IDENTIFICATION AND CHARACTERIZATION OF ... - SIPaV

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Journal of Plant Pathology (2010), 92 (3), 803-806

Edizioni ETS Pisa, 2010

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SHORT COMMUNICATION IDENTIFICATION AND CHARACTERIZATION OF XANTHOMONAS ARBORICOLA pv. CORYLINA CAUSING BACTERIAL BLIGHT OF HAZELNUT: A NEW DISEASE IN POLAND J. Pulawska1, M. Kaluzna1, A. Kolodziejska2 and P. Sobiczewski1 1Research

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

SUMMARY

In the years 2007 and 2009 leaf spots and cankers were observed on different hazelnut cultivars grown in one orchard in central Poland. In both years yellow colony-forming bacteria were isolated from the margin of diseased and apparently healthy tissue. Fifteen isolates tested positive in a PCR assay using primers X1 and X2 specific for bacteria belonging to the genus Xanthomonas. Results of most of the physiological and biochemical features determined in our study agreed with those given in the EPPO standard PM 7/22, but some of them (utilization of L-arabinose, maltose, glycerol, D-xylose, lactose and raffinose) differed. However, they were identical with those of the reference strain Xanthomonas arboricola pv. corylina (Xac) LMG 688. The 15 isolates tested were also identified as Xac on the basis of cellular fatty acid content converted to methyl esters (FAME) as well as in gyrB gene sequence analysis. All isolates showed pathogenic ability on young leaves of potted hazelnut plants cv. Webb’s Prize Cob in a greenhouse test and could be re-isolated from symptomatic tissues. In rep-PCR (BOX, ERIC and REP-PCR) all isolates showed patterns very similar to reference Xac strains (LMG 688, CFBP 1159). Peculiarly, a slight difference in the rep-PCR patterns of isolates recovered in 2007 and 2009 was found. This is the first time that Xac and hazelnut blight are found in and reported from Poland. Key words: BOX, ERIC, FAME, gyrB, hazelnut, pathogenicity test, REP, Xanthomonas arboricola pv. corylina.

In Poland, hazelnut (Corylus avellana) has been traditionally grown by amateur growers in home gardens and small orchards, but recently its commercial production has become more profitable. Infectious diseases are

Corresponding author: J. Pulawska Fax: +48.46 8345375 E-mail: [email protected]

one of the important limiting factors of hazelnut production. A study of Machowicz-Stefaniak and Zalewska (2000) revealed that in Poland hazelnut is attacked by several fungal pathogens such as Monilia spp., Botrytis cinerea, Phyllactinia corylea, Cryptosporiopsis coryli and Cladosporium avellanum. Hazelnut production is more economically significant in countries with a warmer climate where bacterial diseases, mainly bacterial blight induced by Xanthomonas arboricola pv. corylina (Xac), cause severe crop losses (Anonymous, 1986). Bacterial blight appears to be especially dangerous for younger plants, with a mortality rate of up to 10% (Anonymous, 1986, 2004). However, this disease is not widespread in Europe and therefore Xac is classified as a quarantine organism (EPPO List A2). Several years ago a broad and comprehensive monitoring of Polish hazelnut plantations was performed but Xac was not found (Król et al., 2004). Our survey performed in 2007 on hazelnut plantations in central Poland resulted in the detection of a particular disorder on trees of different cultivars grown in one orchard. Angular necrotic lesions were present on the leaves and involucres of shells, accompanied by necroses and cankers on the shoots of the majority of 15-year-old trees. In 2009, similar symptoms on leaves were observed in the same orchard. In a young orchard located in the vicinity, 4-year-old hazelnuts of cv. Webb’s Prize Cob showed shoot necrosis, bud dieback and brown necrotic lesions on the leaves of almost 100% of the plants within 1 ha area. The aim of our study was to determine the etiology of the observed symptoms. From segments of symptomatic tissue of cvs Cosford, Webb’s Prize Cob and Early Long Zeller, small (2-3 mm of diameter), yellow, shiny and rounded bacterial colonies with regular edges were observed after 3 days of growth on YNA medium (Shaad et al., 2001). On King B medium the bacteria did not produce the fluorescent pigment. Twenty colonies were purified and the isolates obtained were tested by PCR using primers X1 and X2 specific for the genus Xanthomonas (Maes, 1993). Fifteen isolates, from which DNA amplification resulted in the expected 480 bp DNA fragment, were subjected to physiological and biochemical tests recommended by the EPPO standard for diagnosis of Xac (Anonymous,


