genomic analysis using independant genomic markers placed the two isolates of
Xanthomonas that were not detected by the primers set, outside the X. a. pv. juglandis group. The pathogenicity of
these strains was checked to validate the identification tools ; They were non pathogenic on walnut plantlets.




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CHARACTERIZATION AND GENETIC DIVERSITY OF PSEUDOMONAS SYRINGAE ISOLATES ORIGINATING
FROM STONE FRUITS AND HAZELNUTS XX USING REPETITIVE PCR AND MLST METHODS


Kałużna Monika1, Ferrante Patrizia2, Sobiczewski Piotr1, Scortichini Marco2

1
  Research Institute of Pomology and Floriculture, Department of Plant Pathology, Pomologiczna 18 str., Skierniewice,
Poland
2
  C.R.A., Istituto Sperimentale per la Frutticoltura, Via di Fioranello 52, 00040 Ciampino Aeroporto, Roma, Italy


Of 33 Pseudomonas syringae isolates originating from stone fruits and hazelnut grown in Poland and Italy, 16 were
classified phenotypically as P. syringae pv. syringae (Pss), 12 as race 1 of P. syringae pv. morsprunorum (Psm) and 5
isolates as race 2 of Psm. Genetic analysis of toxins production showed that out of all isolates 11 of Pss and 1 of Psm
race 2 possessed gene encoding syringomycin (syrB), 3 of Psm race 1 isolates gene encoding coronatine (cfl) and only 3
of Psm race 2 isolates possessed gene encoding yersiniabactin (YBT). BOX and ERIC PCR analyses allowed discrimination
3 separate clusters within tested isolates according to phenotypically characterized pathovars and races. The only
exception was found in case of 2 isolates of Psm race 2 that did not possess YBT. Both rep-PCRs showed that their
patterns were more similar to those obtained for pathovar syringae isolates. Based on rep-PCR fingerprinting 20
representative isolates were selected for analyses of gyrB, gapA, gltA and rpoD genes according to Sarkar and Guttman
(2004). Dendrogram resulting from MLST shows three distinguished clusters consisting of Pss, Psm1 and Psm2
respectively, similarly to data obtained from both rep-PCR methods.


Sarkar S.F. and Guttman D.S.: 2004. Appl. and Environ. Microbiol. 70, 1999-1012




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DIFFERENT EVOLUTIONARY DYNAMICS OF                        PSEUDOMONAS AVELLANAE STRAINS INFERRED BY
MULTILOCUS SEQUENCE TYPING ANALYSIS


Ferrante P., De Luca D., Scortichini M.


C.R.A. – Centro di Ricerca per la Frutticoltura, Via di Fioranello, 52, 00134 Roma, Italy
mscortichini@yahoo.it

In this study we investigated the genetic diversity, occurrence of possible recombination events, natural selection and
selective pressures in 38 Pseudomonas avellanae strains representative of several epidemics of European hazelnut
bacterial canker and decline in northern Greece and central Italy. Internal fragments of four housekeeping genes, namely
gapA, gltA, gyrB and rpoD were sequenced, and a total of 2.3 kb were assessed by means of multilocus sequence typing
analysis. A closely-related pseudomonad, P. syringae pv. tomato DC3000 was used as outgroup. Phylogenetic analysis
performed with both neighbor-joining algorithm and phylogenetic network pointed out three main group of strains, A, B
and C. These group of strains appear as genetically different. Groups A and B are clonal complexes, whereas in group C,
recombination significantly contributes to the variation between strains. Linkage disequilibrium analysis confirm such
findings. RpoD gene appears closely-related to the homologous of P. s. pv. tomato DC3000 and recombinant as revealed
by the phylo-network and by the detection of a recombination breakpoint within the gene sequence. All the genes are
under purifying selection and the codon adaptation index revealed a relevant metabolic versatility of such genes. As a
whole P. avellanae would appear as a new microorganism exploiting a new habitat. In some circumstances clonal
complexes have successfully colonized the host plant. Such complexes have arisen independently one from another in
both countries where the epidemics were observed. The occurrence of recombination events among the strains,
however, yields frequently new intraspecific variability. This pathogen would seem a distinct microbial ecotype, according
to the “ecotype simulation” model.




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SENSITIVE REAL-TIME PCR DETECTION OF XANTHOMONAS ARBORICOLA PV. PRUNI IN SYMPTOMATIC
AND ASYMPTOMATIC PRUNUS SPP.


Palacio-Bielsa A.1*; Cubero J.2; López M.M.3; Collados R.4; Berruete I.1; & Cambra M.A.4

1
 .Centro de Investigación y Tecnología Agroalimentaria de Aragón. Zaragoza, Spain. apalaciob@aragon.es
2
 . Departamento de Protección Vegetal, INIA. Madrid, Spain.
3
 . Centro de Protección Vegetal y Biotecnología, IVIA. Moncada, Valencia, Spain.
4
 . Centro de Protección Vegetal. Zaragoza, Spain.



Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease on Prunus spp., is considered a quarantine
organism in the UE and an A2 pathogen by the EPPO, because the risk it represents for Prunus spp. and its recent
spread in several countries of that region.
Currently, only visual inspections for symptoms are performed to certify plants free of X. arboricola pv. pruni in nurseries.
However, the bacterium can have an epiphytic phase and/or be latent and be transmitted in whole plants or budwood.
Thus, plant propagation material can disperse the pathogen to areas or countries previously free of the disease.
The availability of a highly sensitive, accurate and specific molecular assay for detecting X. arboricola pv. pruni had been
lacking. A conventional PCR protocol, for specific detection of gene sequence for a putative protein in X. arboricola pv.
pruni with similarity to the ABC transporter ATP-binding system, has been recently developed (Pagani, 2004). However,
although this protocol offers a specific approach for detection and identification of the bacterium in symptomatic plants,
it is not sensitive enough for such purposes in symptomless plants.
In recent years, several real-time PCR methods for the detection of plant pathogenic bacteria have been designed and
validated, with significant improvements in speed, specificity and sensitivity. The goal of the present study was to set up
an alternative real-time PCR based on a TaqMan® oligoprobe and primers designed on those sequences reported by
Pagani (2004) to provide a fast, sensitive and reliable test for X. arboricola pv. pruni detection that can be used for
diagnosis as well as for in planta screening of latent infections.
The developed real-time PCR protocol has been validated for detection in naturally infected peach and almond plant
material from the field, as well as in different rootstocks from nurseries, both symptomatic and asymptomatic. In plants
with symptoms, amplification of the pathogen was even achieved from tissue washes without a previous DNA extraction
step. A good correlation between the results of the analysis by real-time PCR and isolation of the bacterium has also
been obtained.




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PHYLOGENETIC DISCRIMINATION OF PSEUDOMONAS SYRINGAE-RELATED PATHOGENS USING THE RPOD
LOCUS


Neil Parkinson

FERA, York , United Kingdom, n.parkinson@csl.gov.uk


MLST analysis has grouped Pseudomonas syringae-related pathogens into five major phylogroups. An amplicon produced
using newly designed primers to conserved regions in the rpoD gene produced a read length of 587 nucleotides. The
locus which encompassed a smaller region from previous studies, was sequenced from all 67 pathovar type strains and
all 137 NCPPB strains designated as „P. syringae undetermined pathovar‟. Together with previously sequenced strains
approximately 40 pathogens with stone fruit and nut hosts have been sequenced using the rpoD locus. All strains
grouped into previously defined MLST phylogroups except for a new phylogroup comprising P. caricapapayae and two
other related pathovars, which had not been included in previous studies. Within the major phylogroups the locus
discriminated a large number of sequevars some of which grouped together into clades.
The phylogeny was sufficiently discriminating to be useful in diagnosis, by confirming whether pathogenic isolates
belong to the same lineage as pathovar type-strains associated with a given host. Potential new pathovars were also
identified. Stone fruit strains pathogenic to Prunus hosts occurred within phylogroups 1, 2 and 3 suggesting
pathogenicity to the genus has arisen independently. Within phylogroup 1, two clades were discriminated each of which
contained several pathogens of different Prunus species. Two clades containing Prunus isolates were also found in
phylogroup 2, whilst other strains did not share close sequence similarity. A larger survey of stone fruit and nut isolates
is needed to improve our knowledge of pathogen lineages, which may be associated with specific pathogen traits, and
also to improve monitoring and epidemiological surveillance of these important and destructive pathogens.




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MALDI-TOF MASS SPECTROMETRY SUBSPECIES-LEVEL RAPID RESOLUTION OF THE COMPLETE GENUS OF
PHYTOPATHOGENIC XANTHOMONAS



Joël F. Pothier1, Valentin Pflüger2, Mauro Tonolla3, Guido Vogel2 and Brion Duffy1,*

1
 Agroscope Changins-Wädenswil ACW, Plant Protection Division, CH-8820 Wädenswil, Switzerland.
2
 Mabritec AG, CH-4002 Basel, Switzerland.
3
 Cantonal institute of microbiology, Microbial Ecology laboratory, Microbiology Unit, BioVeg Dept. UNIGE, CH-6500
Bellinzona, Switzerland



*brion.duffy@acw.admin.ch



Rapid and reliable identification of plant pathogenic bacteria is critical for effective implementation of phytosanitary
measures. Xanthomonas includes a number of economically important and quarantine/select agent taxa at the
subspecies level (i.e., pathovars determined by differential host-plant pathogenicity). Current technology to achieve
sufficient subspecies discrimination is time-consuming and complicated, and is based on semi-selective isolation, PCR and
serology for some taxa, plant bioassays and gene locus sequencing. Here we present a streamlined approach based on
MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) demonstrating its potential
as a „gold standard‟ for first-line identification in plant diagnostics.
A collection of Xanthomonas species and pathovars covering the entire genus was analysed using MALDI-TOF MS and
the resulting phylogenetic tree was compared with that obtained using the current „gold-standard‟, gyrB locus
sequencing. The MALDI-TOF MS tree (constructed based on discriminatory mass spectra patterns) was congruent with
that obtained using gyrB gene sequencing. This supports recent systematic rearrangements of the genus. Moreover, it
demonstrates that MALDI-TOF MS is reliable alternative to more time-consuming and expensive sequencing for
identification at the species and subspecies (pathovar) level.
SuperSpectra™ for each taxa within Xanthomonas were created based on the mass spectra fingerprints for inclusion in
the SARAMIS™ database. These contain information on mass signal signatures for each taxonomic level, and will serve
as a future reference for identification of Xanthomonas. Our MALDI-TOF MS approach offers a streamlined approach for
identification and diversity analysis of one of key groups of plant pathogenic bacteria with phytosanitary and biosecurity
importance.




