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Journal of Pathology (2009), 91 (2), 365-373 Edizioni ETS Pisa, 2009 365

DETECTION OF AVELLANAE AND THE BACTERIAL MICROFLORA OF HAZELNUT AFFECTED BY ‘MORIA’ IN CENTRAL ITALY

S. Loreti, A. Gallelli, D. De Simone and A. Bosco

CRA-PAV, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy

SUMMARY (Campania, Sicily). About 20,000 ha are given over to this crop in the province of Viterbo (Latium). Bacterial The presence of Pseudomonas avellanae and the canker and decline, caused by Pseudomonas avellanae is bacterial flora associated with different hazelnut or- one of the main diseases of hazelnut in Italy and in gans, was monitored from 2004-2007 in two areas of Greece (Scortichini, 2002). It was first observed in north- the province of Viterbo (central Italy). Samples were ern Greece in 1976 (Psallidas and Panagopoulos, 1979), randomly selected from three orchards affected by the where, in a few years the bacterium destroyed young disease known as ‘Moria’ (dieback): symptomatic twigs plantings of the cultivar Palaz (Psallidas, 1987). Over the or branches (2004-2005 and 2007), symptomless suck- last 25 years, in the Viterbo area, this disease, locally ers (2004-2006), leaves and pollen (2006-2007). The known as ‘Moria’ or ‘bacterial dieback’, has destroyed presence of P. avellanae was examined by PCR assay hundreds of hectares (Scortichini and Tropiano, 1994). (PCRWA/WC) and also checked by isolation, enabling us The main symptoms are sudden wilting of foliage, twigs, to verify the reliability of the PCR. The incidence of P. branches, and of the whole tree in spring and summer. avellanae in symptomatic samples ranged from 9 to 38% Losses have been estimated to be about $ 1.5 million per in the areas monitored. P. avellanae was found in up to year (Scortichini, 2002). As a consequence, a national law 3% of symptomless suckers whereas pollen and leaves all was issued to partially compensate farmers whose or- tested negative. PCRWA/WC proved to be more reliable chards were seriously damaged (Scortichini, 2002). The than isolation for the detection of P. avellanae. The asso- causal agent was identified by Varvaro et al. (1990) as a ciation of with different hazelnut organs was as- member of the “true erwiniae” group, and by Scortichini sessed by isolation followed by 16S rDNA sequencing. and Tropiano (1994), and Psallidas (1987) in Greece, as The recovered isolates were closely related to bacteria the bacterium P. avellanae. Several papers showed the mainly associated with the environment, with the excep- primary role of P. avellanae as cause of the disease in the tions of Brenneria quercina and . B. Viterbo province (Scortichini and Tropiano, 1994; Scorti- quercina was rarely recovered, and only from leaf sur- chini and Lazzari, 1996; Scortichini et al., 2000). Recent- faces. On the other hand, P. syringae pv. syringae was fre- ly, the presence of several bacterial species has been re- quently isolated from buds, bark tissue and leaves. A pre- ported in the areas affected by ‘Moria’, in association liminary characterization of this P. syringae population by with the insect Anisandrus dispar (Bucini et al., 2005), but rep-PCR is reported. also with diseased hazelnut (Vuono et al., 2006). However, as reported by different authors, only P. avel- Key words: bacterial canker, dieback, rep-PCR, 16S lanae has so far been isolated from diseased plants (Scor- rDNA, Pseudomonas syringae pv. syringae tichini and Tropiano, 1994; Bosco et al., 2006; Vuono et al., 2006) and has reproduced the disease symptoms when inoculated into hazelnut trees (Scortichini and INTRODUCTION Tropiano, 1994; Scortichini and Lazzari, 1996). Control of the disease is difficult and mainly based The European hazelnut or filbert (Corylus avellana L.) on an integrated approach that includes the use of sev- is widespread throughout Europe and is grown commer- eral procedures, such as sprays with copper compounds cially in Turkey, Spain, Greece, and in several regions of in association with agronomic practices and also inocu- Italy, in the north (Piedmont), centre (Latium) and south lum destruction (Scortichini, 2002; Vuono et al., 2006). The hazelnut cultivars used in the Viterbo province, namely Tonda Gentile Romana and Nocchione, are very susceptible (Scortichini, 1998). One of the best ways to Corresponding author: S. Loreti Fax: +39.06.82070370 avoid the disease is to prevent the introduction of la- E-mail: [email protected] tently infected plants and to produce disease-free nurs- 011_JPP383Loreti_365 25-06-2009 11:41 Pagina 366

366 Hazelnut ‘Moria’ and P. avellanae in Italy Journal of Plant Pathology (2009), 91 (2), 365-373

ery plant material. Thus, diagnostic methods for detect- days. The residual suspension was filtered and cen- ing P. avellanae in symptomatic and symptomless plant trifuged for 10 min at 10,000 rpm to concentrate the material, are important. bacterial cells. The pellet was treated with DNeasy Two molecular methods that permit rapid amplifica- Plant Mini Kit (Qiagen, Germany) according to the tion of a DNA target specific to P. avellanae have been manufacturer’s instructions. developed (Scortichini and Marchesi, 2001; Loreti and Five to six suckers were collected in 2004, 2005 and Gallelli, 2002 ). The PCR method of Loreti and Gallelli 2006 from each plant and pooled to constitute a single

