persicae determined by restriction fragment analysis ... analysed using the software package Gelcompar. ... (Vigouroux, 1967) and from Hawke's Bay, New.
Plant Pathology (1996) 45, 350–357
Relationships between populations of Pseudomonas syringae pv. persicae determined by restriction fragment analysis J. M. YOUNG, D. S. JONES and M. GILLINGS* Manaaki Whenua—Landcare Research, Private Bag 92170, Auckland, New Zealand and *Biological and Chemical Research Institute, PMB 10, Rydalmere, New South Wales 2116, Australia Pseudomonas syringae pv. persicae, the pathogen causing bacterial decline of stone-fruit, was first noted almost simultaneously in France and in New Zealand, together with a closely related pathogen from myrobalan plum in England. The relative similarity of 31 strains from these three countries was examined by comparing DNA restriction endonuclease fragment patterns. Fragment patterns produced from digested DNA samples which were electrophoresed on polyacrylamide gels and visualized by silver-staining, were analysed using the software package Gelcompar. The fragment patterns produced by strains from France and England formed homogeneous but separate groups, while those from New Zealand were relatively heterogeneous. This finding suggests that the New Zealand population of P.s. persicae is older than those found in Europe. Problems in explaining the distribution of the pathogen are discussed.
INTRODUCTION Pseudomonas syringae pv. persicae (Prunier et al., 1970 is a pathogen of particular Prunus species. It was first recorded from the Rhoˆne Valley, France (Vigouroux, 1967) and from Hawke’s Bay, New Zealand in 1967 (Young, 1987a). In France, the pathogen is confined to Prunus persica (peach) and P. persica subsp. nucipersica (nectarine), and in New Zealand it has been isolated from peach, nectarine, and P. salicina (Japanese plum). Since it was first observed, the pathogen has caused serious injury to peach and nectarine trees in both the Rhoˆne Valley (Prunier et al., 1976) and in the Central Otago stone-fruit growing region in New Zealand (Young, 1987a, 1988). A related pathogen was isolated almost simultaneously in 1966 from Prunus cerasifera (myrobalan plum), in Kent, England (Garrett & Crosse, 1967). Following the classification of the pathogen (Prunier et al., 1970), its ecology and control have been extensively investigated (Luisetti et al., 1973; Prunier et al., 1974; Young, 1987a, b, 1988). The origin of the pathogen has been a matter of discussion because of its implication for quarantine. The relationship between populations of P.s. persicae from different countries has been a matter of speculation (Young, 1987a, 1988). Young (1988) reported a comparison of strains of these pathogens Accepted 31 July 1995.
from New Zealand, France, and England. DNA– DNA hybridization results indicated closer affinities between the strains from New Zealand, France, and England than to the pathotype strain of Pseudomonas syringae pv. syringae van Hall 1902. Selected biochemical tests (Young, 1987a, 1988) indicated a number of differences between the populations from different countries, but the level of interstrain variation within each geographic group was so high that these were discounted as insignificant. In attempting to explain the origin of the pathogen, Young (1987a, 1988) considered the possibility that it had evolved recently from an unknown source. More plausible was the suggestion that the pathogen had existed for a long period as a subpopulation of P. syringae in Prunus, increasing to epiphytotic proportions only when new, susceptible cultivars were introduced. Of some interest was the question as to whether the pathogen had been present as independent populations in New Zealand and Europe or had been transported between hemispheres. For completeness, the possibility was also considered that the pathogens in the northern and southern hemispheres had separate polyphyletic origins, arising by independent simultaneous mutation, but this hypothesis was discounted. At that time, 1986–88, there was no simple method by which such questions could be easily answered. Since then, simplified methods for the production and visualization of DNA restriction endonuclease fragment patterns have allowed the