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INTEGRATED USE OF METABOLOMIC AND BIOINFORMATIC TECHNOLOGY TO PROVIDE INSIGHT INTO
THE ADAPTATION OF PSEUDOMONAS SYRINGAE FOR GROWTH IN PLANTS


Gail M. Preston

Department of Plant Sciences, University of Oxford, United Kingdom



The bacterial plant pathogen Pseudomonas syringae colonises plant surfaces and the intercellular spaces between plant
cells, and grows by assimilating metabolites present in leaf exudates and in apoplastic fluid. The ability of P. syringae to
colonise plant leaves depends on the activity of a type III protein secretion system, which injects proteins into plant cells,
and on low molecular weight toxins and phytohormones. However, relatively little is known regarding the physiological
and metabolic adaptations that allow P. syringae to grow in plant tissues. In this talk I will describe how experimental
and bioinformatic analyses of stress tolerance and nutrient assimilation in Pseudomonas can be integrated with
metabolomic analyses of apoplast composition to show how P. syringae is adapted for growth in the plant apoplast, and
to explore the impact of environmental factors on disease development.




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WORKING GROUP 2




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XYLELLA FASTIDIOSA – AN EMERGING THREAT?


Jaap D. Janse

Dept of Laboratory Methods and Diagnostics, Dutch General Inspection Service, Emmeloord, The Netherlands
(jjanse@nak.nl)


The bacterium Xylella fastidiosa, a xylem-inhabiting, vector-transmitted, gram-negative, very slow growing bacterium,
was cultured and properly described for the first time in 1987 in the USA as the cause of Pierce‟s disease (PD) of
grapevine, Vitis vinifera (the disease observed already in 1884) and as the cause of phony peach disease (PPD) in peach,
Prunus persica (disease observed in 1890 in the USA) and in 1993 in Brazil as the cause of citrus variegated chlorosis
(CVC) or citrus X disease of Citrus. Moreover it was found that the bacterium also causes a number of so-called leaf
scorch diseases in Prunus spp.(incl. almond leaf scorch or ALS in P. armeniaca and plum leaf scald or PLS in
P.domestica), Acer spp., Carya illinoinensis (pecan), Coffea arabica (CLC, in Brazil isolated in 1995 and also pathogenic to
Citrus), Hedera helix, Morus rubra, Nerium oleander (OLS), Platanus occidentalis, Quercus spp., Ulmus americana.
Furthermore in Medicago sativa, alfalfa (alfalfa dwarf)and Vinca major (wilting symptoms). Many wild plants may carry
the pathogen with, but more often without showing symptoms, such as grasses, sedges and trees. A list of hosts will be
presented. These diseases occur mainly in tropical/subtropical areas, although leaf scorch diseases also occur in much
colder climate, e.g. oak leaf scorch up to Canada. Several pathogenic varieties have been described, that are often host-
specific (e.g., the PD strain will not cause disease if introduced to peach or plum).The following subspecies have been
described:

-   Xylella fastidiosa subsp. fastidiosa (erroneously named X. f. subsp. piercei) – PD and LSA, strains from cultivated
    grape, alfalfa, almond (two), and maple

-   X. fastidiosa subsp. multiplex – PPD, PLS, strains from peach, elm, plum, pigeon grape, sycamore, and almond (one)

-   X. fastidiosa subsp. pauca – CVC, strains from citrus and probably those of coffee (CLC)

-   X. fastidiosa subsp. sandyi – strains from nerium oleander (OLS)

-   X. fastidiosa supsp. tashke – strains from the ornamental tree Chitalpa tashkentensis.
Vectors are mainly sharpshooters and spittlebugs (Cicadellidae) that lack a latent period, have no transstadial or
transovarial transmission, show persistence in adults, and multiplication in the foregut In N. America main vectors (for
PD unless indicated) are Cuerna costalis (PPD), Draculacephala minerva (green sharpshooter) important also in ALS in
California; Graphocephala atropunctata (blue-green sharpshooter),most important before GWSS; G. versuta (PPD);
Hordnia circellata, most efficient; Homalodisca vitripennis (formerly) coagulate (glas-winged sharpshooter or GWSS); H.
insolita (PPD),Oncometopia nigricans, O. orbona (PPD), Xyphon (formerly Carneocephala) fulgida (red-headed
sharpshooter, For CVC in Brazil Acrogonia terminalis, lays eggs externally on leaves; Dilobopterus costalimai and
Oncometopia fascialis. Local possible vectors for Europe are Cicadella viridis and Philaenus spumarius (meadow spittle
bug).
X. fastidiosa is an emerging threat in the SW USA ( due to recent establishment of the glassy-winged sharpshooter
(GWSS) vector (H. vitripennis), leading to very serious outbreaks of PD in grapevine, ALS and OLS due to a much more
efficient transmission than local vectors). GWSS probably first entered California as eggs in plants. The eggs are
deposited into plant tissues. In C. and S. America X. fastidiosa has become very noxious due to the rapid expansion
(most likely via distribution of infected planting material) of CVC in Citrus (leading to more than a third of all trees in the
area having symptoms of CVC) and CLC in coffee.



                                                              40
41
For Europe there are until now only a few unconfirmed reports of finding X. fastidiosa, viz. from Kosovo, grapevine
(erroneously mentioned as Slovenia in my book Phytobacteriology, Principles and Practice, 2006) or disease symptoms.
viz. from France in grapevine. Since X. fastidiosa has more that 150 hosts and many of them, including Vitis planting
material, were and are imported, the risk of introduction (especially in latent form) must not be underestimated.
Absence of the diseases caused by X. fastidiosa will mainly be due to the absence of suitable vectors. However,
introduction of the pathogen and vectors with plant material can certainly not be excluded. Moreover also local
Cicadellidae (see above) could become (potential) vectors. Therefore X. fastidiosa has the A1 quarantine status in the
EPPO region and H. vitripennis (GWSS) that has a very large host range and also feeds on almond, peach and plum,
was recently put on the EPPO alert list. As in the more Northern parts of the USA, Vitis varieties in Europe are very
susceptible to X. fastidiosa and this is really a risk when a vector would become established that could survive the
winters in S. Europe and would also establish in wild hosts (e.g. wild and domestic plums and wild cherry are
symptomless reservoirs in the USA) and cause spring infections that are most likely to persist over the years. The same
risk holds true for Citrus (sweet oranges, mandarins, and tangerines) and other hosts, such as almond, plum and peach
that are widely grown in (SE and SW) Europe, especially in the warmer Mediterranean basin (where a disease-favourable
combination of warm nights, regular rainfall/high humidity and long growing season is present). Possible ways to prevent
introduction and to control eventual outbreaks will be indicated.
The conclusion is that X. fastidiosa is a real and emerging threat for Europe, not only for Vitis and Citrus but also for
stone fruits (almond, peach and plum) and oleander (e.g. GWSS likes to feed on oleander), that is difficult to prevent
from entering and difficult to control once established, deserving more attention than up till now. Resistance in European
grapes is scarce or even absent, vector control proved not to be very effective in the USA. Cultural practices to keep
plants in optimum condition are of importance, but not sufficient and the use of avirulent strains for cross-protection is
still in its infancy.




                                                            42
43
EPIDEMIOLOGY AND CAUSAL AGENTS OF BROWN APICAL NECROSIS OF WALNUT


C. Moragrega1, J. Matias2, N. Aletà3, M. Rovira2
1
Institute of Food and Agricultural Technology, University of Girona, 17071 Girona, Spain.
2
Departament d'Arboricultura Mediterrània, IRTA, Centre de Mas Bové, PO Box 415, 43280 Reus, Spain
3
Departament de Producció Forestal, IRTA, Torre Marimon, Spain


The brown apical necrosis (BAN) of walnut was observed for the first time in Spain in 1997 and affects the main walnut
production areas. The disease causes a premature walnut fruit drop and consequently a yield reduction. If disease
incidence and severity are high, the total fruit production can be lost. Infections are observed in walnut fruits from fruit
set to harvest, but they are more frequent and severe in early fruit development stages.
Epidemiological studies on BAN were conducted in Spanish walnut orchards during 2006-08 and results are summarized
and are presented here.
Microorganisms associated to BAN were isolated from lesions during the walnut fruit growing period in the three years of
study. Representative isolates were identified and tested for their pathogenicity on detached immature walnut fruits.
The microorganisms isolated from BAN lesions include Xanthomonas arboricola pv. juglandis, different species of
Fusarium (F. semitectum, F. solani, F. chlamydosporium, F. lateritium and F. poae) and Alternaria sp. Among them, only
the bacteria X. arboricola pv. juglandis was consistently isolated from external and internal necrotic walnut fruit tissues
and developed BAN symptoms when inoculated on detached immature walnut fruits. Some species of Fusarium were
pathogenic on walnut immature fruits.


Research was supported by the Spanish Project INIA RTA2005-00104-00-00.




                                                            44
45
INCIDENCE OF FUNGI ASSOCIATED WITH WALNUT BLIGHT


Giraud M.(1), Prunet J.P. (2) and Verhaeghe A. (3)
(1)
      Ctifl, Centre de Lanxade F-24130 Prigonrieux     giraud@ctifl.fr
(2)
      Ctifl, Walnut Experimental Station, Creysse, F-46600 Martel , France jp.prunet.creysse@wanadoo.fr
(3)
      Ctifl, SENuRA, Les Colombières, Chatte, F-38160 St Marcellin , France averhaeghe@senura.com


Some fungi are frequently isolated from typical symptoms of Walnut Blight ( Xanthomonas arboricola pv. juglandis), and
from other necrosis responsible for nut early drops. Study was carried out in the two major regions of Walnut growing,
Quercy and Isère, during two years. Typical symptoms of BAN were not observed. The main fungi recovered from fruits
were Colletotrichum sp., Alternaria sp. and Fusarium sp.. The same mycoflora was also isolated from dormant buds, but
with a dominance of Alternaria sp. and presence of other species (Trichoderma sp., Epicoccum nigrum, Sordaria
fimicola), always associated with X. a. pv. juglandis. These fungi were first suspected to interact with X. a. pv. juglandis
infection in spring, but unfortunately were not recovered from infected flowers, except Alternaria sp.. We are now
considering that they are belonging to the natural epiphytic mycoflora, increasing with necrosis development on fruits in
summer, by secondary infection. However, all drops are not caused only by X. a. pv. juglandis, and it was demonstrated
that the isolated strains of Colletotrichum are pathogenic to walnut fruits.




