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Phylogenetic analysis of Puccinia distincta and P. lagenophorae, two closely related rust fungi causing epidemics on Asteraceae in Europe
Mycol. Res. 107 (1): 15–24 (January 2003). f The British Mycological Society
15
DOI: 10.1017/S0953756202006937 Printed in the United Kingdom.
Phylogenetic analysis of Puccinia distincta and P. lagenophorae, two closely related rust fungi causing epidemics on Asteraceae in Europe
Roland W. S. WEBER1*, John WEBSTER2 and Gu¨nther ENGEL1# 1
Lehrbereich Biotechnologie, Universita¨t Kaiserslautern, Paul-Ehrlich-Str. 23, 67663 Kaiserslautern, Germany. 12 Countess Wear Road, Exeter EX2 6LG, Devon, UK. E-mail : [email protected]
2
Received 23 January 2002; accepted 18 October 2002.
Phylogenetic analyses of the ITS1-5.8S-ITS2 region of the ribosomal RNA gene cluster were carried out with two short-cycled (aecial/telial) European rusts on Asteraceae, Puccinia distincta causing the current pan-European epidemic on Bellis perennis, and P. lagenophorae causing a similar disease on Senecio spp., as well as the macrocyclic P. obscura which alternates between B. perennis (pycnial and aecial host) and Luzula spp. (main host). All three species formed a well-resolved cluster when compared with the ITS sequences of a range of other rust fungi, using both parsimony and distance methods. The sequences of P. distincta and P. lagenophorae differed from each other at three positions whereas P. obscura differed from P. distincta at 37 points. Together with consistent morphological and epidemiological differences across Europe, these data support the recognition of P. distincta as a separate species from P. lagenophorae. Both may be derived from P. obscura, although the precise evolutionary history remains obscure.
INTRODUCTION The occurrence of a severely debilitating rust disease on wild and cultivated forms of the daisy (Bellis perennis) has been noted since about 1996 in western Europe (Scholler 1997, Weber, Al-Gharabally & Webster 1998b, Preece, Weber & Webster 2000) and, more recently, also in south-eastern Europe (Gullino et al. 1999, Mu¨ller 2000, Jurc & Weber 2000, 2001). The rust is short-cycled and autoecious on B. perennis; infections are carried solely by aeciospores, although viable teliospores are also formed under certain conditions (Weber et al. 1998b). The origin and identity of the causal fungus has been the subject of controversial discussions. Because of the similarity of its reduced life-cycle with that of Puccinia lagenophorae Cooke 1884, which infects mainly Senecio spp. and is of Australian origin (Wilson, Walshaw & Walker 1965, Scholler 1994), the daisy rust has been considered to belong to the same species (Scholler 1997, Gullino et al. 1999). However, whereas P. lagenophorae rapidly spread throughout Europe especially on S. vulgaris since its first sighting in 1960 (Viennot-Bourgin 1964, Scholler 1994, Mu¨ller 1995), only very occasional
* Corresponding author. # Present address: St Quentin Ring 49, 67663 Kaiserslautern, Germany.
observations of rusted B. perennis plants were made prior to 1996, and such infections were almost always seen on cultivated host varieties (Preece et al. 2000). Further, uninfected B. perennis populations can still be observed in the immediate vicinity of heavily infected S. vulgaris and vice versa (Jurc & Weber 2001). Moreover, attempts at obtaining reciprocal infections from aeciospores growing on these two hosts failed under experimental conditions (Weber et al. 1998b) ; Wilson et al. (1965) succeeded in their cross-infection experiments but did not observe infections on B. perennis in the field. Thus, if the daisy rust was conspecific with P. lagenophorae, it would have to be recognized as a new race or special form. In addition to the epidemiological differences between the short-cycled rusts on Bellis and Senecio, thorough investigations of their teliospore morphology revealed significant differences which have been consistently observed with material collected across Europe, e.g. in the UK (Weber et al. 1998b), the Czech Republic (Mu¨ller 2000) and Slovenia (Jurc & Weber 2001). These differences are readily detected by routine light microscopy and include a much wider teliospore stalk as well as the presence of one-, two- and three-celled teliospores in the Senecio rust, whereas teliospores of the new daisy rust have narrow stalks and one or two but never three cells. Taken together, the above features were considered sufficient by Weber et al. (1998b) to
Puccinia distincta and P. lagenophorae
16
Table 1. Teliospore dimensions of Puccinia species studied.
One-celled spore body (n=10) One-celled spore stalk2 (n=10) Two-celled spore body (n=25) Two-celled spore stalk (n=25) Three-celled spore body (n=10) Three-celled spore stalk (n=10) 1 2
P. distincta (sample 25)
P. lagenophorae (sample 26)
P. obscura (sample 27)
30.2¡3.5 r16.0¡1.6 mm 5.5¡1.1 mm 40.6¡3.1r19.8¡1.3 mm 6.2¡0.9 mm (not observed) (not observed)
29.0¡3.2r15.3¡1.3 mm 7.1¡1.0 mm 43.1¡4.3r18.7¡1.6 mm 8.0¡1.1 mm 45.3¡2.5r20.8¡2.3 mm 9.3¡0.9 mm
30.1¡2.8r15.2¡1.3 mm 6.4¡0.9 mm 43.1¡4.8r17.0¡1.7 mm 6.6¡0.9 mm (not observed) (not observed)
1
Values given as average¡SD. Measured as stalk diam at the point of attachment to the spore body.
identify the new daisy rust as a species separate from P. lagenophorae, i.e. P. distincta McAlpine 1895 which was reported by McAlpine (1906) to have caused daisy rust epidemics in Australia a century ago. Teliospores of P. distincta are indistinguishable from those of P. obscura J. Schro¨ter 1877, a macrocyclic rust alternating between daisies and Luzula spp. Grove (1913) speculated that P. distincta might have arisen from P. obscura by acquiring the ability to complete its life-cycle on the formerly aecial host, B. perennis. One very powerful method to investigate phylogenetic relationships between fungi is the comparison of selected DNA sequences. For closely related species, the internal transcribed spacer (ITS) regions which separate the genes encoding the 18S, 5.8S and 26S ribosomal RNAs, are commonly chosen because of their high degree of variability. This tool has already been applied to rust fungi by several research groups (e.g. Zambino & Szabo 1993, Kropp et al. 1997, Vogler & Bruns 1998, Pfunder, Schu¨rch & Roy 2001), and here we describe and compare the ITS1-5.8S-ITS2 sequences obtained from P. distincta on B. perennis, P. lagenophorae on S. vulgaris, and P. obscura on B. perennis and Luzula campestris. The unexpected result that P. distincta is phylogenetically closer to P. lagenophorae than to P. obscura is discussed.