(2002) (hereafter indicated as PCRWA/WC) is based on sample. Small portions of the bark including the cambi- the amplification of a 350 bp fragment of the hrpW um (3-5 mm) were aseptically removed in a helicoidal gene of P. avellanae encoding a harpin protein (Loreti et progression along each sucker and were macerated in al., 2001). After being tested on bacterial colonies and saline (5 ml). The macerate was filtered and centrifuged on experimentally infected hazelnut twigs, the method for 10 min at 10,000 rpm to concentrate the bacterial was successfully applied in preliminary experiments to cells. The pellet was resuspended in 1 ml saline and 0.1 detection of P. avellanae in naturally infected twigs or ml aliquots were spread onto NSA medium as described branches and in infected symptomless suckers (Bosco et above. Buds were collected both from symptomatic and al., 2006). symptomless samples, along each twig, branch or sucker This study characterizes the bacterial isolates associ- and macerated in 5 ml saline. The macerate was filtered ated with hazelnut decline in central Italy: (i) to estimate and centrifuged, and the pellet obtained was resuspend- the current presence of P. avellanae in symptomatic ed and treated for isolation as described above. twigs and branches and in symptomless suckers, several Leaves were collected throughout 2006 and 2007. years after the severe outbreak of the disease reported About 20 leaves per sample (45 samples in all) were re- by Scortichini and Tropiano (1994); (ii) to determine if moved from each partially wilted tree. The leaves col- P. avellanae is present on or in leaves and pollen, to as- lected were green and not wilting. Each sample was sess whether the bacterium resides on the leaves or is washed by gently shaking in sterile distilled water (50- spread by pollen; (iii) to catalogue the bacterial flora 70 ml) for 1 h. Aliquots of 0.1 ml and ten-fold (10-1 and present in different organs of plants affected by ‘Moria’. 10-2) serial dilutions were spread onto the NSA medium This last aspect was considered because recently, several and incubated as described above. bacterial species other than P. avellanae, have been re- Pollen was collected from catkins in February 2006 ported in ‘Moria’-affected areas (Vuono et al., 2006). and 2007 from partially wilted or apparently healthy The final aim of this study was to verify the reliability plants (33 samples in total) and about 0.2-1.7 g were

and specificity of PCRWA/WC, for large-scale detection of washed by gently shaking in sterile distilled water (10 P. avellanae infected samples. ml) for 3 h. An aliquot (0.1 ml) and ten-fold serial dilu- tions (10-1 and 10-2) were spread onto NSA medium and incubated as above. For PCR amplification, 0.2 mg MATERIALS AND METHODS pollen was triturated in liquid nitrogen, and nucleic acids were extracted with the DNeasy Plant Mini Kit Sampling and sample preparation. Three orchards (Qiagen, Germany) according to the manufacturer’s rec- were sampled, all located in the Viterbo province. In ommendations. particular from 2004 to 2006 two orchards in Vico Ma- trino (VM) and Campo Rosano (CR1), respectively, and Detection and characterization of Pseudomonas avel- in 2007 a neighbouring orchard in Campo Rosano lanae. Detection of P. avellanae was performed either (CR2), because no disease symptom was observed in the from levan-positive colonies grown in isolation (NSA

VM and CR1 orchards in 2007. medium) or from plant tissue using PCRWA/WC. After Samples were collected from adult trees from March growth of each bacterial colony on NSA medium for 48 h to October depending on the development of symp- at 27°C, 10 µl aliquots of bacterial suspension prepared

toms. Symptomatic twigs or branches were collected in sterile double-distilled water at OD660 nm = 0.06 (cor- from plants showing partial or total wilting. One or responding to about 5x107 CFU ml-1), were used in

more symptomatic twigs or branches were collected PCRWA/WC. Strain NCPPB38 of P. avellanae was used as from each plant to constitute a single sample. Small por- positive control. Water was used as negative control. All tions of tissue (3-5 mm), including the cambium, were colonies that were PCR-positive were compared with ref- aseptically removed at the margin of spots or necrotic erence strains NCPPB38 and BPIC631T, by repetitive-se- areas and were macerated in 1-2 ml of sterile saline quence PCR (rep-PCR) (Louws et al., 1994) of bacterial (0.85% NaCl in distilled water). Aliquots (0.1 ml) of the suspensions prepared as above. suspension and of ten-fold dilutions (10-1 and 10-2) were Ten microliters of DNA from samples extracted with spread on nutrient agar (Oxoid) with 5% sucrose DNeasy Plant Mini Kit (Qiagen, Germany), of sympto- (NSA). The plates were incubated at 25-27°C for 3-4 matic twigs or branches, symptomless suckers, healthy 011_JPP383Loreti_365 25-06-2009 11:41 Pagina 367