                                                             46
47
ETIOLOGICAL APPROACH TO BROWN APICAL NECROSIS ON WALNUT FRUITS IN TURKEY


Hatice Ozaktan1 Senem Akat1 Ahmet Akkopru1 Mine Erdal2


1
University of Ege Faculty of Agriculture Dept.of Plant Protection 35100,Bornova-İzmir/Turkey.
2
Ministry of Agriculture and Rural Affairs, Agricultural Quarantine Directorate, Izmir/Turkey


Brown apical necrosis (BAN) is a disease that causes walnut fruit drop and reduces the yield of walnut in commercial
orchards in Marmara Region (Northern west part of Turkey) of Turkey. The disease causes a premature walnut fruit drop
and a yield reduction. In 2009, study was carried out in a walnut orchard located in Marmara Region of Turkey to
substantiate the etiology of BAN. In this work the microorganisms associated and symptom evolution, disease progress
and cultivar susceptibility have been monitored in a walnut orchard from June to August from external to internal.
Infections in young fruits observed in July affected pericarp tissues and reached seed whereas early infections (June)
and late infections (August) of walnut fruits localized only pericarp tissues. According to our isolation and pathogenicity
results from external and internal tissues of walnut fruits of all tested cultivars, the microorganisms associated to BAN of
walnut included Xanthomonas arboricola pv.juglandis, isolated from all walnut fruit affected tissues in more than %30
fruits, Alternaria alternata isolated in 7% fruits mainly from pericarp tissues, Fusarium spp (F.moniliforme) isolated from
pericarp and seed in 3% fruits. On the other hand, X.arboricola pv.juglandis was isolated from seed and endocarp tissues
of healthy appearing walnut fruits samples at the rate of 5%. A different degree of sensibility to BAN among walnut
cutivars in Turkey has been noticed. The cultivars “Hartley”, “Vina”, “Rendede” and “Bilecik” showed a high incidence
while “Howard” and “Şebin” were less susceptible.




                                                            48
49
EPIDEMIOLOGY OF XANTHOMONAS ARBORICOLA PV. PRUNI ON PEACH IN FRANCE

Alain Garcin, Jérôme Vibert, Ctifl, France

Aurélie Leclerc, Ctifl/Université d‟Avignon, France



Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap) is one of the most serious diseases in Prunus orchards
in France as well as all over the world. Direct control methods are quite limited making, it urgent to find ways of limiting
Xap infection and distribution. To develop a mathematical model for prediction of Xap infection risk on peach trees, the
Ctifl is working on environmental parameters of infection that must be determined. First, observations on healthy peach
trees growing in a contaminated orchard were made, and then we studied the relationship between the infection and
weather conditions: only rain provokes an infection when it‟s combined with a wetness period lasting longer than 4
hours, but we don‟t know the duration and quantity of rainfall necessary for infection to take place. The first results show
that infection is possible from 12°C and the incubation period is from about 350 to 500 degree day after the rain when
infection takes place. Some of this information will be used to develop the model and others parameters must be
incorporated such as the varietal sensibility and the level of inoculum from the previous year.




                                                            50
51
EPIDEMIOLOGY OF WALNUT BLIGHT IN TASMANIA, AUSTRALIA


Michael D. Lang, Katherine J. Evans*

Tasmanian Institute of Agricultural Research (TIAR), University of Tasmania, 13 St Johns Avenue, New Town, Tasmania
7008, Australia
*Corresponding author: Katherine.Evans@utas.edu.au


Introduction
The island state of Tasmania produces a broad range of horticultural crops; the main perennial crops are wine grapes,
apples, cherries, apricots and walnuts. There has been significant expansion of the area of these crops, with the
exception of apples, in the last 10 years. Tasmania has a maritime, cool-temperate climate with average daily maximum
temperatures in the warmest and coolest months of 22 and 12oC, respectively. Rainfall is frequent in spring and early
summer. Bacterial diseases of economic importance include bacterial canker of cherries, caused by Pseudomonas
syringae, and walnut blight, caused by Xanthomonas arboricola pv. juglandis. In time, more diseases may become
problematic as recent plantings mature or quarantine barriers are breached.
             Walnuts with symptoms resembling those of walnut blight have been present in Tasmania since early
European settlement (Rodway 1912); however, the cause was not confirmed until recently (Lang and Evans 2009). In
Tasmania, walnut blight has led to 70% crop loss in non-treated trees in some years (Lang et al. 2006); thus, to protect
susceptible fruits, copper-based bactericides are applied at weekly intervals from bud-burst for 4–5 weeks, and then at
7–14 day intervals depending on the weather, for up to ten applications per year. Two copper tolerant isolates were
identified among a broad sample of 34 isolates from commercial orchards with a short history of copper use (Saravanan
2007). The potential for selection of copper tolerant bacterial populations and accumulation of this heavy in soil means
that reduction of copper inputs is a key objective toward sustainable control of walnut blight in Tasmania (Lang et al.
2006).
             Objectives of this study were (a) to understand the temporal progression of blight incidence in walnut fruit
in non-treated plots, (b) to quantify the relationship between blight incidence and nut yield, (c) to investigate the impact
of spray timing on blight incidence and nut yield and (d) to initiate development of a weather-based decision rule for
timing copper-based sprays.


Methods
Randomized, complete block trials were conducted using single tree plots ( n = 6) of cultivars Vina and Franquette over
four years in northern (wet climate) and eastern (dry climate) regions of Tasmania. The number and timing of
applications of a copper-mancozeb formulation, Mankocide® DF (0.5 kg product/100 L water, applied at a spray volume
of 1,000 L/ha), varied among treatments. For non-treated trees, disease incidence was assessed on the same 100 fruit
from fruit set to harvest, or until a fruit fell. Crop yield in non-treated plots was defined as the percentage of the 100
fruit present at fruit set that produced marketable nuts at harvest.


Types of epidemic
Daily cumulative disease incidence was analyzed as a function of time to obtain a model of „best fit‟. In 2004-05 and
2006-07, disease progression was described best by the monomolecular model, whereas in the relatively wet season of
2005-06, disease progression was described well by the logistic or Gompertz model, depending on the site. A single
model was then developed for all blight epidemics to allow disease prediction. This process, to be presented, revealed
two types of epidemic, which, for practical purposes, were labeled „Type I‟ and „Type II‟ epidemics.




                                                            52
53
Blight incidence and nut yield
In non-treated plots, blight incidence on fruits at half full-size diameter accounted for 97% of the variation in crop yield
(Fig. 1), when non-blighted fruit that fell were removed from the data set.


Spray timing
In 2004-2005 (Type I epidemic), applications of copper at bud burst and one week later reduced blight incidence
significantly (P < 0.05), from 43% (non-treated) to 6.5%. Further applications of copper did not improve blight control.
There was another Type I epidemic in 2006-07, when there was no significant difference between non-treated (8%
blight incidence) and copper-treated plots.         In 2005-2006 (Type II epidemic), nine weekly applications of copper from
bud burst reduced blight incidence significantly, although control was commercially unacceptable. Blight incidence was
reduced from 79% (non-treated) to 44% and the yield (marketable nuts) was increased from 3% (non-treated) to 25%
(P < 0.05).


Weather-based decision rule
A weather variable called „daily rain intensity‟ (mm rainfall/hours of leaf wetness) was accumulated for the interval
representing the previous 17 to 24 degree days (Tmin = 1oC; Tmax = 28oC). The accumulated daily rain intensity
accounted for 67% of the variance in the percentage increase in blight incidence per day (Fig. 2). A decision rule for
timing copper-based bactericides was developed from this relationship and the results of model testing in field trials
conducted in 2008-09 will be presented. In short, the number of spray applications was reduced for the same level of
blight control as observed for a full, calendar-based spray program.


                   100                                                                                   5.0
                                        y = -1.0027x + 101.24                                                        y = 0.5372x + 0.4584
                                                                        Percentage new blighted fruits




                                              R2 = 0.97                                                                    R2 = 0.67
                    80                                                                                   4.0
   Nut yield (%)




                    60                                                                                   3.0
                                                                                   per day




                    40                                                                                   2.0

                    20
                                                                                                         1.0

                     0
                         0   20    40       60      80     100                                           0.0
                                                                                                               0.0      1.0     2.0      3.0     4.0     5.0       6.0
                             Disease incidence (% )                                                                  Daily rain intensity (rain (mm) / LWRD (h))

Figure 1. Relationship between nut yield and blight                   Figure 2. Relationship between percentage increase in
incidence on fruits at half full-size diameter.                       blight incidence per day and daily rain intensity (rain
                                                                      (mm)/leaf wetness (h)) accumulated in the previous 17-
                                                                      24 degree days.




                                                                 54
55
Conclusions
In the absence of copper treatment, half full-size diameter walnut fruits with blight tended not to produce a marketable
nut. There is potential to reduce the number of applications of copper-based bactericides for Type I epidemics. A model
for the temporal progression of blight incidence was utilised to develop an empirical, weather-based decision rule for
timing applications of copper. This decision rule needs further refinement, calibration and validation.




References
Lang, M.D. and Evans K.J. (2009) Characters of the walnut blight bacterium in Tasmania. Australian Nutgrower 23: 35-
          39.
Lang, M.D., Hills, J.L. and Evans, K.J. (2006) Preliminary studies towards managing walnut blight in Tasmania. Acta
          Horticulturae 705: 451–456.
Rodway, L. (1912) Bacterial black spot of walnut. Agricultural Gazette pp. 85–86
Saravanan, U. (2007) Copper sensitivity in Tasmanian populations of the walnut blight pathogen Xanthomonas arboricola
          pv juglandis MSc. thesis. University of Tasmania, Australia.

Acknowledgements
This project was supported by the Australian Government through Horticulture Australia Limited in partnership with
Webster Walnuts and Agronico Pty Ltd.