MATERIALS AND METHODS Origin of samples Collection details of samples of rust infections used for ITS sequencing are given in Table 2. Voucher material for each sample is kept at the Department of Biotechnology, University of Kaiserslautern, and in the 1 Royal Botanic Gardens Kew (K). The GenBank sequences of other rust fungi used for phylogenetic comparison are listed in Table 3. Morphological identification of rust fungi Infections by Puccinia distincta, P. lagenophorae and P. obscura were identified by their macroscopic symptoms (Weber et al. 1998a, Jurc & Weber 2000). When present, telial material was examined by light microscopy, especially for the presence of three-celled teliospores and for the width of the teliospore stalk (Weber et al. 1998b, Jurc & Weber 2001). Teliospore dimensions of German
samples were measured and drawn for comparison with those of the published British (Weber et al. 1998b) and southern European samples (Mu¨ller 2000, Jurc & Weber 2001). Preparation of material Spores were collected from 5–10 aecia or telia of a fresh or dried leaf using a fine needle. The spores were suspended in a small drop of distilled water and allowed to soak for 30 min, followed by air-drying of the drop. The spore deposit was crushed between two microscope slides which had been treated previously by wiping with dichloromethylsilane, soaking in 0.01 M HCl (2 min), and washing in distilled water. Crushed spores were suspended in 50 ml extraction buffer (Zambino & Szabo 1993) containing 89 mM Tris, 45 mM boric acid, 50 nM EDTA and 1% (v/v) b-mercaptoethanol. The suspension was then transferred to an Eppendorf tube containing 1 ml 1 % (v/v) Tween-20, and boiled for 15 min. A 100-fold dilution was prepared in extraction buffer without b-mercaptoethanol and stored at x20 xC until required for amplification. PCR amplification of ITS sequences Amplification of the ITS1-5.8S-ITS2 stretch of DNA was achieved with a thermocycler (PCR Sprint, Hybaid, UK) using primers ITS5 and ITS4 which bind to conserved flanking regions of the 18S and 26S rDNA, respectively (White et al. 1990). The PCR mixture (100 ml) contained 0.5 mM of each primer (MWG Biotech, Ebersberg), 0.2 mM of each deoxynucleotide (Sigma, St Louis, MO), 5 ml diluted spore extract, and 0.025 U Taq polymerase mlx1 in the appropriate reaction buffer (both from Sigma). Following an initial pre-heat at 94 x for 1 min, a total of 38 amplification cycles was performed, each cycle consisting of 30 s at 94 x, 1 min at 45 or 52 x, and 1 min at 72 x. This was followed by a final extension time of 4 min at 72 x. Amplified bands of approx. 700 base pairs (bp) size were separated on a 1% (w/v) agarose gel, excised, and purified using the GENECLEAN II1 kit (Bio 101, Vista, CA) according to the manufacturer’s instructions. Cloning and sequencing of PCR products PCR products were either sequenced directly or ligated into the pGEMTeasy vector (Promega, Madison, WI).
R. W. S. Weber, J. Webster and G. Engel 1
17 2
20 m
Figs 1–2. Teliospores of Puccinia distincta from Bellis perennis (Fig. 1) and P. lagenophorae from S. vulgaris (Fig. 2). Spores were freshly collected on the University of Kaiserslautern campus and mounted directly in water. The two haploid nuclei have fused in some but not all teliospore cells.
Fig. 3. The complete ITS sequence of Puccinia distincta sample 1 (K(M) 92018). This sequence was conserved in all P. distincta samples except for the German sample 14 (K(M) 92033) which differed at position 205 (deletion of a single T). The sequence of P. lagenophorae differed at positions 61 (C instead of A), 570 (C instead of T) and by the insertion of a C after position 191.
For ligation, a mixture (20 ml) of 25 ng vector, 35 ng DNA and 6 U T4 DNA ligase in the appropriate buffer (both from Promega) was incubated overnight at r.t. Transformation of chemically competent Escherichia coli XL1-Blue cells (Stratagene, La Jolla, CA) was performed as described by the supplier. Transformants carrying an insert were selected on LB agar containing
ampicillin (200 mg lx1) within 24 h of incubation at 37 x, and grown subsequently in 5 ml and 100 ml volumes of liquid LB medium with ampicillin for 18 h at 37 x and 180 rpm on an orbital shaker. Plasmids were purified with the NUCLEOBOND AX 100 kit (Macherey & Nagel, Du¨ren, Germany) according to the manufacturer’s instructions.
Puccinia distincta and P. lagenophorae
18
Table 2. Material of rust fungi sequenced in the current work.
Sample
Species/host
Puccinia distincta 1 Aecia on cult. B. perennis (Asteraceae) 2
Telia on cult. B. perennis
4
Aecia on wild B. perennis
5
Aecia on cult. B. perennis
7
Aecia on wild B. perennis
9
Aecia on wild B. perennis
10
Aecia on wild B. perennis
11 13 14
Aecia on wild B. perennis Aecia on wild B. perennis Aecia on cult. B. perennis
21
Aecia on wild B. perennis
23
Aecia on wild B. perennis
24
Aecia on cultivated B. perennis
25
Aecia/telia on wild B. perennis
Puccinia lagenophorae 6 Aecia on S. vulgaris (Asteraceae) 8
Aecia on S. vulgaris
12
Aecia on S. vulgaris
15
Aecia/telia on S. vulgaris
22
Aecia on S. vulgaris
26
Aecia/telia on S. vulgaris
Puccinia obscura 16 Aecia on wild B. perennis 18
Telia on L. campestris (Juncaceae)
27
Uredinia/telia on L. campestris
Other rust fungi 19 P. poarum (aecia) on Tussilago farfara (Asteraceae) 20 P. graminis (telia) on Agropyron repens (Poaceae)
Herbarium accession no.
GenBank1 accession no.
Exeter, Devon, UK (Jan. 1999). Inoc. R. W. S. Weber Exeter, Devon, UK (Jan. 1999). Inoc. R. W. S. Weber Malton, N. Yorks., UK (4 Sept. 1998). P. M. Booth Westcliff-on-Sea, Essex, UK (25 Feb. 1999). L. J. Lundie Cholsey, Oxon, UK (13 Oct. 1999). C. T. Ingold Cardigan Bay, Wales, UK (Sept. 1999). T. F. Preece Llynclys, Salop, UK (18 Sept. 1999). T. F. Preece Perpignan, France (7 Oct. 1999). W. B. Jones Neuers, France (24 Sept. 1999). W. B. Jones Kaiserslautern, Germany (19 Oct. 1999). R. W. S. Weber Ljubljana, Slovenia (13 July 2000). D. Jurc Vignola nr. Modena, Italy (17 June 2000). P. Davoli Wiegersen nr. Buxtehude, N. Germany (10 Sept. 1999). R. W. S. Weber Kaiserslautern, Germany (3 Aug. 2002). R. W. S. Weber
K(M) 92018
AF468040
Exeter, Devon, UK (16 Aug. 1999). J. Webster Cardigan Bay, Wales, UK (26 Sept. 1999). T. F. Preece Wiegersen nr. Buxtehude, N. Germany (10 Sept. 1999). R. W. S. Weber Kaiserslautern, Germany (16 Oct. 1999). R. W. S. Weber Ljubljana, Solvenia (14 Sept. 2000). D. Jurc Kaiserslautern, Germany (3 Aug. 2002). R. W. S. Weber
K(M) 92025
Collection
K(M) 92019 K(M) 92020 K(M) 92021 K(M) 92026 K(M) 92028 K(M) 92029 K(M) 92030 K(M) 92032 K(M) 92033 K(M) 102614 K(M) 102615 K(M) 102616 K(M) 102617
AF468041
K(M) 92027 K(M) 92031 K(M) 92034 K(M) 102618 K(M) 102619
Mardon Down, Devon, UK (24 Sept. 1999). J. Webster Grimspound, Dartmoor, Devon, UK (16 Sept. 1999). J. Webster Kaiserslautern, Germany (3 Aug. 2002). R. W. S. Weber
K(M) 92035
Lago Garda, Italy (1 Oct. 1999). J. Webster
K(M) 92037
AF468043
Exeter, Devon, UK (22 Oct. 1998). R. W. S. Weber
K(M) 92038
AF468044
The standard primers T7 and SP-6 were used for the sequencing reaction using plasmid DNA. These bind to either end of the multiple cloning site of the pGEMTeasy vector, thereby enabling the sequencing of the entire PCR product for each clone. For each rust sample, several independent plasmid clones were selected. Purified plasmids were sequenced independently in our laboratory with the Long-ReadTM Tower (Visible
AF468042
K(M) 92036 K(M) 102620
Genetics, Toronto) and externally by MWG Biotech. In the case of ambiguities, a second and, if necessary, further plasmid preparations were sequenced. Additionally, the PCR product was sequenced directly by MWG Biotech using primers ITS5 and ITS4. Bases were assigned and sequences proof-read by eye using CHROMAS 1.62 (Technelysium, Helensvale, Qd).