Journal of Plant Pathology (2009), 91 (2), 365-373 Loreti et al. 367

plants as negative control, and from pollen were used Detection and characterization of Pseudomonas sy- for PCRWA/WC amplification. ringae pv. syringae. Levan-positive colonies, that were negative to the P. avellanae-specific PCR assay were 16S rDNA characterization of bacterial colonies as- characterized by applying the following tests, according sociated with hazelnut. Colonies with the more com- to Lelliot and Stead (1987): presence of oxidase, soft rot mon types of morphology were selected from the vari- activity on potato slices, presence of arginine dihydro- ous plant organs collected, and were characterized by lase, hypersensitivity reactions in tobacco leaves sequencing the 16S rDNA. The 16S rDNA was ampli- (LOPAT tests), metabolism of glucose, gelatine liquefac- fied using 6F and 1510R primers or V1.1 and V3.2 tion, aesculin and arbutin hydrolysis, nitrate reduction, primers (Van der Meer et al., 1998). PCRs were carried ice nucleation activity, fluorescence on medium B of out in a 50 µl reaction mixture containing 0.2 mM King et al. (1954). Presence of the syrB gene, which is re- µ dNTPs, 2 mM MgCl2, 0.5 M of each primer, 2.5 units quired for the synthesis of syringomycin, was tested ac- of Taq DNA polymerase (EuroClone, Italy), and 1x cording to Sorensen et al. (1998). Pathogenicity tests PCR buffer (EuroClone, Italy). Bacterial suspensions were done on lemon (Citrus limon L.) fruits and on adult

were prepared in sterile double distilled water at OD660 plants of pear (Pyrus communis L.) and lilac (Syringa vul- nm = 0.06 (corresponding to about 5x107 CFU ml-1) af- garis L.). On lemons, the test was done inoculating the ter growth on NSA medium for 48 h at 27°C. A 10 µl sterilized fruits (two fruits for each bacterial isolate) by aliquot was first denatured for 10 min at 100°C and wounding the epidermis with a sterile needle and plac- used in PCR as the template source. The PCR thermal ing about 10-20 µl of bacterial suspension (108 CFU/ml) profile consisted of an initial denaturation step (94°C, 3 on the wound. The lemons were then placed in a humid min), followed by 30 cycles at 94°C (30 sec), 55°C (1 chamber for 48 h at room temperature. Lilacs and pears min), 72°C (90 sec) and a final elongation step of 10 were inoculated by pricking the leaf lamina with a sterile min at 72°C. Five µl of the amplification products (am- needle, and placing about 10-20 µl of the suspension plicons) were analysed on 1% (w/v) agarose gels cast (107 CFU ml-1) on the wound. For each isolate, five dif- and run in TAE buffer (0.04 M Tris, 0.001 M EDTA, ferent leaves were inoculated. The inoculations were 0.02 M acetic acid), stained with ethidium bromide and made in spring, in field conditions. Pathogenic reactions photographed under UV illumination. Amplicons were were observed up to 7-10 days after inoculations. P. sy- purified using Nucleospin Extract II Kit (Macherey- ringae pv. syringae strain NCPPB3869 was used as posi- Nagel, Germany) and sequenced at Genelab-ENEA- tive control. P. avellanae NCPPB38, P. syringae pv. coryli Casaccia (S. M. Di Galeria, Italy). The sequences were NCPPB4273T and distilled water were used as negative analysed with the BLAST 2.2.18 program (Zheng et al., controls. 2000), using default parameter values, to give the per- Sixty five P. syringae isolates collected from different centage homology with known sequences in the EMBL hazelnut organs and several P. syringae strains isolated database. from other plant species (Table 1) were typed by repeti- tive-sequence PCR (rep-PCR) using ERIC and BOX

Table 1. List of Pseudomonas syringae pv. syringae isolates used in rep-PCR assay. The source, origin, year of isolation and dendro- gram group of each isolate are also shown.

Dendrogram Host Isolates and source (a) Origin and year of isolation group Corylus avellana From ISPaVe1365 to 1372 Italy-Latium-Viterbo (Campo Rosano-CR1), 2006 (A) From ISPaVe1374 to 1379 Italy-Latium-Viterbo (Vico Matrino), 2006 (A) From ISPaVe1380 to 1388 Italy-Latium-Viterbo (Campo Rosano-CR2), 2007 (C) From ISPaVe1389 to 1393 Italy-Latium-Viterbo (Vico Matrino), 2007 (C) From ISPaVe1394 to 1431 Italy-Latium-Viterbo (Campo Rosano-CR2), 2007 (C) ISPaVe14a-P6-P11 Italy-Latium-Viterbo (Capranica), 1998 (B)

DPP381; DPP77 Italy-Sardinia, --- (A) Laurus nobilis NCPPB3869 Italy-Umbria, 1992 (C) Citrus limon ISPaVe1146 Italy-Campania-Napoli, 1999 (D) Pyrus communis ISPaVe1205 Italy, 2000 (F) ISPaVe1066 Italy-Latium, 1994 (F) B301 - J. DeVay UK,--- (E) Prunus avium ISPaVe1065 --- (E) Prunus domestica ISPaVe1203 Italy-Latium, 2000 (F) Actinidia deliciosa ISPaVe010 Italy-Latium, 1991 (D) ----- B-382 USA (D) a ISPaVe = CRA, Centro di Ricerca per la Patologia Vegetale, Rome (Italy); DPP = Dipartimento per la Protezione delle Piante, University of Sassari (Italy); J. DeVay, Department of Plant Pathology, University of California, Davis, CA, USA. 011_JPP383Loreti_365 25-06-2009 11:41 Pagina 368