                                                             56
57
WORKING GROUP 3




      58
59
ETHNOBOTANY OF PEACH, CHERRY AND WALNUT


M. Petriccione

CRA- Unità di Ricerca per la Frutticoltura, Via Torrino, 3 - 81100 Caserta- Italy


Ethnobotany is the study of the relationships between plants and cultures. People have always depended on plants for
their primary needs (i.e. food, timber, medicines, warmth) and have learned their uses in a natural way.
Ancient herbals represent an important and neglected source of information on the methods used by pre-scientific
people to select and manipulate plants for exploiting their edible and medicinal properties. In this context, I have re-
examined the historical background of peach, cherry and walnut cultivation and uses.
The botanical name of peach (Prunus persica (L.) Batsch) refers to the putative country of origin, Persia (i.e. Iran), and
Linné (1758) first named the species based on this opinion (Amygdalus persica). Only in the 19th century was the Far
East geographical origin (western China) finally acknowledged, written records and archaeological evidence date peach
domestication at least as far as back as 3.000 BC. Peaches were among the first fruit crops domesticated in China. They
moved to Persia along silk trading routes. Greeks and especially Romans spread the peach throughout Europe and
England starting in 300-400 BC. Peaches came to the new world with explorers of the 16th - 17th centuries, with
Portuguese introducing it to South America and the Spaniards to North America.
In the Chinese culture the peach is a symbol of human longevity in Taoist mythology was the old man (Shou Lu) who
appears in illustrations with his finger stuck into the suture of a fuzzy peach, perhaps to symbolize the way to attain a
long life. In Roman mythology the peach was the fruit of Venus, and Albertus Magnus believed that peaches were
aphrodisiac. A peach with a leaf attached symbolizes the union of the heart and tongue, hence truth. Peaches were used
by ancient Egyptians as offerings to the God of Tranquility.
Sweet cherry (Prunus avium) originated in the area between the Black and Caspian Seas of Asia Minor and was probably
carried to Europe prior to human civilization by birds. Cultivation is believed to have begun with the Greeks, and was
perpetuated by the Romans, which lead to its spread throughout Europe. There, trees were planted along roadsides and
were grown for their timber as well as their fruit. Sweet cherries were brought to the United States with English colonists
in 1629 and later were introduced to California by Spanish missionaries.
Sour cherry (Prunus cerasus) derives from a natural cross between P. avium and P. fruticosa (ground cherry). The
geographic ranges of the two species overlap in northern Iran and Turkmenistan, which is the center of origin of sour
cherry. From there, sour cherry followed a similar course to Europe as sweet cherry, and it came to North America with
English settlers.
Cherry stems have long been recognized as having diuretic properties and they are found in prescriptions dating back to
the Middle Ages. Cherry bark infusions were used to treat colds and flu, while the fruit and juice were considered to be
an effective remedy for gout, aches and arthritis. Modern research has shown that cherries actually do contain natural
anti-inflammatory agents.
The Persian walnut (Juglans regia L.), is a traditional nut of Old World agriculture. It grows wild in mesic, temperate,
deciduous forests of the Balkan, North Turkey, South Caspian Region, Caucasus and Central Asia. It reappears in the
Thien Shan province of Western China.
The walnut tree is known since antiquity, besides its fruits and timber, as source of substances harmful to other living
things, but also for his deep roots in folklore and mythology. Such was the baneful reputation of the walnut in the south
of Italy, that is called "witches tree".




                                                               60
61
Walnut provides amongst the earliest recorded suggestion of allelopathy since the tree was considered a source of
substances harmful to other organisms by Roman scholars Varro. and Pliny the Elder, who wrote treatises on agriculture
dealing with the methods of good crop husbandry.




                                                         62
63
TRANSFERRING EXPERTISE WITH APPLE DEFENCE BIOCHEMISTRY TO STONE FRUIT AND NUTS


Heidi Halbwirth*, Karl Stich
Technische Universität Wien, Institut für Technische Biowissenschaften, Getreidemarkt 9/1665, A-1060 Vienna, Austria;
*corresponding author: hhalb@mail.zserv.tuwien.ac.at




Transferring expertise with apple defence biochemistry to stone fruit and nuts is possible in two ways: First, it can be
investigated whether defence mechanism found in apple are also relevant for other fruit crops. Second, successful plant
protection measures could be evaluated for their potential to act also on relevant host-pathogen systems in stone fruits
and nuts.
In many host plants, pathogen resistance against a wide range of fungi and bacteria is connected to the presence of
phenolic compounds, in particular flavonoids [Treutter 2006]. Antimicrobial compounds either are synthesized de novo as
a response to infection or attempted pathogen invasion (phytoalexins) or constitutively stored in plant cells in
anticipation of a pathogenic attack (phytoanticipins) [Dakora and Phillips 1996]. In apple, several compounds of the
polyphenol pathway are involved in pathogen defence including dihydrochalcones, specific organic acids, flavanols
(synonyms: proanthocyanidins, tannins) and flavonols. The majority of these compounds can be also found in stone fruit
and nut – depending on physiological, biotic and abiotic factors [Jay-Allemand et al., 1986, Halbwirth et al., 2009].
Studies on polyphenol formation frequently investigate either polyphenol accumulation or related gene expression or
related enzyme activities. However, experience with apple has shown that a simultaneous consideration of more than
one of these biosynthetic levels is important. Whereas gene expression studies and polyphenol analysis are widely used
methods, the determination of enzyme activities is not always that simple, and sometimes require sophisticated protocols
[Claudot and Drouet, 1992a; Halbwirth et al., 2009]. To estimate the possibility to assess these enzyme activities in
stone fruits and nuts, we have tested different species and tissues for the detectability of polyphenol enzymes. Our
studies showed that a modified Claudot and Drouet [1992a] protocol which was adapted to the presence of polyphenol
compounds, is frequently the superior protocol for the investigations of various fruit tissues [Halbwirth et al., 2009].
Enyzme activities in fruits seem to be generally lower than in leaves and flowers. Previous studies have indicated that
early enzymes in the pathway are frequently rate-limiting [Claudot and Drouet, 1992b, Strissel et al., 2005].




                                                            64
65
References
Claudot A. C., Drouet A. (1992a), Phytochemistry 31, 3377-3380
Claudot A. C., Drouet A. (1992b), Bulletin de Liaison - Groupe Polyphenols , 16, 75-78.
Dakora F. D. & Phillips D. A., (1996), Physiological and Molecular Plant Pathology 49, 1-20.
Halbwirth H. et al. 2009, J. Agric. Food Chem. 57, 4983-4987-
Jay-Allemand, C. et al. (1986), Bulletin de Liaison - Groupe Polyphenols 13 580-2.
Strissel T. et al. (2005) Plant Biology 7: 677-685.
Treutter, Dieter. (2006), 4(3), 147-157




                                                             OH                                OH                                   OH


                  O            O                                                               PAL
                                    +    CoAS                                 HO
              3
                      OH     SCoA                                                                         HCOO          NH2
                                                   O                               O

                  Malonyl-CoA             p-Coumaroyl-CoA                     Cinnamic acid                    Phenylalanine

                              CHS CHI
                                                       OH                              OH                                      OH

                      HO            O                        HO          O                     HO          O
                                                       FHT                               FLS
                                                                               OH                                  OH
                              OH    O                              OH    O                           OH    O

                               Naringenin                         Dihydrokaempferol                  Kaempferol


                                                                          DFR
                                                                                        OH

                                                             HO          O
               Epicatechin              Cyanidin                                                          Catechin
                                                                                  OH
                                                                    OH   OH

                                                                  Leucopelargonidin




Figure: Selected part of the flavonoid pathway




                                                                             66
67
STUDIES ON RESISTANCE OF ALMOND VARIETIES TO HYPERPLASTIC CANKER CAUSED BY PSEUDOMONAS
AMYGDALI AND IDENTIFICATION OF GENE HOMOLOGUES OF HRPZ AND HRPW, POTENTIAL MOLECULAR
TARGETS FOR INHIBITING INFECTION PROCESS


M.C. Holeva1*, P.E. Glynos1, C. Karafla1, D.C. Stylianides2 and A.E. Voloudakis3

1
Laboratory of Bacteriology, Benaki Phytopathological Institute, Kifissia, Greece; 2Pomology Institute, Naoussa, Greece;
3
Laboratory of Plant Breeding and Biometry, Department of Crop Science, Agricultural University of Athens, Greece.



„Hyperplastic canker‟ of almond, Prunus dulcis (Mill.) Webb, caused by Pseudomonas amygdali, is the most destructive
bacterial disease of this fruit tree in Greece. We have recently initiated studies on the pathosystem „almond-P. amygdali‟,
aiming at identifying genetic sources of plant resistance, as well as molecular means to inhibit bacterial infection.
The response to artificial inoculation with P. amygdali was investigated on four almond varieties, namely, the varieties:
„Kathigitou Raptopoulou‟ (8 trees), „Alkyon‟ (5 trees), „Loran‟ (10 trees) and „Ferragnes‟ (5 trees). The experimental
plants, obtained from the Pomology Institute (Naousa, Greece), were two-year old, grafted on bitter almond seedlings
and were planted in 20-liter pots at the Benaki Phytopathological Institute in Kifissia (Attica, Greece) in winter (December
2007). Plants were artificially inoculated in the following autumn (November 2008) with a greek strain of P. amygdali
(BPIC 71) isolated by Dr P. Psallidas. The inoculations were carried out by placing 25 μl of a BPIC 71 suspension culture
grown overnight in LB (aprox. 1.5 x 109 cfu/ml): a) on leaf scars immediatedly after detaching the leaves forcibly by
hand; four leaf scars per shoot were inoculated, and b) into a wound of the bark made with a sterile scarpel; two or
three wounds along each shoot, at about 10 cm distance between them, were inoculated. Each of the above inoculation
methods, described by Psallidas and Stylianides (1985) was applied to two shoots per tree. By mid-August, upon visual
examination of the experimental trees, the wound inoculated shoots of all four varieties exhibited symptoms, while
shoots inoculated at leaf scars were asymptomatic.
In addition to screening almond varieties for disease resistance, the molecular basis of pathogenicity of P. amygdali was
investigated. Specifically, the presence of hrp genes encoding harpin proteins (HrpZ and HrpW) known to be involved in
virulence of other Pseudomonas phytopathogens was explored. In this regard, it was found that the DEG1/DEG4
degenerate oligonucleotides as well as the W1/W2 oligonucleotides, reported to amplify hrpZ sequences of P. syringae
pathovars (Loreti et al., 2001) and hrpW sequences of P. s. pv. tomato (Charkowski et al., 1998), respectively, amplified
also sequences of the expected sizes from BPIC 71. Cloning and sequencing of the amplification products are under way
to confirm the presence of hrpZ and hrpW homologous genes in P. amygdali; functional analysis of these genes will
follow. Since the hrp gene cluster codes for a highly specialized protein secretion system („Type III‟), often required for
bacterial virulence, it is currently considered as an attractive target for development of antimicrobial compounds.
Therefore, identification of hrp genes in P. amygdali might contribute to the development of compounds for inhibiting
hyperplastic canker in almond effectively.


References
       Charkowski A.O. et al. 1998. Journal of Bacteriology, 180:5211-5217.
       Loreti S., Sarrocco S. and Gallelli. 2001. Journal of Phytopathology, 149:219-226.
       Psallidas P.G. and Stylianides D.C. 1985. CIHEAM- Options Mediterraneennes, n.1985-I:107-110.