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Characterization of Polish X. a. pv. corylina isolates


Table 1. Phenotypic characteristics of bacteria isolated from hazelnuts with blight symptoms. Lp. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Test

According to EPPO standard* + + + + + + + + + + + + + +

Reference strain LMG 688 + + + + + + + + + + + + + +

Tested Xanthomonas isolates** + + + + + + + + + + + + + +

L-arabinose D-arabinose Glucose Galactose Mannose Sucrose Maltose Trehalose Cellobiose Glycerol L-xylose D-xylose Rhamnose Lactose Raffinose Adonitol Mannitol Inuline Sorbitol Dulcitol Erythritol Oxidase Eskulin hydrolysis Starch hydrolysis Growth at 35°C Tobacco hypersensitivity 27. Growth on SQ + + + medium * Anonymous, (2004) ** Fifteen isolates from hazelnuts classified to genus Xanthomonas on the basis of PCR with primers X1 and X2 (Maes, 1993)

2004) and described by Lelliott and Stead (1987) and Lee et al. (1992). Most of the tested features of all isolates corresponded to the standard. However, using the minimal medium (Hugh and Leifson, 1953), the utilization of some carbon compounds such as L-arabinose, maltose, glycerol, D-xylose, lactose and raffinose was found to differ from those reported in literature (Anonymous, 2004). Although these features were not in agreement with the EPPO standard, they were congruent with those of the reference strain Xac LMG 688, isolated from C. avellana in the USA (Table 1). Tested isolates were also characterized by gas chromatography (GC) using the MIDI system (Microbial Identification System, USA) on the basis of cellular fatty acids converted to methyl esters (FAME), according to the standard and recommended procedure (Operating manual ver. 4.5, MIDI, USA). Afterwards, FAME were analysed by Sherlock MIS software using the aerobe method and compared with TSBA library version 5.0. It was found that the similarity index of tested isolates to Xac ranged from 0.295 to 0.423 and it was the most probable match. Although, X. arboricola consists of many

pathovars, as a species is very homogenous in terms of fatty acid composition. The diversity of fatty acid content of X. arboricola is comparable to that of single-host species, such as X. albilineans (Vauterin et al., 1996). The isolates were subsequently further identified on the basis of the sequence of the gyrB gene fragment. Their DNA was amplified with primers XgyrPCR2F and Xgyrrsp1 (Parkinson et al., 2007) and sequences were compared to those available in GeneBank using the BLAST N program. The gyrB sequences were 100% similar to the sequences of Xac strains ICMP 5726 and NCPPB 935 (accession Nos EU499002.1, EU 258235.1). Parkinson et al. (2007, 2009) and Young et al. (2008) showed that the gyrB gene phylogeny of bacteria belonging to the genus Xanthomonas has greater resolution than those based on 16S rDNA or 16S-23S rDNA. In the genus Xanthomonas, the gyrB locus also found to be discriminating at the species but not always at the pathovar, which can indicate a more rapid host specialization than housekeeping gene evolution (Parkinson et al., 2009). We compared all that was available in GenBank for gyrB gene sequences of different pathovars of X. arboricola, finding that the similarity