368 Hazelnut ‘Moria’ and P. avellanae in Italy Journal of Plant Pathology (2009), 91 (2), 365-373

primers according to Louws et al. (1994). Tests were avellanae was detected in about 38% of samples tested done in duplicate. The clearly resolved bands, present by PCR (Table 2B). Comparison of the results obtained in both amplification gels, were scored and recorded. by isolation and by PCR, shown in Table 2B, showed Similarity coefficients were determined using Dice’s co- that only 8% P. avellanae was detected by isolation, efficient (Dice, 1945). Cluster analysis was performed compared to 38% by PCR. according to the unweighed pair-group method with av- erage linkages (UPGMA) using the NTPSYSpc soft- Detection of P. avellanae on symptomless suckers, ware (version 2.11j; Exeter Software, NY) (Rohlf, leaves and pollen. The presence of P. avellanae detected 2000). A dendrogram was constructed using the tree by culturing on samples of symptomless suckers ranged display option (TREE). from 0 to 3% (Table 2C). PCR performed on samples shown by isolation to be P. avellanae-positive, always confirmed the isolation results. P. avellanae was not de- RESULTS tected by culturing in 33 pollen or 45 leaf samples test- ed, and PCR using DNA template extracted from Detection of P. avellanae on symptomatic samples. pollen samples gave negative results. The presence of P. avellanae (number of infected sam- ples divided by number of samples tested) was meas- Occurrence of bacterial colonies associated with ured for twigs or branches showing wilting (Table 2). P. hazelnut organs. Different bacterial colonies were isolat- avellanae was detected in 13% and 9% of samples in ed from wilted twigs, branches, symptomless suckers 2004 and 2005, respectively. The pathogen was detected (bark tissue and buds), leaves and pollen. All the isolates by culturing at levels as low as 2% on symptomatic tis- selected gave the expected 16S rDNA amplification sue collected up to June 2005 (data not shown). In Sep- products, and sequencing of the partial 16S rRNA gene tember 2005, P. avellanae was detected in 0 and 19% of followed by BLAST alignment with known GenBank se- 37 samples collected, by culturing and PCR respectively. quences, revealed their relationship. The maximum To confirm the culture-based result all the colonies identity percentage obtained by BLAST alignment, the grown in isolation were tested by PCR. No amplifica- accession numbers of the best matching 16S rDNA se- tion products were obtained. Moreover, during 2007 P. quences and the presumptive bacterial genera or species are shown in Table 3. Among the levan-positive isolates there were bacteria which belong to, or are closely relat- Table 2. Pseudomonas avellanae and Pseudomonas syringae pv. ed to the following genera or species: Pseudomonas trivi- syringae detection in symptomatic samples (twigs and branch- alis or poae, P. libaniensis, Brenneria quercina, es) collected from 2004 to 2005 (A), in (2007) (B), and on Roseomonas terpenica, Microbacterium and Arthrobacter. symptomless samples (suckers) from 2004 to 2006 (C). Sam- ples were collected from March to October of each year, de- The levan-negative isolates were related to the following pending on the development of symptoms for symptomatic genera or species: Aeromicrobium, Sanguibacter, Mi- samples. The number of infected samples divided by the total crobacterium, Roseomonas, Bacillus, Staphylococcus, number of samples and the corresponding percentage value Pseudomonas, Frigobacterium and Arthrobacter. (in brackets) is reported for each sampling. For P. avellanae the results of comparison among isolation and specific PCR are also reported for 2007 (B). Detection and characterization of Pseudomonas sy- (WA-WC) ringae pv. syringae. The levan-positive colonies, negative (A) Symptomatic twigs or branches in the P. avellanae-specific PCR assay and different from Pseudomonas avellanae P. syringae pv. syringae the levan positive isolates mentioned above, showed the

Isolation PCR(WA-WC) Isolation following characteristics. All isolates were fluorescent 2004 2/15 (13%) ND 2/15 (13%) on KB medium. LOPAT tests resulted in the following: 2005 8/87 (9%) ND 2/87 (2%) oxidase-negative, potato soft rot-negative, arginine dihy- drolase-negative, and tobacco hypersensitivity-positive. (B) Symptomatic twigs or branches In addition, they oxidatively metabolised glucose, hy- Pseudomonas avellanae P. syringae pv. syringae drolyzed arbutin and aesculin, and liquefied gelatine Isolation PCR Isolation (WA-WC) but did not reduce nitrates. Ice nucleation was positive 2007 5/63 (8%) 24/63 (38%) 47/63 (71%) at -5 and -10°C. They belonged to LOPAT group IA sensu Lelliot et al. (1966). Extensive necrotic reaction (C) Symptomless suckers on lemon fruits at the inoculation site and darkening of Pseudomonas avellanae P. syringae pv. syringae the leaf lamina on lilacs and pears were observed for Isolation PCR(WA-WC) Isolation 2004 2/72 (3%) ND 9/72 (12.5%) these strains. No symptoms were seen on the negative 2005 2/99 (2%) ND 7/99 (7%) control plants. All isolates gave the syrB amplicon by 2006 0/30 (0%) ND 15/30 (50%) PCR amplification. Based on these results, these bacteri- ND: not done al colonies were classified as P. syringae pv. syringae. An 011_JPP383Loreti_365 25-06-2009 11:41 Pagina 369

Journal of Plant Pathology (2009), 91 (2), 365-373 Loreti et al. 369

Table 3. List of the more abundant bacterial isolates recovered from hazelnut, their organ of isolation, the putative as- signed genera or species on the basis of 16S rDNA sequencing and BLAST alignment, their accession number and the respective identity value.