R. W. S. Weber, J. Webster and G. Engel
19
Table 3. Details of GenBank sequences used in phylogenetic comparisons. Species/host
Source
GenBank accession no.
Cronartium ribicola on Pinus (Pinaceae) or Ribes (Grossulariaceae) Helicobasidium sp. on Weigelia hortensis (Caprifoliaceae) Peridermium harknessii on Pinus spp. (Pinaceae) Puccinia aberrans on Smelowskia calycina (Brassicaceae) Puccinia carduorum on Carduus thoermeri (Asteraceae) Puccinia codyi on Smelowskia borealis (Brassicaceae) Puccinia consimilis on Arabis holboellii var. pinetorum (Brassicaceae) Puccinia coronata f. sp. avenae on Rhamnus (Rhamnaceae) or Avena (Poaceae) Puccinia drabae on Draba longipes (Brassicaceae) Puccinia monoica on Arabis microphylla (Brassicaceae) Puccinia sorghi on Oxalis (Oxalidaceae) or Zea (Poaceae) Puccinia striiformis f. sp. tritici on Triticum aestivum (Poaceae) Puccinia thlaspeos on Thlaspi alpestre (Brassicaceae) Pucciniastrum goeppeltianum on Abies grandis (Pinaceae) or Vaccinium (Ericaceae) Uromyces fabae on members of the Fabaceae
Vogler & Bruns (1998)
L76499
N. Matsumoto, M. Arakawa, H. Nakamura & Y. Uetake (unpubl.) Vogler & Bruns (1998)
AB056576 L76510
Kropp et al. (1997)
PAU88231
Berthier et al. (1996)
U57351
Kropp et al. (1997)
PCU88233
B. A. Roy (unpubl.)
AF182992
L. J. Szabo (unpubl.)
AY114290
Kropp et al. (1997)
PDU88229
B. A. Roy (unpubl.)
AF182996
L. J. Szabo (unpubl.)
AY114291
L. J. Szabo (unpubl.)
AY114292
Kropp et al. (1997)
PTU88220
Vogler & Bruns (1998)
L76509
W. H. Chung, T. Tsukiboshi & M. Kakishima (unpubl.) Pfunder et al. (2001)
AB085193 AF180159
Pfunder et al. (2001)
AF180193
Pfunder et al. (2001)
AF180161
Uromyces pisi sensu stricto on Euphorbia (Euphorbiaceae) or Lathyrus pratensis (Fabaceae) Uromyces scutellatus on Euphorbia cyparissias (Euphorbiaceae) Uromyces striatus on Trifolium arvense (Fabaceae)
Comparison of ITS sequences Sequences of different samples of Puccinia distincta, P. obscura and P. lagenophorae were compared using ClustalX (Thompson et al. 1997). The PILEUP program in the Wisconsin package (Genetics Computer Group, Madison) was used to align sequences with those of other rust fungi (gap creation penalty=1, gap extension penalty=0.5) ; these were obtained from a FASTA search, whereby only sequences spanning the entire ITS1-5.8S-ITS2 region were selected (see Table 3). Prior to analysis, all sequences were trimmed to incorporate one base of the 18S rDNA and 19 bases of the 26S rDNA. Where necessary, alignment was corrected manually. Helicobasidium sp. was chosen as the outgroup taxon because this genus has been placed outside the rust group within the Urediniomycetes and is considered to be phylogenetically related to the rusts (Sjamsuridzal et al. 1999). More distant members of the Urediniomycetes, such as the red yeasts Sporobolomyces or Rhodotorula, were tried but rejected as outgroups because their sequences were too far diverged for unequivocal alignment with those of rust fungi. Three rusts not belonging to the Pucciniaceae, i.e. Pucciniastrum
goeppeltianum (Pucciniastraceae), Cronartium ribicola and Peridermium harknessii (both Cronartiaceae) were included as ingroups in order to test the positioning of P. distincta, P. lagenophorae and P. obscura within the Pucciniaceae. Trees were generated by heuristic search with maximum parsimony, using PAUP version 4.0.0d55 (Swofford 1999) incorporated into the Wisconsin Package and accessed online via the HUSAR interface (DKFZ, Heidelberg). Gaps in the sequence alignment were handled as missing data and were omitted from the analysis. Branch swapping by tree-bisection-reconnection was performed, with branches collapsing if the maximum branch length was zero. The most parsimonious tree was sought by random stepwise addition of sequences, setting the limit of saved trees to 1000. Bootstrap support (Felsenstein 1985) was estimated for all tree branches (1000 replications). A phylogenetic distance analysis was also performed. The distance matrix of the aligned sequences was corrected using the method of Jukes & Cantor (1969), and phylogenetic trees were created by the neighbour-joining method (Saitou & Nei 1987). The limit of saved trees was set to 1000, and bootstrap
Helicobasidium sp.
Helicobasidium sp.
5
Puccinia aberrans
Puccinia aberrans
P. codyi
P. codyi
P. monoica
P. consimilis
P. thlaspeos
P. thlaspeos
P. consimilis
P. monoica
P. sorghi
P. drabae
P. drabae
P. sorghi
P. coronata f. sp. avenae
P. coronata f. sp. avenae
P. graminis f. sp. agropyri
P. graminis f. sp. agropyri
P. striiformis f. sp. tritici
P. striiformis f. sp. tritici
Puccinia distincta and P. lagenophorae
4
P. poarum
P. poarum
P. carduorum
P. carduorum P. distincta
P. distincta
P. lagenophorae
P. lagenophorae P. obscura
P. obscura
Uromyces fabae
Uromyces fabae U. scutellatus
U. scutellatus
U. striatus
U. striatus U. pisi
U. pisi
Peridermium harknessii
Peridermium harknessii
Cronartium ribicola
Cronartium ribicola
Pucciniastrum goeppeltianum
Pucciniastrum goeppeltianum
10 substitutions per 100 bp
20
Figs 4–5. Fig. 4. One of the two most parsimonious trees derived from the phylogenetic analysis of nucleotide sequences of the ITS region. The only other equally parsimonious tree differed in that the positions of Puccinia codyi and P. monoica were swapped. Both trees had 1313 steps, with a CI of 0.570, RI of 0.645, and RC of 0.368. Fig. 5. Neighbour-joining tree with the lowest minimum evolutionary score. The tree was generated with PAUP 4.0 using the Jukes–Cantor algorithm with a distance matrix of the ITS1-5.8S-ITS2 sequences of rust fungi.
R. W. S. Weber, J. Webster and G. Engel analyses (1000 replicates) were carried out to assess the robustness of the trees.
RESULTS Morphological features Identification of and distinctions between Puccinia distincta, P. lagenophorae and P. obscura on the basis of disease symptoms and microscopic features were always unequivocal. The dimensions of teliospores of the freshly collected German samples 25–27 are given in Table 1 and were representative of all teliosporebearing material. Drawings of typical teliospores of P. distincta (sample 14) and P. lagenophorae (sample 15) collected three years previously at the same site as samples 25–27 were very similar to those collected elsewhere and showed the typical species-specific features, notably the wide teliospore stalk and the presence of three-celled teliospores only in P. lagenophorae (Figs 1–2). Properties of sequences The complete genomic DNA sequences between the primers ITS5 and ITS4 were obtained for 14 samples of Puccinia distincta, six of P. lagenophorae, and three of P. obscura. All P. distincta samples, which had been collected in the UK (England and Wales), France, Germany, Slovenia and Italy, were identical to each other with the exception of the German sample 14 which lacked 1 bp at position 205 (Fig. 3). This deletion was not found in sample 25, collected at the same site three years later. All six P. lagenophorae sequences from the UK, Germany and Slovenia were identical to each other, as were the three P. obscura sequences from England and Germany. Using P. distincta as a reference for a careful base-by-base comparison, P. lagenophorae showed the following three points of difference ; change from A to C at position 61, insertion of a single C at 191, and change of T to C at 570 (Fig. 3). In contrast, when taking multi-bp substitutions or deletions as a single mutational event, the sequence of P. obscura differed from that of P. distincta at 37 points (alignments available upon request from R.W.S.W.). These differences between the three rusts were found even in samples 25–27, collected on the same day within about 200 m of each other. The sequence of P. distincta including the two primers had a length of 677 bp except for sample 14 (676 bp), that of P. lagenophorae 678 bp, and that of P. obscura 670 bp. In P. distincta, the 3k end of 18S rDNA contained 54 bp, the ITS1 region 209 bp, the 5.8S rDNA 156 bp, the ITS2 region 197 bp, and the 5k end of the 26S rDNA 61 bp (see Fig. 3). In addition to the above three species, the sequence alignment used for phylogenetic analysis included P. graminis (sample 20), P. poarum (sample 19) and 18 sequences obtained from GenBank1 (Table 3).