Relationships between populations of P.s. persicae interpretation of recent phylogenetic relationships based on random mutational changes in the bacterial chromosome (Broadbent et al., 1992; Gillings & Fahy, 1993). In this study, we report on the relationships between the pathogens found in New Zealand, France, and England by comparing DNA restriction endonuclease fragment patterns. MATERIALS AND METHODS Strains Strains were from the International Collection of Micro-organisms from Plants (ICMP), Auckland (Table 1). Cultures were maintained on slopes of yeast-extract phosphate salts agar (YPA) containing (g/l): NH4Cl, 0.5; KCl, 0.2; MgSO4.7H2O, 0.2; K2HPO4, 1.0; yeast extract (Difco), 3.0; agar (Davis), 12; in de-ionized water. All cultures were incubated at 258C and stored at 88C. DNA extraction and digestion Bacteria were grown as lawn cultures (48 h at 258C) on King’s medium B (King et al., 1954) and DNA was extracted and digested using endonuclease Hae III (Life Technologies Inc., Gaithersburg, MD, USA) following the method of Gillings & Fahy (1993). Hae III was selected for use in restriction endonuclease analysis following a preliminary study of the use of Alv I, Cfo I, Dpn I, Hha I, Mbo I and Rsa I which suggested that patterns generated using Hae III had the greatest number of fragments of large molecular weight with a higher usable information content. Polyacrylamide gel electrophoresis Gel slabs (3 mm 2 16 cm 2 24 cm) were prepared using 5% polyacrylamide (30 : 1 acrylamide: bisacrylamide), 0.125% ammonium persulphate, and 0.04% TEMED in 40 mM Tris–acetate, 1 mM EDTA adjusted to pH 7.2 with acetic acid. Samples were electrophoresed for 18 h at 80 mA using a Hoefer SE 660 vertical slab gel apparatus. Molecular weight markers of 1 kb (Life Technologies Inc., Gaithersburg, MD, USA) and 100 bases (Pharmacia, KB, Uppsala, Sweden) were included to allow normalization of fragment patterns between gels. Silver staining Gels were fixed in freshly prepared solution comprising 50% ethanol and 10% acetic acid in a
351
Table 1 Pseudomonas syringae pv. persicae from New Zealand, France and England used in restriction analysis.
Pseudomonas syringae pv. persicae ICMP Host Location No.
Isolation date
2126 7090 7092 7099 8780 5786 3680 6766 6769 7094 7095 7097 7098 7467 8386 8390 8391 8419 8783 8784
Japanese plum Japanese plum Japanese plum peach peach nectarine nectarine nectarine nectarine nectarine nectarine nectarine nectarine nectarine nectarine nectarine nectarine nectarine nectarine nectarine
New Zealand Hawkes Bay Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra Alexandra
1967 1980 1980 1980 1984 1977 1972 1979 1979 1980 1980 1980 1980 1980 1982 1982 1982 1982 1984 1984
3980 3979 4982 4992 4997 5044 5058 5846
peach peach peach peach peach peach peach peach
France Angers Arde´che Arde´che Arde´che Arde´che Arde´che Gard Gard
1968 1969 1969 1969 1970 1969 1974 1974
3707 3708 3709
myrobalan myrobalan myrobalan
England Kent Kent Kent
1966 1966 1966
sealed perspex container for 1 h on an orbital shaker, followed by fixing in 10% ethanol and 1% acetic acid for 1 h. Gels were then treated with freshly prepared 12 mM AgNO3 for 1 h in the dark. Gels were then rinsed thoroughly in three changes of 200 ml distilled water for 10 min. Finally, gels were rinsed in a change of developer (0.74 M KOH, 0.25% formaldehyde, pre-cooled to below 108C), then re-immersed and allowed to develop for 20 min, aspirated to remove surplus developer, rinsed with de-ionized water, and bands were allowed to develop to completion.