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ranged from 95.7% to 100%. Only gyrB gene sequences of bacteria belonging to the pv. corylina were identical to those of our isolates. The fifteen isolates were included in a routine test with primers Y17CoF and Y17CoR (Pagani, 2004) developed for identification of Xanthomonas arboricola pv. pruni (Xap) This test is used in our laboratory for all isolates with “Xanthomonas-like” colony morphology that are recovered from stone fruit trees. All Xac isolates, including the reference strain LMG 688, produced a single band with characteristic length of 943 bp, a size identical to that of Xap strain PD 740. Primers Y17CoF and Y17CoR were originally tested for their specificity with 167 non Xap strains. In all reactions no amplification products were observed by Pagani (2004) who, however, did not include Xac isolates in her tests. On the other hand, it is known that pvs corylina, juglandis and pruni classified as X. arboricola, form a highly related group and may have a common ancestor (Vauterin and Swings, 1997). The pathogenicity of isolates was analyzed by infiltration of bacteria into leaves, using a 107 CFU/ml cell suspension of a 2-day-old culture grown on YNA medium. Very young hazelnut leaves of 1-year-old potted trees of cv. Webb’s Prize Cob. grown in a glasshouse were used. Strain LMG 688 was used as a positive control and sterile water as a negative control. Shoots with infiltrated leaves were covered with plastic bags for 48 h. After 20 days, brown, necrotic spots of 3-5 mm in size were observed at the places in which leaf infiltration with the tested isolates and strain LMG 688 had taken place. No symptoms occurred on leaves infiltrated with water. To fulfill the Koch’s postulates, bacteria were re-isolated from symptomatic plant tissues and their identity confirmed as Xanthomonas, based on colony morphology and PCR amplification with X1 and X2 primers. This way of testing for pathogenicity was found most appropriate for our use. The test recommended by EPPO (Anonymous, 2004) based on inoculation of buds of hazelnut trees growing in the field from October to June, proved not to be efficient under Polish climatic conditions. The buds and shoots were inoculated twice in the field, once in October 2007 and a second time in May 2008, but these two tests were unsuccessful. The temperatures in autumn were too low for disease development. The same type of weather conditions prevailed in the spring of 2008 (dry and not very warm). In Poland, from October to June and particularly during winter, the weather is too cold to obtain results from the recommended field pathogenicity test. Three methods of repetitive PCR were used for analysis of our Xac isolates and reference strains LMG 688 and CFBP 1159. Fingerprints were obtained using BOX, ERIC and REP primers according to Louws et al. (1994) and Versalovic et al. (1991, 1994), respectively. Products obtained were separated in 1.5% agarose gel and stained

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with ethidium bromide. Each obtained band was scored as present (1) or absent (0) for all isolates. A dendrogram with a distance matrix was constructed using the Jaccard’s coefficient and UPGMA algorithm. Rep-PCR allowed differentiation of the studied isolates. A higher variability was shown by REP- and BOXPCR than ERIC-PCR. With ERIC- and BOX-PCR small differences in patterns were found between isolates from 2007 and those from 2009. These differences resulted in different clusters of these isolates (Fig. 1). Only when using REP-PCR these isolates clustered independently from the year of isolation. No relationship was found between amplification pattern and the plant organ from which bacteria were isolated (data not shown). The tree constructed with concatenated data from all three repetitive analyses showed that the isolates studied differed slightly from each other. Two groups of isolates were shown to be identical with all performed methods. One group consisted of isolates: RIPF X09, RIPF X11, RIPF X13 and RIPF X15 and the other of RIPF X23, RIPF X25 and RIPF X26 (Fig. 1). Genetic and phenotypic characterization done by Scortichini et al. (2002) showed that the population of Xac was quite homogenous and that there was no relationship to geographic origin. However, slight differences in Rep-PCR patterns of isolated bacteria were observed. In our study, as well as in that by Scortichini et al. (2002), the type strain of the pathovar corylina CFBP 1159 (=NCPPB 935), which originates from Corylus maxima, produced the most distinct fingerprint patterns. This strain differed also because of the atypical negative gelatin liquefaction and was not virulent to C. avellana plants. Because of these discrepancies, the question of the validity of its type strain status for X. arboricola pv. corylina arose (Scortichini et al., 2002).

Fig. 1. Dendrogram of genetic similarity of Xanthomonas arboricola pv. corylina isolates constructed on the basis of combined data set of ERIC-, REP- and BOX-PCR primers using UPGMA analysis and Jaccard’s coefficient. Year of isolation was indicated next to the isolate names.