21 Including the gap matrix, the total length of aligned and cut sequences was 700 bp, comprising 18S rDNA (1 bp), ITS1 (264 bp), 5.8S rDNA (156 bp), ITS2 (260 bp) and 26S rDNA (19 bp). The matrix contained 259 constant characters, 105 variable but parsimony-uninformative and 336 variable and parsimony-informative characters. The mean nucleotide composition of all sequences showed a high A–T content (67.7%). Phylogenetic analysis Two most parsimonious trees (Fig. 4) were obtained (length, 1313 steps ; consistency index CI, 0.570 ; retention index RI, 0.645 ; rescaled consistency index RC, 0.368). The grouping of Puccinia distincta with P. lagenophorae, as well as the grouping of these two species with P. obscura, were shown in both most parsimonious trees and were supported by bootstrap values of 99.2 % and 93.8 % (respectively) from 1000 computed parsimony trees (Fig. 6). The distance tree with the smallest minimum evolution score (1.969) is shown in Fig. 5. The consensus tree of 1000 samples (Fig. 6) supports the grouping of P. distincta with P. lagenophorae (99 %), and of these two species with P. obscura (95.8 %). Good bootstrap support from parsimony (90.1 %) and distance (89 %) analyses was obtained also for the grouping of P. carduorum with these three rusts. Among the other rust fungi used in the analysis, strong phylogenetic groupings were observed in both types of analysis for the three non-Pucciniaceae (i.e. Peridermium harknessii, Cronartium ribicola and Pucciniastrum goeppeltianum), and the four rust species associated with Euphorbiaceae/Fabaceae (namely Uromyces fabae, U. pisi, U. scutellatus and U. striatus). Five rusts associated with Brassicaceae (i.e. P. aberrans, P. codyi, P. consimilis, P. monoica and P. thlaspeos) also formed a moderately well-supported cluster, with a bootstrap support of 82.3 % in the parsimony analysis. In contrast, the cereal rusts could not be unequivocally separated from those on Brassicaceae in that P. sorghi and P. coronata f. sp. avenae were grouped weakly with the latter.
DISCUSSION The current study has shown that there were only three differences in the ITS sequences between Puccinia lagenophorae on Senecio vulgaris and P. distincta on Bellis perennis which were, however, consistently present in all British and continental European samples examined. The homogeneity of our data extends to the fact that within either species, all sequences were identical with the exception of the German P. distincta sample 14 which differed at only one position from all other German samples and those from all other countries. This is in agreement with the known epidemiological data for both species which are recent arrivals in Europe, having caused epidemics only within
Puccinia distincta and P. lagenophorae
22 Helicobasidium sp.
82.3
P. thlaspeos P. monoica P. sorghi P. drabae
65.6 69.9 97.0 65.8
Puccinia aberrans P. codyi P. consimilis
87.3 74.0
P. coronata P. graminis P. striiformis
82.3 73.3 65.9 62.2 53.8 84.5
53.6 81.8
81.0 68.2
P. poarum P. carduorum
89.4 90.1
99.2 93.8
99.3 95.6 95.2
87.5
100 100
P. distincta P. lagenophorae P. obscura Uromyces fabae U. scutellatus U. striatus U. pisi Perid. harknessii Cron. ribicola
87.9 89.0
99.0 95.8
89.7 93.8 96.3
88.0
99.6 100
Pucciniastrum goeppeltianum
Fig. 6. Parsimony (left) and distance (right) 50 % majority rule consensus trees of the ITS1-5.8S-ITS2 sequences of rust fungi. Values associated with branch-points indicate the percentage (bootstrap support) from 1000 bootstrapped trees at which the branches clustered together.
the past 40 years. Longer-established rust populations might have been expected to show larger variations between different isolates of the same species (Pfunder et al. 2001). The consistent differences between P. distincta and P. lagenophorae indicate that interbreeding does not occur in nature, thus supporting our previous contention that the rust on daisies should be regarded as a species distinct from P. lagenophorae, which makes its naming by McAlpine (1906) particularly apt in retrospect. Differences between these two species can be regularly observed in terms of current (Jurc & Weber 2001) and historical (Preece et al. 2000) field distribution data. Briefly, the European epidemic of P. lagenophorae on Senecio spp. arose in 1960 whereas the new daisy rust became a pan-European epidemic only from 1996 onwards. The comparative measurements of teliospore morphology (Figs 1–2) and dimensions (Table 1) are based on material collected within less than 200 m on two occasions separated by a three-year interval. Their excellent agreement with data reported previously for British and south-east European specimens (Weber et al. 1998b, Mu¨ller 2000, Jurc & Weber 2001) supports the temporal and spatial identity of P. distincta and P. lagenophorae as different species. Koike & Scholler (2001) noted the occurrence of a new rust on B. perennis in North America which they regarded as identical with one on Senecio spp. which is also a recent arrival there. Under controlled conditions, these authors succeeded in infecting B. perennis with rust inoculum obtained from S. vulgaris, as had already
been demonstrated by Wilson et al. (1965) in Britain. At present, it is difficult in the absence of morphological or DNA sequence information to relate the identity of the American daisy rust to the European rust. An examination of rusts on Bellis and Senecio worldwide would be very interesting but may provide only short-term answers as new races or species seem to spread rapidly. Compared with the small ITS sequence differences between P. lagenophorae and P. distincta, the phylogenetic distance between these rusts and P. obscura was considerably greater (37 points of difference). Nonetheless, all three rusts formed a highly coherent cluster in both types of phylogenetic analysis used, and were wellseparated from another rust of Asteraceae, P. poarum (aecial host, Tussilago farfara). The clustering of correlated short- and long-cycled species of grass rusts was observed by Zambino & Szabo (1993) who found that they differed by about 5–10 bp. In contrast, the same authors found that ITS sequences of different special forms of any one species of grass rust varied by only 1–3 bp. In other well-established rusts, a similar distance has been found between different isolates of the same species (Vogler & Bruns 1998, Pfunder et al. 2001), whereas Vogler & Bruns (1993, 1998) found that microand macrocyclic pine rust fungi (e.g. Peridermium harknessii, Cronartium quercuum f. sp. banksiana) had identical ITS sequences but were nonetheless given different names based on differences in the morphology and life-cycles. Numerical base-pair differences, therefore, have to be interpreted within the biological context
R. W. S. Weber, J. Webster and G. Engel of the species concerned. In the case of the recent European arrivals P. lagenophorae and P. distincta, a consistent difference of 3 bp is clearly more significant than it would be between two long-established rust species showing a certain degree of intra-species variation. Rusts with a reduced life-cycle on one host plant are considered to be derived from macrocyclic heteroecious precursor species (Malloch 1995). This relationship is called Tranzschel’s Law if the teliospore state of the derived species is produced on the aecial host of the ancestral species (Shattock & Preece 2000). If we accept a common phylogenetic origin between P. obscura, P. distincta and P. lagenophorae based on Tranzschel’s Law (Weber et al. 1998b), P. distincta is a demicyclic (-opsis type) derivative of P. obscura as indicated by their sharing of a common aecial host (B. perennis) and close morphological similarity and dimensions of their teliospores (Grove 1913). Concerning the relationship of P. lagenophorae to the other two species, our results indicate that P. distincta and P. lagenophorae are more closely related to each other than either is to P. obscura, and this could be taken to imply that P. lagenophorae, like P. distincta, has arisen from P. obscura. There are, however, some obvious difficulties with this hypothesis relating to the place and time where the required evolutionary steps might have occurred. The available evidence suggests that P. lagenophorae is an Australasian species which is known in Europe only since 1960 (Wilson & Walshaw 1963, Wilson & Henderson 1966). In contrast, P. obscura seems to be a long-established European species (Plowright 1889, Grove 1913). P. distincta was first described on cultivated daisies in Australia in 1895 (McAlpine 1906), and we have recently verified that P. obscura was also present in Australia on B. perennis at that time (Weber et al. 1998b). It is possible that P. obscura was introduced into Australia in the mid-nineteenth century, and that P. distincta and P. lagenophorae arose there and were inadvertently reintroduced into Europe on two separate occasions. However, these speculations may never be resolved due to our lack of information on the historical distribution of hosts and pathogens. It would be interesting to compare the sequences of a wider range of rusts on Asteraceae, especially those placed in synonymy with P. lagenophorae by Wilson et al. (1965).