Isolate code Hazelnut Putative bacterial species or genera(a) Max identity (b) Accession No. (c) of organ matching sequence Levan positive isolates NC1 branch Roseomonas terpenica 100% AM503920.2 VM29-7g1 bud Microbacterium sp. 99% DQ985071 VM4-7p pollen Arthrobacter sp. 99% AJ785761 M8-10b sucker Microbacterium sp. 99% EF204433 M26-5g2 sucker Pseudomonas sp. 100% EF540515 CR2-FG2.1 leaf Brenneria quercina 100% EF534571 CR2-FG4.1 leaf Pseudomonas poae/ 100% AJ492829/ Pseudomonas trivialis AJ492831.1 CR2-FG10.1 leaf Pseudomonas libaniensis 100% AF057645.1 Levan negative isolates M14-15p pollen Staphylococcus sp. 100% DQ076322 VM2-6p pollen Arthrobacter sp. 100% AY370617 CR1p pollen Staphylococcus sp. 99% EU446158.1 CR3p-2 pollen Sanguibacter sp. 98% AB242791.1 CR3p-3 pollen Pseudomonas sp. 100% EU375001 VM17-9g1 bud Pseudomonas sp. 99% EU595584 VM31-16g bud Pseudomonas sp. 99% AM403728 CRF branch Frigobacterium sp. 100% AY439250 CRN branch Bacillus sp. 100% AM910175 M15-11 twig Staphylococcus sp. 100% EU925623 M25-16 twig Microbacterium sp. 100% DQ398000 VM26-7g3 sucker Aeromicrobium sp. 100% AM990810

a Bacterial species or genera assignment based on sequence similarity only; bIdentity value, calculated by BLAST analysis; c Accession number of the best matching sequence.

exception was isolate ISPaVe1376, which was ice-nucle- (similarity of 68%). Groups D, E, F included strains iso- ating and syrB negative. lated from other woody plants (similarity ranging from The presence of P. syringae pv. syringae from sympto- 41 to 60%). Rep-PCR analysis demonstrated a high ge- matic and symptomless hazelnut samples is shown in nomic variability among the isolates of P. syringae pv. sy- Table 2. On leaves, P. syringae pv. syringae was detected ringae from hazelnut: groups A, B, and C, showed a de- in 6 (13%) out of the 45 samples. gree of similarity ranging from 37 to 47% only. Isolates DNA fingerprints were obtained by rep-PCR from all from hazelnut seemed to be grouped together mainly in the P. syringae pv. syringae isolates shown in Table 1. The relation to the period of isolation: all the isolates from PCR products obtained ranged from 200 bp to 3000 bp. 2007 in group C, the isolates collected in 1998 in group A representative gel is illustrated in Fig. 1. A total of 40 B, and isolates from 2006 in group A, with the exception clearly resolved BOX and ERIC bands were used for of isolate ISPaVe1376 (which belongs to group B). No cluster analysis. Six main groups were highlighted by clear relationship between isolates and the area of isola- UPGMA analysis (Fig. 2). Isolates from hazelnut tion was observed, since isolates from Vico Matrino and grouped in three out of the six main groups of the den- Campo Rosano (CR1 orchard) grouped in both groups drogram (groups A, B, C). Group A included 15 isolates A and C (Table 1). Lastly, the organ of isolation did not (44% similarity) all isolated from hazelnut in 2006. seem to influence the clustering of isolates (Fig. 2): Group B included only four isolates (74% similarity), group A mainly included isolates from buds (of which three of which were isolated in 1998. Group C included there were 10), from bark tissue (4), and only 1 from the majority of P. syringae isolates collected from hazel- leaf; group B included isolates from bark tissue (3) and nut in 2007. The reference strain NCPPB3869 isolated only 1 from buds; group C contained isolates from bark from Laurus nobilis also belonged to the same group C (47) and leaves (4). 011_JPP383Loreti_365 25-06-2009 11:41 Pagina 370

370 Hazelnut ‘Moria’ and P. avellanae in Italy Journal of Plant Pathology (2009), 91 (2), 365-373

DISCUSSION The percentage of P. avellanae presence on sympto- matic branches or twigs varied in different years from 9- Our findings on bacterial canker and decline of 13% (2004-2005) to 38% (2007) depending also on the hazelnut in the province of Viterbo include: (i) P. avel- orchards monitored. The orchard in Campo Rosano lanae, causal agent of the disease, was present in symp- (CR2) monitored in 2007 showed more severe symp- tomatic and symptomless vegetating organs; (ii) P. avel- toms compared to the orchards in Vico Matrino and lanae was not found on leaves or pollen; (iii) PCR was Campo Rosano (CR1), monitored in 2004 and 2005. effective for monitoring presence of the bacterium; (iv) Scortichini (2002) reported that the disease has resulted a bacterial flora was associated with different organs of in the mortality of more than 40,000 trees in central hazelnut affected by ‘Moria’; (v) P. syringae pv. syringae Italy. There may have been a reduction in the presence was recovered from diseased hazelnut plants and char- of this pathogen in these areas, as showed by the cur- acterized. rent monitoring of the bacterium, compared with the We proved the presence of P. avellanae in symptom- situation of about 10 years ago. This situation might be less suckers. The large-scale dissemination of P. avel- the result of timely management of the disease in these lanae through the distribution of latently infected suck- areas in recent years. Failure to isolate P. avellanae from ers, used for propagation, was previously deduced on leaves and pollen suggests that it does not reside in the the basis of the spread of P. avellanae from the northern phyllosphere and is not spread by pollen.