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Characterization of Polish X. a. pv. corylina isolates

Our results allowed identification and characterization of Xac as the causal agent of bacterial blight of hazelnt for the first time in Poland. ACKNOWLEDGEMENTS

The authors wish to thank M. Scortichini (Istituto Sperimentale per la Frutticoltura, Rome, Italy) for supplying the strain of Xac and for his advices and J.D. Janse (Dutch General Inspection Service, The Netherlands) for his advice concerning pathogenicity tests and for the critical analysis of our results. This work was conducted within the framework of COST Action 873 and it was supported by Polish Ministry of Science and Higher Education Grant 118/N-COST/2008/0. REFERENCES Anonymous, 1986. Data sheet on quarantine organisms, 134: Xanthomonas campestris pv. corylina (Miller et al. 1940) Dye 1978. Bulletin OEPP/EPPO Bulletin 16: 13-16. Anonymous, 2004. EPPO Diagnostic protocols for regulated pests - PM 7/22, Xanthomonas arboricola pv. corylina. Bulletin OEPP/EPPO Bulletin 34: 155-157. Hugh R., Leifson E., 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. Journal of Bacteriology 66: 24-26. Król E., Machowicz-Stefaniak Z., Zalewska E., 2004. Bakterie uszkadzajace owoce leszczyny (Corylus avellana L.) uprawianej w poludniowo-wschodniej Polsce. Acta Scientiarum Polonorum, Hortorum Cultus 3: 75-84. Lee Y.A., Hildebrand D.C., Schroth M.N., 1992. Use of quinate metabolism as a phenotypic property to identify members of Xanthomonas campestris DNA homology group 6. Phytopathology 82: 971-973. Lelliott R.A., Stead D.E., 1987. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific Publications, Oxford, UK. Louws F.J., Fulbright D.W., Stephens C.T., de Bruijn F.J., 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains gen-

Received April 15, 2010 Accepted May 9, 2010

Journal of Plant Pathology (2010), 92 (3), 803-806 erated with repetitive sequences and PCR. Applied and Environmental Microbiology 60: 2286-2295. Machowicz-Stefaniak Z., Zalewska E., 2000. Grzyby wystepujace na nadziemnych organach leszczyny (Corylus L.). In: Lisiewska H., Lawrynowicz M. (eds). Monitoring Grzybów, pp. 153-166. Sekcja Mikologiczna PTB, PoznanLódz, Poland. Maes M., 1993. Fast classification of plant-associated bacteria in the Xanthomonas genus. FEMS Microbiology Letters 113: 161-166. Parkinson N., Aritua V., Heeney J., Cowie C., Bew J., Stead D., 2007. Phylogenetic analysis of Xanthomonas species by comparison of partial gyrase B gene sequences. International .Journal of Systematic and Evolutionary Microbiology 57: 2881-2887 Parkinson N., Cowie C., Heeney J., Stead D., 2009. Phylogenetic structure of Xanthomonas determined by comparison of gyrB sequences. International .Journal of Systematic and Evolutionary Microbiology 59: 264-274. Pagani M.C., 2004. An ABC transporter protein and molecular diagnoses of Xanthomonas arboricola pv. pruni causing bacterial spot of stone fruits. Ph.D. Thesis, North Carolina State University, Raleigh, NC, USA. Schaad N.W., Jones J.B., Chun W., 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd Ed. APS Press, St, Paul, MN, USA. Vauterin L., Swings J., 1997. Are classification and phytopathological diversity compatible in Xanthomonas? Journal of Industrial Microbiology and Biotechnology 19: 77-82. Vauterin L., Yang P., Swings J., 1996. Utilization of fatty acid methyl esters for the differentiation of new Xanthomonas species. International Journal of Systematic Bacteriology 46: 298-304. Versalovic J., Koeuth T., Lupski J.R., 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research 19: 6823-6831. Versalovic J., Schneider M., De Bruijn F.J., Lupski J.R., 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular and Cellular Biology 5: 25-40. Young J.M., Park D.C., Shearman H.M., Fargier E., 2008. A multilocus sequence analysis of the genus Xanthomonas. Systematic and Applied Microbiology 31: 366-377.