352
J. M. Young et al.
Relationships between populations of P.s. persicae Gel scanning and analysis Developed gels were sandwiched between clear acetate sheets (roller transport cleanup film; Eastman Kodak Co., Rochester, New York) and scanned using a Mikrotek Scanmaker IIXE fitted with an adapter to capture transparent images. Images were captured to TIF files using Aldus Photostyler. Data was analysed using Gelcompar software (Applied Maths, Kortrijk, Belgium). A reconstructed normalized gel image of all strains was produced (Fig. 1). A similarity matrix based on the Pearson product–moment correlation coefficient was generated which was analysed by the Unweighted Pair Group Method using Arithmetic averages (UPGMA) and a dendrogram was produced (Fig. 2). The Neighbour Joining algorithm of Saitou & Nei (1987) was also applied to produce a diagram which indicates phylogenetic relationships (Fig. 3). RESULTS Digestion of P.s. persicae DNA with Hae III produced complex fragment patterns (Fig. 1). Hae III-generated DNA fragments were smaller than c.1900 bases, and below c.700 bases formed a confluent series or smear. Analysis therefore was restricted to the size range 700–1900 bases. A problem was encountered because the 1-kb and 100-base ladders did not align as would be expected. For example, there were regularly six bands of the 100 bases ladder between the 1018base and 1636-base bands of the 1-kb ladder, and more than 10 100-base bands between the 1636 and 2036 bands. Furthermore, between gels, adjacent fragments did not maintain the same relative positions within gels (Jones & Young, 1995, unpublished observations). Consequently, lengths of fragment sizes were estimated using only the 1kb ladder as a comparative standard. For the analysis using Gelcompar, both ladders were used as arbitrary internal markers. Inspection of the dendrogram of clusters produced by UPGMA indicated that strains from England and France formed separate homogeneous clusters with greater than 72% similarity. New Zealand strains were more heterogeneous, most clustering at 70%, and two strains sharing only 45% similarity to other New Zealand strains (Fig. 2).
353
Strains from England were an outlying group at 52%. The strains from France shared similarity with New Zealand strains at 62%. Using Neighbour Joining (Fig. 3), strains from France and England formed independent internal subsets and New Zealand strains formed two independent groups. DISCUSSION Pseudomonas syringae pv. persicae has been reported in France and New Zealand. A pathogen sharing the same distinctive colony characteristics and high level of similarity in DNA–DNA hybridization studies was also found in England. A comparison of the restriction endonuclease fragment patterns of these three pathogenic populations with those of seven other pathovars of P. syringae shows that they form a single cluster (J.M.Y., D.S.J., M.G., unpublished data), suggesting that they comprise a single taxonomic group. On the basis of these data, we believe that the strains from myrobalan plum from England should be classified in P.s. persicae. Proof that they are identical in their pathogenic interactions or that they should be distinguished as races of P.s. persicae will depend on the results of comprehensive pathogenicity tests, on peach, nectarine, and on Japanese and myrobalan plum, using strains from all countries. Restriction endonuclease fragment patterns have been used extensively to compare the relationships of closely related plant pathogens (Hartung & Civerolo, 1987; Lazo et al., 1987; Gillings & Fahy, 1993). The method described here involves analysis of the band pattern of DNA fragments greater than 700 bases. Restriction endonuclease cleavage sites are assumed to occur at random in the bacterial chromosome and therefore mutations are more likely to be detected as changes in length in these longer fragments. The longer fragments have the capacity to accumulate more mutations over time than the smaller fragments, and therefore to give a more sensitive measure of mutation rate. In this study we set out to explain the restriction endonuclease fragment patterns produced in populations of P.s. persicae from France, England and New Zealand and hence to investigate the origins of these populations. This discussion is based on our analysis of the dendrogram produced by UPGMA cluster analysis (Fig. 2) as indicating the overall
Fig. 1 Normalized gel image of Hae III restriction endonuclease cleaved DNA of strains of P. syringae pv. persicae from New Zealand, France and England.
354
J. M. Young et al.
Fig. 2 Dendrogram of endonuclease restriction patterns of strains of P. syringae pv. persicae from New Zealand (nz) and France (f), and England (uk), analysed using UPGMA. Dotted lines indicate confidence limits.