to southern orchards in the Latium region (Scortichini Our PCRWA/WC assay was found to be useful for mon- and Tropiano, 1994). We have now confirmed the possi- itoring P. avellanae. The availability of an efficient diag- bility of latent contamination of suckers. nostic method is the basis for an adequate monitoring system and the control of disease, especially for a case like ‘Moria’, that cannot be controlled directly but most- ly through preventive measures. Two main aspects high- lighted the efficacy of this method: (i) if applied on pure bacterial cultures, it enabled us to distinguish P. avel- lanae colonies from others with a levan-positive mor- phology on the NSA medium used for the isolation; (ii) if applied directly to infected tissue, it permits rapid de- tection of the bacterium. Comparison of the results ob- tained by our PCR and by culturing showed that PCR- positive samples were frequently isolation-negative. In a previous study, Loreti and Gallelli (2002) showed that false-negatives are obtained with both techniques. From naturally infected hazelnut plants, PCR was more effec- tive than isolation for the detection of P. avellanae. This may depend on the presence of P. syringae pv. syringae – frequently recovered from diseased tissue – that even if present as a secondary colonizer in a naturally diseased tissue, could interfere or compete in planta or in the nu- trient medium with the growth of the causal agent. Finally, this study showed that the majority of the re- covered levan-positive or levan-negative isolates, from several hazelnut organs, appeared to be closely related to some bacteria present in the environment, water, soil or vegetation (Dabboussi et al., 1999; Behrendt et al., 2003; Jiang et al., 2006; Rijavec et al. 2007; Stevens et al., 2007; Coorevits et al., 2008), which do not play a Fig. 1. Agarose gel electrophoresis of ERIC (a) and BOX (b) major role as pathogens. An exception is B. quercina, fingerprinting patterns from genomic DNA of Pseudomonas syringae pv. syringae isolates, showing some of the different the cause of bark canker and drippy nut in Quercus spp. profiles obtained. Lane 1: ISPaVe1365; lane 2: ISPaVe1368; in Spain (Biosca et al., 2003) and of drippy nut disease lane 3: ISPaVe1370; lane 4: ISPaVe1371; lane 5: ISPaVe1373; in live oaks in California (Hildebrand and Schroth, lane 6: ISPaVe1376; lane 7: ISPaVe1377; lane 8: ISPaVe1378; 1967). This bacterium has been obtained from inside lane 9: ISPaVe1379; lane 10: ISPaVe1386; lane 11: IS- and outside the Anysandrus dispar (Scolytidae) collected PaVe1392; lane 12: ISPaVe1411; lane 13: ISPaVe1432; lane in the Viterbo areas affected by ‘Moria’ (Bucini et al., 14: DPP381; lane 15: DPP77; lane 16: ISPaVeP6; lane 17: IS- PaVeP11; lane 18: ISPaVeP14a. M: molecular marker Gene 2005). We stress that only one isolate of this species was RulerTM 100 bp DNA ladder plus (Fermentas, Lithuania). isolated during our study and, among the different ma- 011_JPP383Loreti_365 25-06-2009 11:42 Pagina 371

Journal of Plant Pathology (2009), 91 (2), 365-373 Loreti et al. 371 strains isolated from other plant species reported in s coefficient. The isolate number is followed by the following P. syringae isolates from hazelnut and other P. syringae pv. pv. Pseudomonas syringae Dendrogram of genetic similarity 65 Table 1. The similarity is the result of combined data sets ERIC and BOX primer using UPGMA Dice ’ Table isolates without any code were isolated from bark tissue (twig or branch). The scale at the bottom indi- codes according to the isolation plant organ : -l, leaf; -b, bud. The ISPaVe cates the degree of genetic similarity among isolates. Fig. 2. 011_JPP383Loreti_365 25-06-2009 11:42 Pagina 372