                                                             68
69
IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF BACTERIAL AGENTS OF STONE FRUITS AND
WALNUTS IN LITHUANIA


Daiva Burokiene

Institute of Botany, Zaliuju Ezeru Str. 49, LT-08406, Vilnius, Lithuania, daiva.burokiene@botanika.lt



The detection, identification and molecular characterization of most harmful bacterial diseases of stone fruits and walnuts
were performed.
Specimens from symptomless plum, sour and sweet cherry trees as well as those exhibiting bacterial canker symptoms
were collected in stone fruit orchards in various regions of Lithuania. Seventy seven Pseudomonas syringae-like isolates
were obtained from plant material – leaves, branches, shoots. The isolates were identified using biochemical and
physiological tests (Gram- and catalase reaction, fluorescent pigment on King‟s medium B, metabolism of glucose,
LOPAT, GATTa), pathogenicity on tobacco and host plants. Identification with PCR was applied for cfl gene, which is
involved in production of phytotoxin coronatine (primers cfl1 and cfl2), for yersiniabactin (primers PSYE2 and PSYE2R)
and syringomycin (primers syrB1 and syrB2). According to the tests, only 21 strains were identified as P. syringae. 14
strains were determined as P. s. pv. syringae, 4 strains – P. s. pv. morsprunorum race 1, 3 strains – P. s. pv.
morsprunorum race 2.
Molecular characterization using PCR MP for Pseudomonas syringae strains was performed. Insignificant differences
among P. s. pv. syringae strains were estimated, but PCR profiles with P. s. pv. morsprunorum (race 2) strains from
Lithuania were different from typical strain (CFBP 3800).
Five walnut species (Juglans cinerea, J. mandshurica, J. regia, J. nigra, J. cordiformis) and two hybrids (J. x bixbyi, J x.
quadrangulata) grown in Lithuania were inspected for Xanthomonas arboricola pv. juglandis (Xaj), the causal agent of
walnut blight. According to the diseases symptoms on leaves, branches and fruits, colony morphology on YDC medium
and tested characteristics (Gram-, oxidase and catalase reactions and growth on potato slices), 59 Xaj-like isolates were
obtained from diseased walnuts. But only 8 strains gave PCR amplification product (~480 bp) with Xanthomonas genus
specific primers (X1 and X2). Further, according to biochemical tests (growth at 35 ºC, aesculin and starch hydrolysis,
mucoid growth on YDC and SQ media, pathogenicity on tobacco plants and host plants) these strains were identified as
Xaj.
Preliminary results were obtained using REP-PCR for Xaj. The strains from Lithuania and Poland were compared, and no
visible differences between the strains were found. Detailed investigations are needed for more accurate results, and
PCR with other primers – BOX and ERIC – is under examination.




                                                            70
71
PHENOLIC COMPOUNDS AS POTENTIAL MARKERS FOR WALNUT BLIGHT RESISTANCE -PRESENT RESULTS
OF SLOVENE RESEARCH

A. Solar1, T. Dreo2, M. Mikulič-Petkovšek1, A. Likozar1, M. Šuštaršič2, R. Veberič1, L. Matičič2, M. Ravnikar2, F. Štampar1

1
Chair for Fruit Growing, Agronomy, Biotechnical Faculty, University of Ljubljana, Slovenia
2
National Institute of Biology, Ljubljana, Slovenia


Phenols play an important role in plant defense as agents that protect the plant from pathogens and insect pests. In
walnut fruits, the content of several phenolic compounds were analysed with the aim of evaluating the possible
involvement of phenolics in walnut blight resistance. At the Gf+30 phenophasis, healthy walnuts taken from adult trees
of six cultivars were artificially inoculated with a Spanish reference strain of Xanthomonas arboricola pv. juglandis
according to the method developed by Moragrega (Aleta et al., 2001). After three weeks‟ incubation, disease incidence
was proved in all the studied cultivars, while its severity depended on the cultivar. In very susceptible cultivars
(Seifersdorfski and Šampion), and very low susceptible cultivars (Erjavec, Fernette), respectively, the disease severity of
artificially inoculated walnuts corresponded to the severity assessed at the same sampling date „ in situ‟.
The results were then linked to the phenolic compounds analysed in healthy (H) and artificially inoculated fruits (A), and
also in the fruits, inoculated with sterile distilled water (negative control, N). 18 phenolic compounds belonged to
different groups were detected in all cultivars. The interacting impact of the tissue and cultivar was found in most
phenols. Xaj infection caused increased synthesis of catechin, chlorogenic acid, p-coumaric acid, and rutin, which
appeared in significantly higher contents compared to negative control in all cultivars. In the least susceptible cv.
Fernette, more quercetin-3-O-galactoside (hyperin) was found in the negative control than in healthy, as well as in
inoculated fruits. Healthy fruits of low susceptible Erjavec contained significantly more fluoroglucinol, caffeic acid,
quercetine-rhamnoside and siringic acid than A and N treatments.
Following phenolics‟ contents of the walnuts sampled in the orchard, more clear and consistend differentiation between
healthy and naturally infected fruits was found at the Gf, and Gf+30 phenophases. At the Gf+45, the phenolics‟ synthesis
was more affected by the health status of the fruits, and by the cultivar. In the most susceptible Seifersdorfski, a
significant decrease in syringic acid from the Gf+30 towards the Gf+45 stage, might influence very strong Xaj infection
(62,5 %) in the orchard observed at the Gf+30 stage. Some other connections between seasonal fluctuations in the
concentrations of phenolic compounds and the Xaj severity assessed „in situ‟ will be discussed, as well.




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73
MOLECULAR CHARACTERIZATION OF WALNUT AND EVALUATION OF XANTHOMONA ARBORICOLA                                            PV.
JUGLANDIS DAMAGES IN MURCIA, SPAIN


L. Ruiz, D. Frutos, G. Lopez, A. Fuentes.

Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA).
30.150- La Alberca, Murcia, Spain


Molecular markers provide a genetic finger printing own of each cultivar or selected genotype, independently of ambient.
In this way, it has been characterized a walnut tree (J. regia L.) collection planted in El Chaparral, Murcia, containing 57
accessions. First, it had been analyzed 12 accessions in order to select the most useful molecular markers. To do that 32
SSR (Simple Sequence Repeat) developed in walnut have been used. Based on the results of this analysis, it had been
selected 19 of these 32 polymorphic SSRs useful for walnut molecular characterization. In a second step these 19 SSRs
had been used for characterize the 57 walnuts accessions, what it had allowed to identify unequivocally the accessions
analyzed. Such identification had allowed connecting variability and genetic relationships of the accessions. It had been
identify 97 alleles in the different accessions, what represents an average of 5 alleles per locus. The discrimination power
of analyzed loci ranged from 0.36 for the locus WGA79 and 0.90 for locus WGA276, with an average discrimination
power per locus of 0,74. Loci with bigger discrimination power have been WG09, WGA69, WGA202, WGA276, WGA321
and WGA349. Heterocigosity of the analyzed plant material ranged between 0.11 for cv. Santa Cruz, selected in
Cantabria, and 0.95 for the Californian cv. Sunland. Heterocigosity of the Spanish walnut materials had range between
0.11 and 0.74, with an average of 0.51. The Californian accessions have shown a heterocigosity ranging from 0.37 to 0.
95, been its average 0.55.
The second part of this work intended to connect the genetic proximity and the tolerance or susceptibility to
Xanthomonas arborícola p.v. juglandis (Xaj). With this purpose, symptoms on leaves and shoots caused by Xaj were
recorded in 30 old walnut trees of the prior collection planted in 1984. In 2009 such trees had been treated with copper
sulfate and Mancozeb in spring and summer.
Xaj damages were classified in categories from 0 to 4 (0 when symptoms are totally lacking, 1 for 1% of surface
affected, 2 for 10%, 3 for 50% and 4 for values up to 80%). Leaves, shoots and fruits data were recorder on 27 of July
and 13 of August.     No significant differences were found between dates for shoot damages. However, leaves damages
were significantly higher on 27 of July. Perhaps this can be explained if a significant amount of damaged leaves were
fallen and consequently no recorded on August, 13.
Five groups of varieties were detected according to shoots damages and ten groups according to leaf damages. Cultivars
with very low damages were Nugget, Trinta, De Arriba, Badajoz and Eureka did not present damages on leaves. On the
other end, Alcade 4, Franquette, Chico, Pyne, Sendra and 23 B-1 showed the biggest degree of damages. This last
selection is extremely sensitive to Xaj. It was not possible to connect the tolerance or susceptibility levels of some
varieties with the tolerance or susceptibility levels of their progenies.




                                                               74
75
WORKING GROUP 4 +CoP




         76
77
RECENT ADVANCES ON THE EPIDEMIOLOGY AND CONTROL OF THE BACTERIAL BLIGHT OF WALNUT,
INCITED BY XANTHOMONAS ARBORICOLA PV. JUGLANDIS


D. Giovanardi1, D. Dallai1, E. Cozzolino2 and E. Stefani1.

1
 Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio Emilia, via J.F. Kennedy 17, 42100
Reggio Emilia, Italy. 2Az. Agr. San Martino, San Martino in Strada (FC).
 E-mail: emilio.stefani@unimore.it


The bacterial blight of walnut caused by Xanthomonas arboricola pv. juglandis (Xaj) is an emergent disease, which might
severely affect walnut orchards. Symptoms are visible on all aerial parts of the host plant and particularly on leaves and
nuts; the disease develops more rapidly during springtime, causing spots on leaves and immature fruits, followed by the
formation of small cankers on leaf petioles and twigs. Affected fruits fall down throughout the growing season, with a
peak within mid-may and mid-june. The primary source of the inoculum is available very early in springtime, since
evasion is likely to occur from small overwintering cankers present on twigs as soon as buds and twigs are developing.
The primary inoculum is spread by wind-driven rain droplets and by pollen.
Our study confirms the spread by pollen, but catkins seems to become infected during their spring development from
bacteria oozing out from small twig cankers. Female flowers are not contaminated before pollination, and become
infected during pollination and/or during spring rain. Copper resistance has been studied on a wide collection of over 150
strains isolated in Romagna during 2007-2009: 83% of the collection proved to be tolerant to copper, whereas 36%
proved to be highly resistant.
Control strategies are difficult to implement and are based on the timely effective use of copper compounds with
emphasis on spring treatments. In order to bypass copper resistance the use of alternative molecules as resistance
inducers is under evaluation, coupled with a reduced use of copper.