ACKNOWLEDGEMENTS We thank Philip M. Booth, Paolo Davoli, C. Terence Ingold, William B. Jones, Dusˇ an Jurc, Leslie J. Lundy and Thomas F. Preece for collecting rust samples, and we are most grateful to Heidrun Anke (Institute of Biotechnology and Drug Research IBWF, Kaiserslautern) and Timm Anke (University of Kaiserslautern) for their support.
REFERENCES Berthier, Y. T., Bruckart, W. L., Chaboudez, P. & Luster, D. G. (1996) Polymorphic restriction patterns of ribosomal internal
23 transcribed spacers in the biocontrol fungus Puccinia carduorum correlate with weed host origin. Applied and Environmental Microbiology 62 : 3037–3041. Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. Grove, W. B. (1913) British Rust Fungi. Cambridge University Press, Cambridge, UK. Gullino, M. L., Bertetti, D., Luongo, I., Frausin, C. & Garibaldi, A. (1999) Attacchi di ruggine su margheritina (Bellis perennis) e prova di lotta chimica. Informatore Fitopatologica 49: 52–55. Jukes, T. H. & Cantor, C. R. (1969) Evolution in protein molecules. In Mammalian Protein Metabolism (H. N. Munro, ed.): 21–123. Academic Press, New York. Jurc, D. & Weber, R. W. S. (2000) First report of Puccinia distincta McAlpine, the new European rust on daisies (Bellis perennis L.), from Croatia. Natura Croatica 9: 225–236. Jurc, D. & Weber, R. W. S. (2001) Puccinia distincta and Puccinia lagenophorae, two rust fungi of Asteraceae recently introduced into Slovenia. Zbornik Predavanj in Referatov Slovenskega Posvetovanja o Varstvu Rastlin 5 : 146–154. Koike, S. T. & Scholler, M. (2001) First occurrence of a rust fungus on English daisy (Bellis perennis) in North America. Plant Disease 85: 562. Kropp, B. R., Hansen, D. R., Wolf, P. G., Flint, K. M. & Thomson, S. V. (1997) A study on the phylogeny of the dyer’s woad rust fungus and other species of Puccinia from crucifers. Phytopathology 87: 565–571. Malloch, D. (1995) Fungi with heteroxenous life histories. Canadian Journal of Botany 73 : S1334–S1342. McAlpine, D. (1906) The Rusts of Australia: their structure, nature and classification. R. S. Brain, Melbourne. Mu¨ller, J. (1995) Australischer Rostpilz Puccinia lagenophorae auch in der Tschechischen Republik und in Ungarn. Czech Mycology 48: 161–168. Mu¨ller, J. (2000) Epidemie australske´ rzi Puccinia distincta na sedmikra´ska´ch v Cˇeske´ republice. Mykologicke´ Listy 75: 8–15. Pfunder, M., Schu¨rch, S. & Roy, B. A. (2001) Sequence variation and geographic distribution of pseudoflower-forming rust fungi (Uromyces pisi s. lat.) on Euphorbia cyparissias. Mycological Research 105: 57–66. Plowright, C. B. (1889) A Monograph of British Uredineae and Ustilagineae. Kegan Paul & Trench, London. Preece, T. F., Weber, R. W. S. & Webster, J. (2000) Origin and spread of the daisy rust epidemic in Britain caused by Puccinia distincta. Mycological Research 104: 576–580. Saitou, N. & Nei, M. (1996) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425. Scholler, M. (1994) Puccinia lagenophorae in Deutschland: Anmerkungen zur Einwanderung, Verbreitung und O¨kologie. Verhandlungen des Botanischen Vereins zu Berlin-Brandenburg 127: 177–189. Scholler, M. (1997) Rust fungi on Bellis perennis in Central Europe: delimitation and distribution. Sydowia 49: 174–181. Shattock, R. C. & Preece, T. F. (2000) Tranzschel revisited: modern studies of the relatedness of different rust fungi confirm his Law. Mycologist 14: 113–117. Sjamsuridzal, W., Nishida, H., Ogawa, H., Kakishima, M. & Sugiyama, J. (1999) Phylogenetic positions of rust fungi parasitic on ferns: evidence from 18S rDNA sequence analysis. Mycoscience 40: 21–27. Swofford, D. L. (1999) PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, MA. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882. Viennot-Bourgin, G. (1964) La rouille australienne du Se´necon. Revue de Mycologie 29 : 241–258.
Puccinia distincta and P. lagenophorae Vogler, D. R. & Bruns, T. D. (1993) Use of molecular characters to identify holomorphs : an example from the rust genus Cronartium. In The Fungal Holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics (D. R. Reynolds & J. W. Taylor, eds): 237–245. CAB International, Wallingford. Vogler, D. R. & Bruns, T. D. (1998) Phylogenetic relationships among the pine stem rust fungi (Cronartium and Peridermium spp.). Mycologia 90 : 244–257. Weber, R. W. S., Webster, J., Wakley, G. E. & Al-Gharabally, D. H. (1998a) Puccinia distincta, cause of a devastating rust disease of daisies. Mycologist 12: 87–90. Weber, R. W. S., Al-Gharabally, D. H. & Webster, J. (1998b) Puccinia distincta, cause of the current daisy rust epidemic in Britain, in comparison with other rusts recorded on daisies, P. obscura and P. lagenophorae. Mycological Research 102 : 1227–1232. White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.
24 In PCR Protocols: a guide to methods and applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds): 315–322. Academic Press, San Diego. Wilson, M. & Henderson, D. M. (1966) British Rust Fungi. Cambridge University Press, Cambridge, UK. Wilson, I. M. & Walshaw, D. (1963) A new rust disease on groundsel. Nature 200 : 383. Wilson, I. M., Walshaw, D. F. & Walker, J. (1965) The new groundsel rust in Britain and its relationship to certain Australasian rusts. Transactions of the British Mycological Society 48: 501–511. Zambino, P. J. & Szabo, L. J. (1993) Phylogenetic relationships of selected cereal and grass rusts based on rDNA sequence analysis. Mycologia 85 : 401–414.
Corresponding Editors: J. Sugiyama & D. L. Hawksworth
15
DOI: 10.1017/S0953756202006937 Printed in the United Kingdom.