372 Hazelnut ‘Moria’ and P. avellanae in Italy Journal of Plant Pathology (2009), 91 (2), 365-373

trices or organs monitored, it was recovered only from Pseudomonas trivialis sp. nov., Pseudomonas poae sp. nov. leaf surfaces. It is possible that this bacterium is resident and Pseudomonas congelans sp. nov. International Journal in the area, though the part it may play is not clear. of Systematic and Evolutionary Microbiology 53: 1461-1469. There was quite a high incidence of P. syringae pv. sy- Biosca E.G., González R., López-López M., Soria S., Montón ringae, in all organs tested. This bacterium has been C., Pérez-Laorga E., López M.M., 2003. Isolation and previously reported form hazelnut-growing areas of characterization of Brenneria quercina causal agent for bark canker and drippy nut of Quercus spp. in Spain. Phy- Latium, Sardinia, Campania and Sicily (Scortichini et topathology 93: 485-492. al., 2002; Siscaro et al., 2006; Fiori et al., 2006) causing Bosco A., Gallelli A., Loreti S., 2006. Diagnosi e monitoraggio twig or branch dieback, but rarely the complete and di Pseudomonas avellanae agente della ‘moria’ del nocciolo sudden death of the tree. Thus, it is much less virulent nel Viterbese. Petria 16: 103-114. than P. avellanae. However, up to now, P. syringae pv. Bucini D., Balestra G.M., Pucci C., Paparatti B., Speranza S., syringae, has never been reported in the Viterbo area af- Proietti-Zolla C., Varvaro L., 2005. Bio-ethology of Anisan- fected by ‘Moria’ (Scortichini et al., 2002). Its associa- drus dispar F. and its possible involvement in dieback (Mo- tion with leaves and buds suggests an epiphytic lifestyle ria) diseases of hazelnut (Corylus avellana L.) plants in in this area, even though the bacterium was also found Central Italy. Acta Horticolturae 686: 435-443. to be endophytic within symptomatic twigs and symp- Cirvilleri G., Bonaccorsi A., Scuderi G., Scortichini M., 2005. tomless suckers. Potential biological control activity and genetic diversity of On the other hand, an endophytic and potentially Pseudomonas syringae pv. syringae strains. Journal of Phy- pathogenic strain of P. syringae pv. syringae was recently topathology 153: 564-666. isolated from a healthy wild Corylus avellana tree in the Coorevits A., De Jonghe V., Vandroemme J., Reekmans R., Frosinone province, Latium (Scortichini and Loreti, Heyrman J.,Messens W., De Vos P., Heyndrickx M., 2008. 2007). The rep-PCR that we used to type this bacterial Comparative analysis of the diversity of aerobic spore-form- ing bacteria in raw milk from organic and conventional population, is able to characterize bacterial species at dairy farms. Systematic and Applied Microbiology 31: 126- the strain level (Louws et al., 1994) and has already 140. been used to type P. syringae pv. syringae strains (Little Dabboussi F., Hamze M., Elomari M., Verhille S., Baida N., et al., 1998; Scortichini et al., 2003; Cirvilleri et al., Izard D., Leclerc H., 1999. Pseudomonas libanensis sp. nov., 2005; Natalini et al., 2006; Vicente and Roberts, 2007). a new specie isolated from Lebanese spring waters. Interna- As previously shown by Scortichini et al. (2003), diversi- tional Journal of Systematic Bacteriology 49: 1091-1101. ty was observed among isolates from the same host Dice L.R., 1945. Measures of the amount of the ecological as- (hazelnut) as well as among isolates from the same area. sociation between species. Ecology 26: 297-302. There may also be a correlation among isolates from the Fiori M., Loru L., Marras P.M., Virdis S., 2006. Le principali same year of isolation. P. syringae pv. syringae isolates avversità del nocciolo in Sardegna. Petria 16: 71-88. were more frequently recovered in 2007 than in previ- Hildebrand D.C., Schroth M.N., 1967. A new species of Er- ous years, and all have an identical fingerprint profile. winia causing the drippy nut disease of live oaks. Phy- This group contains isolates from bark and from leaves, topathology 57: 384-397. confirming that the same bacterial population is present Jiang C.Y., Wang B.J., Zhou Y.G., Liu S.J., 2006. Roseomonas as endophyte and epiphyte. lacus sp. nov., isolated from fresh water like sediment. In- We did find a higher incidence of this potential ternational Journal of Systematic and Evolutionary Microbi- pathogen in the areas affected by ‘Moria’, compared to ology 56: 25-28. some years ago. At the moment, we cannot assume that King E.O., Raney M.K., Ward D.E., 1954. Two simple media this P. syringae population is pathogenic. Its potential for the demonstration of pyocianin and fluorescin. Journal of Laboratory and Clinical Medicine 44: 301-307. pathogenicity and possible interactions with P. avellanae on C. avellana, will be the aim of further studies. Lelliot R.A., Billing E., Hayward A.C., 1966. A determinative scheme for the fluorescent plant pathogenic Pseudomonas. Journal of Applied Bacteriology 29: 471-489. Lelliot R.A., Stead D.E., 1987. Methods in Plant Pathology, ACKNOWLEDGEMENTS Vol. 2. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific Publications, Oxford, UK. The authors would like to thank M. Galli for his help Little E.L., Bostock R.M., Kirkpatrick B.C., 1998. Genetic in monitoring. This research was funded by the characterization of Pseudomonas syringae pv. syringae CO.RI.BIO project “Ricerche sul nocciolo finalizzate al- strains from stone fruits in California. Applied and Envi- l’ottenimento di produzioni biologiche di qualità”. ronmental Microbiology 64: 3818-23. Loreti S., Sarrocco S., Gallelli A., 2001. Identification of hrp genes, encoding harpin, in Pseudomonas avellanae (Psalli- REFERENCES das) Janse et al. Journal of Phytopathology 149: 219-226. Loreti S., Gallelli A., 2002. Rapid and specific detection of Behrendt U., Ulrich A., Schumann P., 2003. Fluorescent virulent Pseudomonas avellanae strains by PCR amplifica- pseudomonads associated with the phyllosphere of grasses; tion. European Journal of Plant Pathology 108: 237-244. 011_JPP383Loreti_365 25-06-2009 11:42 Pagina 373