similarities and differences in the band patterns. In this work, we have compared the restriction patterns produced by a single enzyme. We would not expect the general conclusions made here to be affected by investigations with other restriction enzymes. The French strains are probably more homogeneous than is indicated by the dendrogram analysis, inspection of which suggested that the strains could be divided into three groups (Fig. 2). The apparent division is almost certainly an artefact of the different rates of migration of fragments described in Results in connection with the molecular weight ladders. Strains from France share similarities of at least 90%. The heterogeneity expressed by New Zealand strains is a reflection of differences between strains on common gels. The restriction endonuclease fragment patterns reported
in this study show that pathogens from France and England form homogeneous groups embedded within the more heterogeneous New Zealand population. The strains investigated here, from England and France, are from single host species, myrobalan plum and peach respectively, while those from New Zealand are from three plant taxa—nectarine, peach and Japanese plum. Examination of the relationship of New Zealand strains shows that these strains may be loosely grouped according to host source. However, the extensive heterogeneity of restriction endonuclease fragment patterns from nectarine strains from New Zealand contrasts with the homogeneity of the patterns from peach from France (Table 1, Fig. 2). Therefore variation in these restriction endonuclease fragment patterns
Relationships between populations of P.s. persicae
355
Fig. 3 Dendrogram of endonuclease restriction patterns of strains of P. syringae pv. persicae from New Zealand (nz) and France (f), and England (uk), analyzed using Neighbour Joining.
appears not to be due to cultivar selection. Furthermore, most strains were gathered in New Zealand and France over a period of 6–7 years in small geographical regions. From this, we conclude that these factors also do not explain the different patterns observed. The most probable explanation is that differences are due to random mutations in DNA sequences which have accumulated with time, giving expression to the relative age of each population since its separation from an ancestral line. The relative homogeneity of the strains from France indicates a recent origin, and the heterogeneity of New Zealand strains consequently indicates that they could not have been derived from the population in France. The sample of strains from England is too small for useful generalizations to be made. However, its homogeneity and greater difference from strains from other sources indicate that the English population is not the origin of the pathogen. If the
pathogen identified as P.s. persicae has a monophyletic origin then, unless an ancestral population is found elsewhere in the world, our data suggests that this pathogen had its origins in New Zealand and not in the northern hemisphere. As yet, strains have not been found in the New Zealand population which correspond to those found in England and France. If the New Zealand population is so heterogeneous that individual (clonal) populations are small, proof of this relationship between the populations may not be easily demonstrated by the discovery of strains common to the northern and southern hemispheres. Although the temporal relationship of the separate populations seems unambiguous, the synchronous appearance of the disease needs further consideration. In New Zealand, the appearance of the bacterial decline was associated with the introduction and rapid expansion in cultivation of yellow-fleshed Prunus persica cultivars selected in
356
J. M. Young et al.
the United States. In England, the disease was associated with a small number of clones of myrobalan plum but included an old selection. In France, the disease was found on a wide variety of yellow- and white-fleshed peach and nectarine cultivars. Although many of these were from the United States, several cultivars are indigenous and were in grown in France prior to the appearance of the disease (R. Saunier, INRA, Bordeaux, 1994, personal communication). The simultaneous appearance of the disease in recently introduced and in old French cultivars hints at the recency of introduction of the pathogen to France. The data presented give an indication of the recent phylogenetic relationships within a pathovar, but we doubt that these data are adequate for a rigorous phylogenetic analysis. The Neighbour Joining algorithm was developed specifically to show phylogenetic relationships (Saitou & Nei, 1987). For the purposes of the analysis it must be assumed that the strains represent a monophyletic population. In general, the algorithm produces a dendrogram (Fig. 3) which seems to be a plausible representation of the relationships between strains, if it is treated as an unrooted tree, showing the northern hemisphere strains as a subpopulation developing during the evolution of the New Zealand population. The problem in proposing New Zealand as the geographic source of ancestral P.s. persicae relates to the absence of likely ancestral hosts of the pathogen in this country, and to its transmission from that country. Prior to the colonization of New Zealand by peoples from Europe and the introduction of exotic plants in the 19th century, there were only a small number of endemic rosaceous genera in the flora, as hypothetical alternative hosts for the pathogen. Prunus spp. were introduced to southern New Zealand late last century. Past disease investigations (Pennycook et al., 1989), as well as recent systematic surveys (J. M. Young, E. H. C. McKenzie, M. J. Fletcher & J. P. Wilkie, unpublished data) have produced updates of P. syringae, distinguishable from P.S. persicae, from four endemic host species. A plausible ancestral host to P.s. persicae in New Zealand is therefore unknown. Furthermore, if New Zealand is the ancestral source of P.s. persicae, then the pathogen must have been transmitted from this country to those representing its present distribution. Apart from the exchange of small amounts of research material under quarantine, we are unaware of any large-scale dissemination of stone-fruit planting material from New Zealand to the northern hemisphere.