                                                             78
79
STSM ACTIVITIES




       80
81
SURVEYS FOR XANTHOMONAS ARBORICOLA PV. PRUNI IN SERBIA AND IMPLEMENTATION OF
DETECTION PROCEDURES AND CERTIFICATION SCHEME

          1                2           1                  2
Stefani E. , A. Obradoviš , D. Dallai and K. Gasiš

1
 Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio Emilia, via J.F. Kennedy 17, 42100
Reggio Emilia, Italy;
2
 Plant Pathology Dept. University of Belgrade, Nemanjina 6, 11080 Belgrade - Zemun, Serbia. E-mail:
emilio.stefani@unimore.it


A visit to Serbia was organised after an invitation of the Faculties of Agriculture of the Universities of Belgrade and Novi
Sad. The main purposes of the visit were the organisation and implementation of surveys for Xanthomonas arboricola pv.
pruni (Xap) in Serbian orchards and nurseries and the implementation of diagnostic procedures. A group of Serbian
scientists and PhD students participated to the activities, from the Universities of Belgrade and Novi Sad and from the
Institute of Plant Protection, Ministry of Agriculture.
The mission was based on 3 points:


    1.    Field and nursery inspections and sample collection
    2.    Technical lectures on the disease and its causual agent
    3.    Lab training on the detection and identification of Xap


As regards field activities, orchards and nurseries were inspected in northern Serbia, at the Hungarian border
(Autonomous Province of Vojvodina), in Fruška Gora, around the Municipality of Smederevo (Central-eastern Serbia) and
in the Topola area and surrounding municipalities (Central Serbia). Inspections where done in peach and plum orchards
and nurseries.
Lab training started with the isolation from symptomatic tissue and purification of suspect colonies on suitable agar
media. Additionally, a plant extract was prepared for immunofluorescence.
The preliminary characterisation of putative Xap colonies was achieved by the following methods:


    1.    Purification and colony observation on suitable agar media
    2.    Hypersensitive response (HR) on bean pods
    3.    Copper resistance
    4.    rep-PCR with BOX primers
    5.    Indirect immunofluorescence antibody staining (IFAS), used both on plant extract and on pure cultures.


Results were successful: we proved that field samples did not result in the isolation and purification of putative Xap
colonies. One of the sample gave colonies resembling Pseudomonas syringae pv., which is quite common in Serbia.
Hypersensitive response on bean pods, resistance to copper, rep-PCR, immunofluorescence antibody staining (IFAS)
gave very good results using pure cultures from an official collection.
Thus, field inspections followed by lab analyses confirmed that Xap was not present in visited orchards and nurseries.
Indeed, according to a Pest Risk Assessment procedure (see: ISPM No. 11 (2004), Pest risk analysis for quarantine pests
including analysis of environmental risks and living modified organisms) both the probability of entry of the pest and its
potential for establishment and spread appear to be LOW. As a matter of fact, most of plum varieties cultivated in Serbia
belong to Prunus domestica (the European plum), which is known to be quite tolerant to Xap infections. Differently,



                                                              82
83
peach varieties visited in orchards are known to be less tolerant or quite susceptible. During round tables organised with
some nurserymen it came out that importation of stone fruit planting material in not so widespread, and from Countries
where Xap is either not present or not widespread.
Field and lab activities were successful, since a classical procedure for the diagnosis of fruit/leaf bacterial spot of stone
fruit was followed and commented by the participants and trainees. Trainees had chances to actively participate through
questions, manual work, discussion, etc. during the whole week. Both laboratories were sufficiently equipped to
adequately perform the detection of the bacterial leaf/fruit spot and canker of stone fruits and the identification of its
causal agent, Xanthomonas arboricola pv. pruni.


Three technical lectures have been given in a plenary room during the STSM. Their titles were:


   1.     Bacterial canker/leaf spot of stone fruits (Xanthomonas arboricola pv. pruni)
   2.     Identification and population studies of Xanthomonas arboricola pv. pruni
   3.     A nursery certification scheme for Xanthomonas arboricola pv. pruni


The diseases and its causal agent, Xanthomonas arboricola pv. pruni, were described in details and commented, having
reference to the Serbian horticultural situation. Particular emphasis was given to symptom description and epidemiology
of the disease on plum and peach. The scheme published as EPPO standard procedure for the certification of stone fruit
planting material was also described in details, as published by the EPPO Bullettin (2006, PM 7/64).
The mission was completed as agreed with the hosting institutions, namely: the Faculty of Agriculture of the University of
Belgrade and the Faculty of Agriculture of the University of Novi Sad. Future collaboration with the hosting Institutions
and exchange of students/young scientists are envisaged, both on bacterial diseases of stone fruits and nuts and on
other topics related to regulated bacteria.




                                                             84
85
NEW OCCURRENCE OF XANTHOMONAS ARBORICOLA PV. CORYLINA ON EUROPEAN HAZELNUT IN SERBIA

A. Šališ1, K. Gašiš1, M. Ivanoviš1, A. Obradoviš1, P. Ferrante2, M. Scortichini2
1
Department of Plant Pathology, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade-Zemun,
Serbia. E-mail: aleksao@agrif.bg.ac.rs
2
C.R.A. - Centro di Ricerca per la Frutticoltura, Via di Fioranello, 52. I-00134 Roma, Italy.


Hazelnut production in Serbia is considered small crop production of limited importance in national agriculture.
Consequently, that resulted in low interest in pathology of this crop. However, novadays hazelnut production is growing
very fast, with commercial orchards arising quickly over the country. Increase in production, import and movement of
planting material, bring in focus hazelnut diseases, especially those of phytosanitary concern. Regarding bacterial
diseases of hazelnut, there was only one report in last 50 years indicating presence of Xanthomonas corylina in Serbia.
Besides, Pseudomonas avellanae and P. s. pv. coryli are known to be present in close proximity of our country and
therefore represent a risk for this actively growing production. During the orchard surveys in summer of 2008, we
observed spotting and blighting of leaves and necrosis of nut shells on hazelnut plants near Belgrade. In order to study
etiology of the symptoms, samples were taken for laboratory analysis.
From the diseased tissue shiny, yellow, bacterial colonies were isolated on nutrient agar (NA) plates. Ten colonies were
subjected to single-cell purification procedure and maintained for further testing. The strains induced hypersensitive
reaction of tobacco and pepper leaves, as well as bean pods. On GYCA medium strains formed characteristic light yellow,
round, mucoid colonies after 48 h at 27oC, resembling growth of Xanthomonas arboricola pv. corylina, causal agent of
hazelnut bacterial blight. Reference strain of X. a. pv. corylina NCPPB 3037 was used in all assays as a positive control.
The isolates were gram and oxidase negative and catalase positive. They hydrolyzed starch, aesculin and gelatin,
produced acid from glucose, sucrose, and maltose but not from sorbitol. All strains grew at 35°C. Rep-PCR fingerprinting
of genomic DNA using BOX primers showed identical patterns of investigated strains with reference strain NCPPB 3037.
Homogeneity of characteristics among the strains as well as compared with the reference strain of X. a. pv. corylina
indicated that they belong to this bacterium.
In spite of lack of information about this hazelnut pathogen, we assume it was continually present in Serbia either on
cultivated or wild hazelnut and this new record came as a consequence of increased interest on hazelnut bacterial
diseases.




                                                              86
87
FIELD SURVEY AND MICROORGANISMS ASSOCIATED WITH APICAL NECROSIS OF WALNUT

Senem Akat

Department of Plant Protection. Faculty of Agriculture. University of Ege. 35100 Bornova/ Izmir. Turkey



The STSM took place at the Institute of Food and Agricultural Technology of University of Girona (Spain). The main aim
of this STSM was to learn sampling isolation and monitoring techniques of the casual microorganims associated with BAN
(Brown Apical Necrosis) and Apical necrosis of walnut fruits, besides to symptom evolution and disease progress in
walnut commercial orchards in Tarragona and Girona, Spain.
The BAN has been considered as a complex disease in which different microorganisms, bacteria and fungi are implied. So
we sampled some walnut fruits from Spanish different orchards in the NE and SE of Catalonia.
In the period of STSM we visited a commercial walnut orchard in Girona to observe symptoms of BAN and walnut blight
in fruits and to collect samples for isolation in the laboratory. Additionally fruits collected in Tarragona walnut orchards at
Gf and Gf+15 stages with lateral and apical necrosis were analyzed. I provided practical experience about symptom
determination the disease in walnut orchard survey.
In order to differentiate BAN from typical walnut blight apical necrosis walnut fruits were externally and internally
analyzed and tissues affected were described. Microorganisms (fungi and bacteria) were isolated from infected walnut
tissues in general and selective media (PDA, TMM, YDC). Identification of microorganisms was based on morphological
traits and growth on the media. Results indicated that the main microorganism associated to BAN and walnut blight
affected fruits is the bacteria Xanthomonas arboricola pv. juglandis since it was isolated in 100 % of samples where
microorganisms were present. Only in one sample Fusarium spp. was also recovered.




                                                              88
89
MONITORING SURVEYS IN ITALY FOR XANTHOMONAS ARBORICOLA PV. PRUNI (XAP) IN PEACH AND
PLUM ORCHARD


Katarina Gasic

Faculty of Agriculture. Department of Phytomedicine. Belgrade, Serbia



Thanks to COST Action 873, I spent my STSM at the Department of Agricultural and Food Sciences, University of Modena
and Reggio Emilia, Italy, during the period 1st – 30th June, 2008. The scientific objective of this mission was to conduct
monitoring surveys in Italy for Xanthomonas arboricola pv. pruni (Xap) in peach and plum orchards, and including
monitoring for copper-resistance in the pathogen population. The training objective of this mission was to acquire
experience in methods for detection, diagnosis, identification and surveying of this EPPO quarantine pathogen. Xap has
not yet been detected in Serbia, and the training from this STSM will be applied to conducting surveys in Serbia to assess
the actual situation in the coming years. In addition to that, I also participated in an Integrated Pest Management
practices that are already in place in Italy.
During my stay I participated in field inspections of peach and plum orchards in collaboration with the local Extension
Service, in order to study the symptoms of bacterial spot caused by Xap on peach, nectarine and plum. Particular
attention was put on other diseases with symptoms that might be confused with those caused by Xap. Visits were done
with experienced inspectors in several orchards located in an area between Bologna, Ravenna and Rimini.
During field inspections symptomatic plant material, mainly leaves and fruits showing spots and necrotic areas, were
collected for further analysis in lab. The isolation of bacteria from symptomatic plant material was done on GYCA
(Glucose Yeast Calcium-carbonate Agar) medium and incubated for 48 – 72h. After that, we continued with purification
of the colonies and tested for hypersensitive response (HR) on bean pods. Positive HR response was visible after 1 – 2
days. Characterization of strains was also done by extracting DNA and using it as target for rep-PCR with REP, BOX and
ERIC primers. Total soluble cell proteins were also extracted, in order to identify the putative Xap colonies on the basis of
their protein fingerprints.
Copper resistance of bacterial strains was also tested on NSA (Nutrient Sucrose Agar) containing different concentrations
of copper (40, 60 and 100 ppm). We detected some copper resistant strains present in orchards.