Journal of Plant Pathology (2009), 91 (2), 365-373 Loreti et al. 373

Louws F.J., Fullbright D.W., Stephens C.T., de Bruijn F.J., groups: Pseudomonas avellanae and strains resembling P. 1994. Specific genomic fingerprints of phytopathogenic syringae pv. syringae. Applied and Environmental Microbi- Xanthomonas and Pseudomonas pathovars and strains gen- ology 68: 476-484. erated with repetitive sequences and PCR. Applied Envi- Scortichini M., 2002. Bacterial canker and decline of Euro- ronmental Microbiology 60: 2286-2295. pean hazelnut. Plant Disease 86: 704-709. Natalini E., Rossi M.P., Barionovi D., Scortichini M., 2006. Scortichini M., Marchesi U., Dettori M.T., Rossi M.P., 2003. Genetic and pathogenic diversity of Pseudomonas syringae Genetic diversity, presence of the syrB gene, host prefer- pv. syringae isolates associated with bud necrosis and leaf ence and virulence of Pseudomonas syringae pv. syringae spot of pear in a single orchard. Journal of Plant Pathology strains from woody and herbaceous host plants. Plant 88: 219-223. Pathology 52: 277-286. Psallidas P.G., Panagopoulos C.G., 1979. A bacterial canker of Scortichini M., Loreti S., 2007. Occurrence of an endophytic, filbert in Greece. Phytopathologische Zeitschrift 94: 103-111. potentially pathogenic strains of Pseudomonas syringae in Psallidas P.G., 1987. The problem of bacterial canker of symptomless wild trees of Corylus avellana L. Journal of hazelnut in Greece caused by Pseudomonas syringae pv. Plant Pathology 89: 431-434. avellanae. Bulletin OEPP/EPPO Bulletin 17: 257-261. Siscaro G., Longo S., Catara V., Cirvilleri G., 2006. Le princi- Rijavec T., Lapanje A., Dermastia M., Rupnik M., 2007. Isola- pali avversità del nocciolo in Sicilia. Petria 16: 59-70. tion of bacterial endophytes from germinated maize ker- nels. Canadian Journal of Microbiology 53: 802-808. Sorensen K.N., Kim K.H., Takemoto J.Y., 1998. PCR detection of cyclic lipodepsinonapeptide-producing Pseudomonas sy- Rohlf F.J., 2000. NTSYS-PC, Numerical and Mul- ringae pv. syringae and similarity of strains. Applied and En- tivariate Analysis System, Version 2.11j. Exeter Software, vironmental Microbiology 64: 226-230. Setauket, NY, USA, Scortichini M., Tropiano F.G., 1994. Severe outbreak of Stevens H., Brinkhoff T., Rink B., Vollmers J., Simon M., Pseudomonas syringae pv. avellanae on hazelnut in Italy. 2007. Diversity and abundance of Gram positive bacteria Journal of Phytopathology 140: 65-70. in a tidal flat ecosystem. Environmental Microbiology 9: 1810-1822. Scortichini M., Lazzari M., 1996. Systemic migration of Pseudomonas syringae pv. avellanae in twigs and young Van der Meer J.R., Werlen C., Nishino S.F., Spain J.C., 1998. trees of hazelnut and symptom development. Journal of Evolution of a pathway for chlorobenzene metabolism Phytopathology 144: 215-219. leads to natural attenuation in contaminated groundwater. Applied and Environmental Microbiology 64: 4185-4193. Scortichini M., 1998. Response to Corylus avellana germplasm to artificial inoculation with Pseudomonas avellanae (Psalli- Varvaro L., Costa A., Balestra G.M., 1990. A new bacterial das) Janse et al. Agricoltura Mediterranea 128: 153-156. disease of Corylus avellana. Proceedings of the 8th Congress Scortichini M., Marchesi U., Angelucci L., Rossi M.P., Dettori of the Mediterranean Phytopathological Union, Agadir M.T., 2000. Occurrence of Pseudomonas avellanae (Psalli- 1990: 469-471. das) Janse et al. and related pseudomonas on wild Corylus Vicente J.G., Roberts S.J., 2007. Discrimination of Pseudomonas avellana trees and genetic relationships with strains isolat- syringae isolates from sweet and wilt cherry using rep-PCR. ed from cultivated hazelnuts. Journal of Phytopathology European Journal of Plant Pathology 117: 383-392. 148: 523-532. Vuono G., Balestra G.M., Varvaro L., 2006. Control of Scortichini M., Marchesi U., 2001. Sensitive and specific de- dieback (“Moria”) of Corylus avellana in central Italy us- tection of Pseudomonas avellanae using primers based on ing copper compounds. Journal of Plant Pathology 88: 16S rRNA gene sequences. Journal of Phytopathology 149: 215-218. 527-532. Zheng Z., Schwartz S., Wagner L., Miller W., 2000. A greedy Scortichini M., Marchesi U., Rossi M.P., Di Prospero P., 2002. algorithm for aligning DNA sequences. Journal of Compu- Bacteria associated with hazelnut decline are of two tational Biology 7: 203-14.

Received September 19, 2008 Accepted December 17, 2008 011_JPP383Loreti_365 25-06-2009 11:42 Pagina 374