The possibility that P.s. persicae had its origins in an as yet unidentified country cannot be excluded although there is no record of the presence of the pathogen elsewhere. Although the United States is a source of many of the cultivars susceptible to P.s. persicae, the absence of any report of the pathogen from this country, where stone-fruit species and their pathogens are the subject of intense study, is strong evidence that the pathogen is not present there. The eventuality that the pathogen is shown to be ancestral in another country would compound the problem of explaining the simultaneous appearance of the pathogen in England, France and New Zealand. Because the populations in England and France are quite distinct from one another the assumption of a monophyletic origin in New Zealand (or elsewhere) implies separate introductions to each country.
ACKNOWLEDGEMENT Funding support through the Foundation for Research, Science and Technology Contract CO-0309 is acknowledged with thanks. REFERENCES Broadbent P, Fahy PC, Gillings MR, Bradley JK, Barnes D, 1992. Asiatic citrus canker detected in pumello orchard in Northern Australia. Plant Disease 76, 824–9. Garrett CME, Crosse, JE, 1967. Preliminary note on a bacterium associated with a disease of Prunus cerasifera. Report of the East Malling Research Station for 1966, 153–4. Gillings M, Fahy P, 1993. Genetic diversity of Pseudomonas solanacearum biovars 2 and N2 assessed using restriction endonuclease analysis of total genomic DNA. Plant Pathology 42, 744–53. Hartung JS, Civerolo EL, 1987. Genomic fingerprints of Xanthomonas campestris pv. citri from Asia, South America and Florida. Phytopathology 77, 282–5. King EO, Ward MK, Raney DE, 1954. Two simple media for the demonstration of pyocyanin and fluorescin. Journal of Laboratory and Clinical Medicine 44, 301– 7. Lazo GR, Roffey R, Gabriel DW, 1987. Pathovars of Xanthomonas campestris are distinguishable by restriction fragment-length polymorphism. International Journal of Systematic Bacteriology 37, 214–21. Luisetti J, Gardan L, Prunier JP, 1973. E´tudes sur les bacte´rioses des arbres fruitiers. VI. E´tude du pouvoir pathoge`ne de Ps. mors-prunorum f. sp. persicae. Influence de la dose d’inoculum. Annales de Phytopathologie 5, 347–53. Pennycook SR, Young JM, Fletcher MJ, 1989. Part IV. Bacterial plant pathogens. In: Pennycook SR, ed. Plant
Relationships between populations of P.s. persicae Diseases Recorded In New Zealand, Vol. 3. Auckland: Plant Diseases Division, Department of Scientific and Industrial Research, 25–83. Prunier JP, Gaignard JL, Gardan L, Luisetti J, Vigouroux A, 1976. Le de´pe´rissement bacte´rien du peˆcher dans le sud-est de la France. Aboriculture Fruitie`re 274, 21–4. Prunier JP, Luisetti J, Gardan L, 1970. E´tudes sur les bacte´rioses des arbres fruitiers. II. caracte´rization d’un Pseudomonas non-fluorescent agent d’une bacte´riose nouvelle du peˆcher. Annales de Phytopathologie 2, 181–7. Prunier, JP, Luisetti, J, Gardan L, 1974. E´tudes sur les bacte´rioses des arbres fruitiers. Essais de lutte chimique contre le de´pe´rissement bacte´rien du peˆcher en France. Phytiatrie-Phytopharmacie 23, 71–88.
357
Saitou N, Nei M, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–25. Vigouroux A, 1967. Nouvelle de´pe´rissement du peˆcher dans l’Arde`che? Phytoma 192, 34–5. Young JM, 1987a. New plant disease record in New Zealand: Pseudomonas syringae pv. persicae from nectarine, peach, and Japanese plum. New Zealand Journal of Agricultural Research 30, 235–47. Young JM, 1987b. Orchard management and bacterial diseases of stone fruit. New Zealand Journal of Experimental Agriculture 15, 257–66. Young JM, 1988. Pseudomonas syringae pv. persicae from nectarine, peach, and Japanese plum in New Zealand. Bulletin OEPP/EPPO Bulletin 18, 141–51.
