Detection of Pseudomonas Avellanae and the Bacterial Microflora of Hazelnut Affected by ‘Moria’ in Central Italy
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011_JPP383Loreti_365 25-06-2009 11:41 Pagina 365 Journal of Plant Pathology (2009), 91 (2), 365-373 Edizioni ETS Pisa, 2009 365 DETECTION OF PSEUDOMONAS 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 bacteria 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 Pseudomonas syringae. 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 plants (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).