Phylogenetic analysis of Puccinia distincta and P. lagenophorae, two closely related rust fungi causing epidemics on Asteraceae in Europe
Roland W. S. WEBER1*, John WEBSTER2 and Gu¨nther ENGEL1# 1
Lehrbereich Biotechnologie, Universita¨t Kaiserslautern, Paul-Ehrlich-Str. 23, 67663 Kaiserslautern, Germany. 12 Countess Wear Road, Exeter EX2 6LG, Devon, UK. E-mail : [email protected]
2
Received 23 January 2002; accepted 18 October 2002.
Phylogenetic analyses of the ITS1-5.8S-ITS2 region of the ribosomal RNA gene cluster were carried out with two short-cycled (aecial/telial) European rusts on Asteraceae, Puccinia distincta causing the current pan-European epidemic on Bellis perennis, and P. lagenophorae causing a similar disease on Senecio spp., as well as the macrocyclic P. obscura which alternates between B. perennis (pycnial and aecial host) and Luzula spp. (main host). All three species formed a well-resolved cluster when compared with the ITS sequences of a range of other rust fungi, using both parsimony and distance methods. The sequences of P. distincta and P. lagenophorae differed from each other at three positions whereas P. obscura differed from P. distincta at 37 points. Together with consistent morphological and epidemiological differences across Europe, these data support the recognition of P. distincta as a separate species from P. lagenophorae. Both may be derived from P. obscura, although the precise evolutionary history remains obscure.
INTRODUCTION The occurrence of a severely debilitating rust disease on wild and cultivated forms of the daisy (Bellis perennis) has been noted since about 1996 in western Europe (Scholler 1997, Weber, Al-Gharabally & Webster 1998b, Preece, Weber & Webster 2000) and, more recently, also in south-eastern Europe (Gullino et al. 1999, Mu¨ller 2000, Jurc & Weber 2000, 2001). The rust is short-cycled and autoecious on B. perennis; infections are carried solely by aeciospores, although viable teliospores are also formed under certain conditions (Weber et al. 1998b). The origin and identity of the causal fungus has been the subject of controversial discussions. Because of the similarity of its reduced life-cycle with that of Puccinia lagenophorae Cooke 1884, which infects mainly Senecio spp. and is of Australian origin (Wilson, Walshaw & Walker 1965, Scholler 1994), the daisy rust has been considered to belong to the same species (Scholler 1997, Gullino et al. 1999). However, whereas P. lagenophorae rapidly spread throughout Europe especially on S. vulgaris since its first sighting in 1960 (Viennot-Bourgin 1964, Scholler 1994, Mu¨ller 1995), only very occasional
* Corresponding author. # Present address: St Quentin Ring 49, 67663 Kaiserslautern, Germany.
observations of rusted B. perennis plants were made prior to 1996, and such infections were almost always seen on cultivated host varieties (Preece et al. 2000). Further, uninfected B. perennis populations can still be observed in the immediate vicinity of heavily infected S. vulgaris and vice versa (Jurc & Weber 2001). Moreover, attempts at obtaining reciprocal infections from aeciospores growing on these two hosts failed under experimental conditions (Weber et al. 1998b) ; Wilson et al. (1965) succeeded in their cross-infection experiments but did not observe infections on B. perennis in the field. Thus, if the daisy rust was conspecific with P. lagenophorae, it would have to be recognized as a new race or special form. In addition to the epidemiological differences between the short-cycled rusts on Bellis and Senecio, thorough investigations of their teliospore morphology revealed significant differences which have been consistently observed with material collected across Europe, e.g. in the UK (Weber et al. 1998b), the Czech Republic (Mu¨ller 2000) and Slovenia (Jurc & Weber 2001). These differences are readily detected by routine light microscopy and include a much wider teliospore stalk as well as the presence of one-, two- and three-celled teliospores in the Senecio rust, whereas teliospores of the new daisy rust have narrow stalks and one or two but never three cells. Taken together, the above features were considered sufficient by Weber et al. (1998b) to
Puccinia distincta and P. lagenophorae
16
Table 1. Teliospore dimensions of Puccinia species studied.
One-celled spore body (n=10) One-celled spore stalk2 (n=10) Two-celled spore body (n=25) Two-celled spore stalk (n=25) Three-celled spore body (n=10) Three-celled spore stalk (n=10) 1 2
P. distincta (sample 25)
P. lagenophorae (sample 26)
P. obscura (sample 27)
30.2¡3.5 r16.0¡1.6 mm 5.5¡1.1 mm 40.6¡3.1r19.8¡1.3 mm 6.2¡0.9 mm (not observed) (not observed)
29.0¡3.2r15.3¡1.3 mm 7.1¡1.0 mm 43.1¡4.3r18.7¡1.6 mm 8.0¡1.1 mm 45.3¡2.5r20.8¡2.3 mm 9.3¡0.9 mm
30.1¡2.8r15.2¡1.3 mm 6.4¡0.9 mm 43.1¡4.8r17.0¡1.7 mm 6.6¡0.9 mm (not observed) (not observed)
1
Values given as average¡SD. Measured as stalk diam at the point of attachment to the spore body.
identify the new daisy rust as a species separate from P. lagenophorae, i.e. P. distincta McAlpine 1895 which was reported by McAlpine (1906) to have caused daisy rust epidemics in Australia a century ago. Teliospores of P. distincta are indistinguishable from those of P. obscura J. Schro¨ter 1877, a macrocyclic rust alternating between daisies and Luzula spp. Grove (1913) speculated that P. distincta might have arisen from P. obscura by acquiring the ability to complete its life-cycle on the formerly aecial host, B. perennis. One very powerful method to investigate phylogenetic relationships between fungi is the comparison of selected DNA sequences. For closely related species, the internal transcribed spacer (ITS) regions which separate the genes encoding the 18S, 5.8S and 26S ribosomal RNAs, are commonly chosen because of their high degree of variability. This tool has already been applied to rust fungi by several research groups (e.g. Zambino & Szabo 1993, Kropp et al. 1997, Vogler & Bruns 1998, Pfunder, Schu¨rch & Roy 2001), and here we describe and compare the ITS1-5.8S-ITS2 sequences obtained from P. distincta on B. perennis, P. lagenophorae on S. vulgaris, and P. obscura on B. perennis and Luzula campestris. The unexpected result that P. distincta is phylogenetically closer to P. lagenophorae than to P. obscura is discussed.
MATERIALS AND METHODS Origin of samples Collection details of samples of rust infections used for ITS sequencing are given in Table 2. Voucher material for each sample is kept at the Department of Biotechnology, University of Kaiserslautern, and in the 1 Royal Botanic Gardens Kew (K). The GenBank sequences of other rust fungi used for phylogenetic comparison are listed in Table 3. Morphological identification of rust fungi Infections by Puccinia distincta, P. lagenophorae and P. obscura were identified by their macroscopic symptoms (Weber et al. 1998a, Jurc & Weber 2000). When present, telial material was examined by light microscopy, especially for the presence of three-celled teliospores and for the width of the teliospore stalk (Weber et al. 1998b, Jurc & Weber 2001). Teliospore dimensions of German
samples were measured and drawn for comparison with those of the published British (Weber et al. 1998b) and southern European samples (Mu¨ller 2000, Jurc & Weber 2001). Preparation of material Spores were collected from 5–10 aecia or telia of a fresh or dried leaf using a fine needle. The spores were suspended in a small drop of distilled water and allowed to soak for 30 min, followed by air-drying of the drop. The spore deposit was crushed between two microscope slides which had been treated previously by wiping with dichloromethylsilane, soaking in 0.01 M HCl (2 min), and washing in distilled water. Crushed spores were suspended in 50 ml extraction buffer (Zambino & Szabo 1993) containing 89 mM Tris, 45 mM boric acid, 50 nM EDTA and 1% (v/v) b-mercaptoethanol. The suspension was then transferred to an Eppendorf tube containing 1 ml 1 % (v/v) Tween-20, and boiled for 15 min. A 100-fold dilution was prepared in extraction buffer without b-mercaptoethanol and stored at x20 xC until required for amplification. PCR amplification of ITS sequences Amplification of the ITS1-5.8S-ITS2 stretch of DNA was achieved with a thermocycler (PCR Sprint, Hybaid, UK) using primers ITS5 and ITS4 which bind to conserved flanking regions of the 18S and 26S rDNA, respectively (White et al. 1990). The PCR mixture (100 ml) contained 0.5 mM of each primer (MWG Biotech, Ebersberg), 0.2 mM of each deoxynucleotide (Sigma, St Louis, MO), 5 ml diluted spore extract, and 0.025 U Taq polymerase mlx1 in the appropriate reaction buffer (both from Sigma). Following an initial pre-heat at 94 x for 1 min, a total of 38 amplification cycles was performed, each cycle consisting of 30 s at 94 x, 1 min at 45 or 52 x, and 1 min at 72 x. This was followed by a final extension time of 4 min at 72 x. Amplified bands of approx. 700 base pairs (bp) size were separated on a 1% (w/v) agarose gel, excised, and purified using the GENECLEAN II1 kit (Bio 101, Vista, CA) according to the manufacturer’s instructions. Cloning and sequencing of PCR products PCR products were either sequenced directly or ligated into the pGEMTeasy vector (Promega, Madison, WI).