                                                             90
91
POSTER SESSION




      92
93
FIRST REPORT ON BLIGHT ON HAZELNUT CAUSED BY XANTHOMONAS ARBORICOLA PV. CORYLINA IN
POLAND


Joanna Puławska1, Monika Kałużna1, Anna Kołodziejska2, Piotr Sobiczewski1

1
 Research Institute of Pomology and Floriculture, ul. Pomologiczna 18, Skierniewice, Poland
2
 Main Inspectorate of Plant Health and Seed Inspection, Central Laboratory, ul. Zwirki i Wigury 73, 87-100 Toruń,
Poland

In 2007, various types of disorders on the organs of about 15-year-old trees of different hazelnut cultivars grown in
central Poland were observed. The symptoms consisted of angular necrotic lesions on leaves, involucres of shells and
shells as well as dieback of twigs and branches. All diseased parts were removed. In 2009, similar symptoms were
detected again in the same orchard but only on leaves. However, in the orchard located in the vicinity, where 4-year-old
hazelnut trees of cv. Webb‟s Prize Cob were grown the necroses on shoots, bud blast and brown lesions on leaves were
observed at high intensity on almost all plants.
In both years from the margin of diseased and apparently healthy tissue, yellow colony-forming bacteria were isolated
on YPGA and King‟s B media. All selected isolates were positive in PCR with primers X1X1 specific for bacteria belonging
to Xanthomonas genus according to Maes (1993). Isolates were characterized using physiological and biochemical tests
recommended by EPPO (Anonymous, 2004). Most of tested features agreed with standard, but differed in utilization of
some carbon sources (L-arabinose, maltose, glycerol, L-xylose, D-xylose, lactose and raffinose) on minimal medium used
(Hayward, 1964). However, identical results were obtained with reference strain Xanthomonas arboricola pv. corylina
(Xac) LMG 688 isolated from Corylus avellana in USA. Tested isolates were also identified as Xac on basis of cellular
fatty acids content converted to methyl esters (FAME) using the MIDI system (Microbial Identification System, USA).
Based on gyrB gene sequence, isolates were identified as Xac. The pathogenicity of isolates was determined by
infiltration of young hazelnut leaves cv. Webb‟s Prize Cob. with water suspension of tested bacteria (10 7 cfu/ml) in a
glasshouse. Strain LMG 688 was used as a positive, whereas sterile water as a negative control. Shoots with infiltrated
leaves were covered with plastic bags for 48h. To fulfill Koch postulates, bacteria were re-isolated from plant tissue
showing disease symptoms 2 weeks after inoculation and their identity was confirmed by colony morphology and PCR
with X1X2 primers.
Analysed isolates showed BOX, ERIC and REP-PCR patterns similar to reference Xac strains (LMG 688, CFBP 1159).
However, patterns of isolates obtained in 2009 were slightly different from those isolated in 2007.
This is the first report on blight on hazelnut caused by Xanthomonas arboricola pv. corylina in Poland.


Anonymous. 2004. Bulletin OEPP/EPPO Bulletin 34, 155-157.
Maes M, 1993. FEMS Microbiology Letters 113, 161-166.
Hayward, A. C. 1964. J. Gen. Microbiol.35287-298




                                                            94
95
EMERGING BIOINFORMATICS TOOLS FOR THE SELECTION OF PHYTOPATHOGEN-SPECIFIC DIAGNOSTIC
MARKERS.


P. Albuquerque1,2, A. Rodrigues2, A.R.S. Marçal2, M.V. Mendes1 and F. Tavares1,2*,


1
 IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal.
2
 FCUP – Faculdade de Ciências, Universidade do Porto, Portugal.
                                                                             *Corresponding author: ftavares@ibmc.up.pt



     Molecular methods of bacterial detection, identification and typing are increasingly acknowledge as specific, reliable
and sensitive approaches to circumvent the specificity and resolution limitations of the traditional culture-based methods
of bacterial diagnostics. Since DNA-loci rather than organisms are detected, these techniques can be used unbiased by
the limitations of culturability. In addition, molecular methods allow to analyse a high number of samples and might
facilitate an early detection of the pathogen, namely in asymptomatic plants, which is of upmost importance for
quarantine procedures.
     The massive number of non-redundant bacterial DNA sequences currently available on databases and the
accessibility to hands-on bioinformatics tools to analyse those sequences, sustain the potential for a rapid identification
of species- and pathovar-specific molecular markers, as recently reviewed by Albuquerque et al. (2009). CUPID (Core
and Unique Protein Identification) (Mazumder et al, 2005) and Insignia (Phillippy et al, 2009) are two web-based
computational pipelines particularly resourceful to retrieve and identify numerous taxa-specific loci or DNA signatures,
i.e. unique genomic regions that might be used as a molecular markers to discriminate closely related taxonomic groups.
     This communication describes a workflow strategy, using these two bioinformatics resources, for in silico
identification of novel molecular markers for Agrobacterium tumefaciens, Pseudomonas syringae pv. syringae and Xylella
fastidiosa. In addition, a custom-made algorithm developed by our group allow to retrieve the sequences overlaps
between CUPID and Insignia databases, which has been shown to increase the prediction reliability of taxa-specific
molecular markers and to facilitate the selection of molecular markers more suitable for a particular detection technique,
e.g. PCR or hybridization profiling.


     Albuquerque P, Mendes MV, Santos CL, Moradas-Ferreira P and Tavares F. (2009) Emerging approaches of DNA-
based methods for bacterial detection and identification. Sci. Total Environ. 407: 3641-3651.

     Mazumder R, Natale DA, Murthy S, Thiagarajan R and Wu CH (2005). Computational identification of strain-,
species- and genus-specific proteins. BMC Bioinformatics 6: 279.

     Phillippy AM, Ayanbule K, Edwards NJ, Salzberg SL. (2009) Insignia: a DNA signature search web server for
diagnostic assay development. Nucleic Acids Res. 37: W229-W234.




                                                            96
97
IMPROVING SENSITIVITY AND ACCURACY OF XANTHOMONAS ARBORICOLA PV. PRUNI DETECTION BY
CONVENTIONAL PCR.


Peñalver J.1; Quesada J.M.1; Roselló M.2; Llop P. 1; Morente M.C. 1; Ferrante P. 3 ; Scortichini M. 3 ; López M. M.1

(1)
       Centro de Protección Vegetal y Biotecnología, IVIA. Moncada, Valencia, Spain.
(2)
      Área de Producción Agroalimentaria. Generalidad Valenciana. Silla, Valencia, Spain.
(3)
      Centro di Ricerca per la Frutticoltura, CRA. Roma, Italy.


The EPPO protocol for diagnosis of Xanthomonas arboricola pv. pruni (2006), the causal agent of the bacterial spot of
stone fruits, only includes isolation and immunofluorescence (using a polyclonal antiserum) for detection of the pathogen
and biochemical tests, protein and fatty acid profiles and pathogenicity tests for identification of the isolates but, it lacks
of a molecular test. A specific molecular technique would be very useful in routine analysis and for confirmation of the
identity.
Recently, a set of primers (Y17CoF and Y17CoR) and an amplification protocol for PCR detection of this pathogen have
been developed.(Pagani, 2004). Several modifications of this protocol were assayed to increase sensitivity using spiked
samples as well as healthy and infected plant material from different hosts. The sensitivity of the protocol was variable
with the different types of plant material and it was optimized by modifying the PCR mix composition and the number of
cycles of amplification. The optimized protocol reaches sensitivity of 102 cfu/ml in almond leaves but, it has a lower
sensitivity in other hosts (i.e. peach).
The specificity was evaluated against a collection of 100 strains of Xanthomonas arboricola pv. pruni from different
origins, 15 other plant pathogenic bacteria and healthy material from several hosts. The results have demonstrated the
specific detection of such pathogen.
In addition, the analyses of infected plant material (plum and almond) from different Spanish regions showed a good
correlation between bacterial isolation and the optimized PCR protocol.




EPPO Diagnostic protocols for regulated pests. Xanthomonas arboricola pv. pruni. (2006). EPPO Bulletin, 36, 129-133.

Pagani, M.C. (2004). An ABC transporter protein and molecular diagnosis of Xanthomonas arboricola pv. pruni causing
bacterial spot of stone fruits. Ph. D. Thesis. 86 pp. On- line: http://www.lib.ncsu.edu/theses/available/etd-10042004-
232356/




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99
FACTORS INFLUENCING WALNUT BLIGHT SYMPTOMS EMERGENCE AND DEVELOPMENT


A. Chevallier1, O. Bray1, JP. Prunet12, M. Giraud2
1
    Station Expérimentale de Creysse – Perrical – 46600 Creysse - France
2
    Centre Technique Interprofessionnel des Fruits & Légumes - Centre de Lanxade - 24130 La Force - France



Walnut blight, due to Xanthomonas arboricola pv juglandis (Xaj), is one of the most important disease of walnut trees
(Juglans regia). It is responsible for significant crop losses which can rich more than 50 % of nuts drops. In South
western France, management of the disease has relied upon 3 to 4 copper compounds sprays applied from bud break to
blooming.
However, this strategy is often ineffective and no other solution has yet been found to fight this disease. Moreover, there
is a lack of knowledge on this disease development. Thus, we tried to better understand the different factors which could
influence the development of walnut blight. We compared two orchards, the first one with a high sensitivity to walnut
blight, and the second orchard with a low one. The results confirm the difference of sensibility to walnut blight of the two
orchards. The climate, quite similar in the two orchards, does not seem to be the most influencing factor of the disease
development. Similarly, water stress measured would not affect the emergence and development of symptoms. Xaj
populations were higher in the sensitive orchard but do not explain the difference of walnut blight sensitivity between
orchards. Finally, soil and orchard management, seem to have a huge influence on walnut blight development. These
factors must also play a role in the production of phenolic compounds (Flavonoids and HCA) which was higher in the low
sensitive orchard. This confirms their protective role against external diseases attacks.


Keywords: Walnut blight, influencing factors, stress, epidemiology, phenolic compounds


Study presented in the 6th International Walnut Symposium - Melbourne 2009 - Australia.




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101
STUDY OF XANTHOMONAS ARBORICOLA PV. JUGLANDIS POPULATION DYNAMIC IN FRENCH WALNUT
ORCHARDS DURING THREE YEARS.