R. W. S. Weber, J. Webster and G. Engel 1
17 2
20 m
Figs 1–2. Teliospores of Puccinia distincta from Bellis perennis (Fig. 1) and P. lagenophorae from S. vulgaris (Fig. 2). Spores were freshly collected on the University of Kaiserslautern campus and mounted directly in water. The two haploid nuclei have fused in some but not all teliospore cells.
Fig. 3. The complete ITS sequence of Puccinia distincta sample 1 (K(M) 92018). This sequence was conserved in all P. distincta samples except for the German sample 14 (K(M) 92033) which differed at position 205 (deletion of a single T). The sequence of P. lagenophorae differed at positions 61 (C instead of A), 570 (C instead of T) and by the insertion of a C after position 191.
For ligation, a mixture (20 ml) of 25 ng vector, 35 ng DNA and 6 U T4 DNA ligase in the appropriate buffer (both from Promega) was incubated overnight at r.t. Transformation of chemically competent Escherichia coli XL1-Blue cells (Stratagene, La Jolla, CA) was performed as described by the supplier. Transformants carrying an insert were selected on LB agar containing
ampicillin (200 mg lx1) within 24 h of incubation at 37 x, and grown subsequently in 5 ml and 100 ml volumes of liquid LB medium with ampicillin for 18 h at 37 x and 180 rpm on an orbital shaker. Plasmids were purified with the NUCLEOBOND AX 100 kit (Macherey & Nagel, Du¨ren, Germany) according to the manufacturer’s instructions.
Puccinia distincta and P. lagenophorae
18
Table 2. Material of rust fungi sequenced in the current work.
Sample
Species/host
Puccinia distincta 1 Aecia on cult. B. perennis (Asteraceae) 2
Telia on cult. B. perennis
4
Aecia on wild B. perennis
5
Aecia on cult. B. perennis
7
Aecia on wild B. perennis
9
Aecia on wild B. perennis
10
Aecia on wild B. perennis
11 13 14
Aecia on wild B. perennis Aecia on wild B. perennis Aecia on cult. B. perennis
21
Aecia on wild B. perennis
23
Aecia on wild B. perennis
24
Aecia on cultivated B. perennis
25
Aecia/telia on wild B. perennis
Puccinia lagenophorae 6 Aecia on S. vulgaris (Asteraceae) 8
Aecia on S. vulgaris
12
Aecia on S. vulgaris
15
Aecia/telia on S. vulgaris
22
Aecia on S. vulgaris
26
Aecia/telia on S. vulgaris
Puccinia obscura 16 Aecia on wild B. perennis 18
Telia on L. campestris (Juncaceae)
27
Uredinia/telia on L. campestris
Other rust fungi 19 P. poarum (aecia) on Tussilago farfara (Asteraceae) 20 P. graminis (telia) on Agropyron repens (Poaceae)
Herbarium accession no.
GenBank1 accession no.
Exeter, Devon, UK (Jan. 1999). Inoc. R. W. S. Weber Exeter, Devon, UK (Jan. 1999). Inoc. R. W. S. Weber Malton, N. Yorks., UK (4 Sept. 1998). P. M. Booth Westcliff-on-Sea, Essex, UK (25 Feb. 1999). L. J. Lundie Cholsey, Oxon, UK (13 Oct. 1999). C. T. Ingold Cardigan Bay, Wales, UK (Sept. 1999). T. F. Preece Llynclys, Salop, UK (18 Sept. 1999). T. F. Preece Perpignan, France (7 Oct. 1999). W. B. Jones Neuers, France (24 Sept. 1999). W. B. Jones Kaiserslautern, Germany (19 Oct. 1999). R. W. S. Weber Ljubljana, Slovenia (13 July 2000). D. Jurc Vignola nr. Modena, Italy (17 June 2000). P. Davoli Wiegersen nr. Buxtehude, N. Germany (10 Sept. 1999). R. W. S. Weber Kaiserslautern, Germany (3 Aug. 2002). R. W. S. Weber
K(M) 92018
AF468040
Exeter, Devon, UK (16 Aug. 1999). J. Webster Cardigan Bay, Wales, UK (26 Sept. 1999). T. F. Preece Wiegersen nr. Buxtehude, N. Germany (10 Sept. 1999). R. W. S. Weber Kaiserslautern, Germany (16 Oct. 1999). R. W. S. Weber Ljubljana, Solvenia (14 Sept. 2000). D. Jurc Kaiserslautern, Germany (3 Aug. 2002). R. W. S. Weber
K(M) 92025
Collection
K(M) 92019 K(M) 92020 K(M) 92021 K(M) 92026 K(M) 92028 K(M) 92029 K(M) 92030 K(M) 92032 K(M) 92033 K(M) 102614 K(M) 102615 K(M) 102616 K(M) 102617
AF468041
K(M) 92027 K(M) 92031 K(M) 92034 K(M) 102618 K(M) 102619
Mardon Down, Devon, UK (24 Sept. 1999). J. Webster Grimspound, Dartmoor, Devon, UK (16 Sept. 1999). J. Webster Kaiserslautern, Germany (3 Aug. 2002). R. W. S. Weber
K(M) 92035
Lago Garda, Italy (1 Oct. 1999). J. Webster
K(M) 92037
AF468043
Exeter, Devon, UK (22 Oct. 1998). R. W. S. Weber
K(M) 92038
AF468044
The standard primers T7 and SP-6 were used for the sequencing reaction using plasmid DNA. These bind to either end of the multiple cloning site of the pGEMTeasy vector, thereby enabling the sequencing of the entire PCR product for each clone. For each rust sample, several independent plasmid clones were selected. Purified plasmids were sequenced independently in our laboratory with the Long-ReadTM Tower (Visible
AF468042
K(M) 92036 K(M) 102620
Genetics, Toronto) and externally by MWG Biotech. In the case of ambiguities, a second and, if necessary, further plasmid preparations were sequenced. Additionally, the PCR product was sequenced directly by MWG Biotech using primers ITS5 and ITS4. Bases were assigned and sequences proof-read by eye using CHROMAS 1.62 (Technelysium, Helensvale, Qd).
R. W. S. Weber, J. Webster and G. Engel
19
Table 3. Details of GenBank sequences used in phylogenetic comparisons. Species/host
Source
GenBank accession no.