Giraud M.(1), Prunet J.P. (2) and Chevalier A. (3)

(1)
      Ctifl, Centre de Lanxade F-24130 Prigonrieux     giraud@ctifl.fr
(2)
      Ctifl, Walnut Experimental Station, Creysse, F-46600 Martel, France    jp.prunet.creysse@wanadoo.fr
(3)
      Walnut Experimental Station, Creysse, F-46600 Martel, France



Walnut blight, caused by Xanthomonas arboricola pv. juglandis is a major disease of Walnut in France, mainly
responsible for necrosis on fruits and nut early drops before harvest. The population dynamic was studied during three
years in South-West of France, with comparison between orchards with different infestation levels. Samples of buds,
leaves, flowers, pollen and fruits were collected from the beginning of April to end of August, and the epiphytic
population size of X. a. pv. juglandis was measured by plating dilutions of water extract on Tween Modified Medium.
Population size broadly varies during the year from 103 to 107 cfu/g, increasing from budbreak, and remains stable after
flowering. The level is a little lower in less infected orchards, suggesting that the expression of disease is more affected
by pedo-climatic conditions than bacterial population level.




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103
COMPLETE GENOME SEQUENCING OF THE STONE FRUIT QUARANTINE PATHOGEN XANTHOMONAS
ARBORICOLA PV. PRUNI



Joël F. Pothier1, Theo H.M. Smits1, Frank-Jörg Vorhölter2, Rafael Szczepanowski2, Alexander Goesmann2, Alfred Pühler2
and Brion Duffy1,*

1
 Division of Plant Protection, Agroscope Changins-Wädenswil ACW, Wädenswil, Switzerland.
2
 Center for Biotechnology (CeBiTec), University of Bielefeld, Germany
*brion.duffy@acw.admin.ch


Background:
Xanthomonas arboricola pv. pruni causes bacterial spot on a wide range of Prunus spp. (e.g. apricot, peach, plum), a
disease reported in almost major stone fruit producing areas of the world that results in significant economic losses.


Objectives:
Using complete genome data, to elucidate the genetic basis for different host specificity and pathogenicity of
Xanthomonas arboricola pv. pruni strain CFBP 5530.

Results:
We generated a draft genome sequence of Xanthomonas arboricola pv. pruni CFBP 5530, and are currently in the gap
closing phase. The organism is predicted to have a circular chromosome of about 5 Mb and one plasmid of about 50 kb
with an overall G+C content of 61.1%. Sequence analysis of a draft annotation of the chromosome and plasmid content
identified putative virulence genes respective of those known in other sequenced xanthomonads. X. arboricola-specific
genes were identified after comparisons with other sequenced Xanthomonas strains.


Conclusions:
Complete genome sequence of the stone fruit quarantine pathogen Xanthomonas arboricola pv. pruni may bring valuable
information about the genetic basis that explains the differing host specificities and pathogenic processes of
Xanthomonas arboricola pv. pruni. This may then offer novel insight toward the development of efficient methods for
detection and prevention of this important plant disease.




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105
EPIDEMIOLOGY OF BACTERIAL DIEBACK OF PEACH (PSEUDOMONAS MORS-PRUNORUM F. SP. PERSICAE)
IN THE CONDITIONS OF VALU LUI TRAIAN FRUIT GROWING AREA


Marioara Trandafirescu1, Mihai Botu2

1
Fruit Tree Growing Research – Extension Station Constanţa, Romania
2
Fruit Tree Growing Research – Extension Station Vâlcea, Romania

Keywords: symptoms, artificial inoculation, inoculums, Prunus persica


Abstract

The bacterial dieback of peach caused by Pseudomonas mors-prunorum f. sp. persicae is a relatively new disease of this
fruit crop (Vigouroux et Blanche, 1967) which has very important consequences over trees and orchard longevity. From
the time it was first signalized (in 1965 in Ardeche-France), this disease was not eradicated, by contrary, new locations
of appearance were emphasized. Such locations were discovered in Romania in 1990 and 1992: Băneasa, Bihor, Caraş-
Severin, Constanţa, Mehedinţi, Oradea, etc. In these areas, the numerous and different problems caused by decline and
dieback of peach were initially attributed to Pseudomonas syringae, which is spread in the peach growing countries
(especially U.S.A.), but the symptomatology of the attack is different. Under these circumstances, in all new foci where
the disease was signaled studies and rigorous research are required. The research carried out in the pedoclimatic
conditions of the Valu lui Traian, fruit growing area were focused on establishing of the attack symptoms of
Pseudomonas mors-prunorum f. sp. persicae, in a manner in which to understand the epidemiologic characteristics of the
disease and to establish the factors that influence the attack in the conditions of the study area. The responsibility of
Pseudomonas mors-prunorum f. sp. persicae in producing bacterial dieback of peach was confirmed by the symptoms
obtained in all seasons on peach trees on the basis of artificial inoculations with this bacteria. Research on the evolution
of the pathologic process in the conditions of Valu lui Traian fruit growing area emphasized the fact that peach reaction
to bacterial infections depend on various factors like cultivar, physiological status, age, infection locus, inoculums
quantity, environmental conditions (temperature, soil), human action, etc.




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107
THE RESEARCH ON BACTERIAL CANKER OF STONE FRUITS IN LATVIA – PRELIMINARY RESULTS



Inga Moročko-Bičevska, Arturs Stalažs and Edīte Kaufmane

Latvia State Institute of Fruit-Growing, Graudu str. 1, Dobele, LV-3701, Latvia, e-mail: Inga.Morocko@lvai.lv



The fruit growing in Latvia has rather old traditions, however the progressive commercial plantations have been planted
only in the last 15 years. The commercially grown stone fruit species in Latvia are plums, sweet and sour cherry. The
total orchard area of stone fruits grown commercially and in home gardens occupies 1870 ha. Peaches and apricots are
also grown, but only in home gardens or in varietal collections in research institutions in insignificant quantity. The
commercial fruit-growing is rather new sector in the national economics therefore targeted and continuous research
programs on stone fruit diseases have not been carried out.
In 2008 a research was started on the occurrence of diseases in the stone fruit orchards in order to detect the pathogens
present and to evaluate the most serious treads for orchards in Latvia. To evaluate the situation in stone fruit orchards
and collect samples for detection of pathogens 26 plum, 31 sour cherry, and 21 sweet cherry commercial orchards and
varietal collections were surveyed in 2008 and 2009. Samples were taken also from apricots and peaches either in home
gardens or varietal collections at six different geographic locations. The general condition of orchards during the surveys
was evaluated and scored by grade 1-5. In total 204 samples from trunks, branches, buds and flowers with indicative
symptoms of bacterial diseases were collected. The possible pathogenic bacteria were isolated from diseased samples by
agar plating on Pseudomonas semi-selective medium (mKB), 5 % Sucrose Nutrient Agar (SNA) and Nutrient Dextrose
Agar (NDA). Single colonies with morphology characteristic to Pseudomonas on particular medium were transfered on
SNA in pure culture. The presumptive identification of isolated bacteria was done based on colony morphology on King‟s
B (KB), SNA and NDA according to Stead (2008).
During the surveys observations indicated severe problems in Prunus orchards and more than 80 % of orchards were
scored with grade 2-3. The most often observed symptoms were different size cankers on trunks and branches, gum
exudates on these cankers, sunken areas girdling the branches and collapsed buds and young leaves in early summer on
infected braches indicating bacterial infections. In total 2058 bacteria were isolated in pure cultures and 716 gram
negative, putative Pseudomonas isolates after presumptive identification on KB, SNA and NDA have been selected for
further tests. The presumptive identification results showed the possible presence of green fluorescent pseudomonads of
group Ia, IVa, Ib, III, IVb and non-fluorecent pseudomonads. Majority of the isolates had colony morphology on KB, SNA
and NDA characteristic to green fluorescent pseudomonads of group Ib, III, IVb. The work is currently continued and
bacterial isolates further characterized by LOPAT and GATTa tests.
          In Latvia, so far, only fungal diseases have been considered of economic importance in Prunus orchards and
bacterial canker was believed to be a minor disease. The preliminary results of current research indicate that bacterial
canker, possibly caused by different pseudomonads, is wide spread in Prunus orchards and should be considered in plant
protection programs.




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109
POPULATION STUDIES OF XANTHOMONAS ARBORICOLA PV. PRUNI AND NEW STRATEGIES TO ITS
CONTROL IN PEACH ORCHARDS



Dallai D.1, N. Parkinson2, D. Giovanardi1 and E. Stefani1.

1
  Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio Emilia, via J.F. Kennedy 17, 42100
Reggio Emilia, Italy.
 2
  The Food and Environment Research Agency (FERA), Sand Hutton, York, YO41 1LZ, UK. E-mail:
emilio.stefani@unimore.it



The population structure of Xanthomonas arboricola pv. pruni (Xap), the causal agent of the bacterial canker/leaf and
fruit spots of stone fruits, has been investigated in order to achieve a deeper insight in this taxon, thus enabling an
improvement of the current diagnostic protocols and a more effective control of the disease. We confirmed the
homogeneity of a vast collection of strains (over 100), isolated in stone fruit orchards during the last decade in Romagna
(Italy). Protein profiling and rep-PCR genetic fingerprints showed no significant differences among the strains: population
homogeneity was also confirmed by phylotyping analysis. Copper resistant strains are present, but not widespread
among the whole population.
The pathogen is regulated (Directive 2000/29/EC, Annex A, Part II, Section II) and is inserted in the EPPO A2 list. An
effective control of it is based both on analysis of propagation material and use of appropriate control strategies in the
field. Based on available DNA sequences, we designed primers and attempted to developed a PCR protocol to be used in
certification schemes. We also conducted field and glasshouse trials on peach, with the aim to effectively control the
disease by using novel molecules, such as Glucohumates or antagonistic bacteria. Results are very promising and
suggest the possibility to implement effective control strategies, where copper compounds and novel molecules are both
used in commercial orchards.




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SUMMARY OF UP TO NOW 3 YEARS ACTIVITIES OF THE CZECH REPUBLIC IN THE PROJECT COST873


Hana MATOUŠKOVÁ, Lenka DOSTÁLOVÁ, Jaroslav HORKÝ

State Phytosanitary Administration, Division of Diagnostics, Slechtitelu 23, CZ-779 00 Olomouc, Czech Republic


   -   Participation on regular meetings, preparation of posters, short term training mission (York), short term scientific
       mission (Belgium – improving our knowledge of bacterial diseases of stone fruits and nuts, comparing diagnostic
       procedures and learning the new ones )
   -   Coordination of the COST´s participants in the CR
   -   Estimation of lipodepsipeptide production on agar media – tested on our collection of strains(Pss isolated from
       various hosts)
   -   In March 2008, participation the Training Workshop „Plant Bacteriology for Phytosanitary Applications“ at the
       Central Science Laboratory , York (a series of lectures and exercises - easier and more accurate identification of
       phytopathogenic bacteria and to improve our expertise – introducing new method in our lab – esculin hydrolysis
       test for comparing of isolates)
   -   training in molecular methods for Xanthomonas diagnostics (INRA Angers, September 2009)




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