Cronartium ribicola on Pinus (Pinaceae) or Ribes (Grossulariaceae) Helicobasidium sp. on Weigelia hortensis (Caprifoliaceae) Peridermium harknessii on Pinus spp. (Pinaceae) Puccinia aberrans on Smelowskia calycina (Brassicaceae) Puccinia carduorum on Carduus thoermeri (Asteraceae) Puccinia codyi on Smelowskia borealis (Brassicaceae) Puccinia consimilis on Arabis holboellii var. pinetorum (Brassicaceae) Puccinia coronata f. sp. avenae on Rhamnus (Rhamnaceae) or Avena (Poaceae) Puccinia drabae on Draba longipes (Brassicaceae) Puccinia monoica on Arabis microphylla (Brassicaceae) Puccinia sorghi on Oxalis (Oxalidaceae) or Zea (Poaceae) Puccinia striiformis f. sp. tritici on Triticum aestivum (Poaceae) Puccinia thlaspeos on Thlaspi alpestre (Brassicaceae) Pucciniastrum goeppeltianum on Abies grandis (Pinaceae) or Vaccinium (Ericaceae) Uromyces fabae on members of the Fabaceae
Vogler & Bruns (1998)
L76499
N. Matsumoto, M. Arakawa, H. Nakamura & Y. Uetake (unpubl.) Vogler & Bruns (1998)
AB056576 L76510
Kropp et al. (1997)
PAU88231
Berthier et al. (1996)
U57351
Kropp et al. (1997)
PCU88233
B. A. Roy (unpubl.)
AF182992
L. J. Szabo (unpubl.)
AY114290
Kropp et al. (1997)
PDU88229
B. A. Roy (unpubl.)
AF182996
L. J. Szabo (unpubl.)
AY114291
L. J. Szabo (unpubl.)
AY114292
Kropp et al. (1997)
PTU88220
Vogler & Bruns (1998)
L76509
W. H. Chung, T. Tsukiboshi & M. Kakishima (unpubl.) Pfunder et al. (2001)
AB085193 AF180159
Pfunder et al. (2001)
AF180193
Pfunder et al. (2001)
AF180161
Uromyces pisi sensu stricto on Euphorbia (Euphorbiaceae) or Lathyrus pratensis (Fabaceae) Uromyces scutellatus on Euphorbia cyparissias (Euphorbiaceae) Uromyces striatus on Trifolium arvense (Fabaceae)
Comparison of ITS sequences Sequences of different samples of Puccinia distincta, P. obscura and P. lagenophorae were compared using ClustalX (Thompson et al. 1997). The PILEUP program in the Wisconsin package (Genetics Computer Group, Madison) was used to align sequences with those of other rust fungi (gap creation penalty=1, gap extension penalty=0.5) ; these were obtained from a FASTA search, whereby only sequences spanning the entire ITS1-5.8S-ITS2 region were selected (see Table 3). Prior to analysis, all sequences were trimmed to incorporate one base of the 18S rDNA and 19 bases of the 26S rDNA. Where necessary, alignment was corrected manually. Helicobasidium sp. was chosen as the outgroup taxon because this genus has been placed outside the rust group within the Urediniomycetes and is considered to be phylogenetically related to the rusts (Sjamsuridzal et al. 1999). More distant members of the Urediniomycetes, such as the red yeasts Sporobolomyces or Rhodotorula, were tried but rejected as outgroups because their sequences were too far diverged for unequivocal alignment with those of rust fungi. Three rusts not belonging to the Pucciniaceae, i.e. Pucciniastrum
goeppeltianum (Pucciniastraceae), Cronartium ribicola and Peridermium harknessii (both Cronartiaceae) were included as ingroups in order to test the positioning of P. distincta, P. lagenophorae and P. obscura within the Pucciniaceae. Trees were generated by heuristic search with maximum parsimony, using PAUP version 4.0.0d55 (Swofford 1999) incorporated into the Wisconsin Package and accessed online via the HUSAR interface (DKFZ, Heidelberg). Gaps in the sequence alignment were handled as missing data and were omitted from the analysis. Branch swapping by tree-bisection-reconnection was performed, with branches collapsing if the maximum branch length was zero. The most parsimonious tree was sought by random stepwise addition of sequences, setting the limit of saved trees to 1000. Bootstrap support (Felsenstein 1985) was estimated for all tree branches (1000 replications). A phylogenetic distance analysis was also performed. The distance matrix of the aligned sequences was corrected using the method of Jukes & Cantor (1969), and phylogenetic trees were created by the neighbour-joining method (Saitou & Nei 1987). The limit of saved trees was set to 1000, and bootstrap
Helicobasidium sp.
Helicobasidium sp.
5
Puccinia aberrans
Puccinia aberrans
P. codyi
P. codyi
P. monoica
P. consimilis
P. thlaspeos
P. thlaspeos
P. consimilis
P. monoica
P. sorghi
P. drabae
P. drabae
P. sorghi
P. coronata f. sp. avenae
P. coronata f. sp. avenae
P. graminis f. sp. agropyri
P. graminis f. sp. agropyri
P. striiformis f. sp. tritici
P. striiformis f. sp. tritici
Puccinia distincta and P. lagenophorae
4
P. poarum
P. poarum
P. carduorum
P. carduorum P. distincta
P. distincta
P. lagenophorae
P. lagenophorae P. obscura
P. obscura
Uromyces fabae
Uromyces fabae U. scutellatus
U. scutellatus
U. striatus
U. striatus U. pisi
U. pisi
Peridermium harknessii
Peridermium harknessii
Cronartium ribicola
Cronartium ribicola
Pucciniastrum goeppeltianum
Pucciniastrum goeppeltianum
10 substitutions per 100 bp
20
Figs 4–5. Fig. 4. One of the two most parsimonious trees derived from the phylogenetic analysis of nucleotide sequences of the ITS region. The only other equally parsimonious tree differed in that the positions of Puccinia codyi and P. monoica were swapped. Both trees had 1313 steps, with a CI of 0.570, RI of 0.645, and RC of 0.368. Fig. 5. Neighbour-joining tree with the lowest minimum evolutionary score. The tree was generated with PAUP 4.0 using the Jukes–Cantor algorithm with a distance matrix of the ITS1-5.8S-ITS2 sequences of rust fungi.
R. W. S. Weber, J. Webster and G. Engel analyses (1000 replicates) were carried out to assess the robustness of the trees.
RESULTS Morphological features Identification of and distinctions between Puccinia distincta, P. lagenophorae and P. obscura on the basis of disease symptoms and microscopic features were always unequivocal. The dimensions of teliospores of the freshly collected German samples 25–27 are given in Table 1 and were representative of all teliosporebearing material. Drawings of typical teliospores of P. distincta (sample 14) and P. lagenophorae (sample 15) collected three years previously at the same site as samples 25–27 were very similar to those collected elsewhere and showed the typical species-specific features, notably the wide teliospore stalk and the presence of three-celled teliospores only in P. lagenophorae (Figs 1–2). Properties of sequences The complete genomic DNA sequences between the primers ITS5 and ITS4 were obtained for 14 samples of Puccinia distincta, six of P. lagenophorae, and three of P. obscura. All P. distincta samples, which had been collected in the UK (England and Wales), France, Germany, Slovenia and Italy, were identical to each other with the exception of the German sample 14 which lacked 1 bp at position 205 (Fig. 3). This deletion was not found in sample 25, collected at the same site three years later. All six P. lagenophorae sequences from the UK, Germany and Slovenia were identical to each other, as were the three P. obscura sequences from England and Germany. Using P. distincta as a reference for a careful base-by-base comparison, P. lagenophorae showed the following three points of difference ; change from A to C at position 61, insertion of a single C at 191, and change of T to C at 570 (Fig. 3). In contrast, when taking multi-bp substitutions or deletions as a single mutational event, the sequence of P. obscura differed from that of P. distincta at 37 points (alignments available upon request from R.W.S.W.). These differences between the three rusts were found even in samples 25–27, collected on the same day within about 200 m of each other. The sequence of P. distincta including the two primers had a length of 677 bp except for sample 14 (676 bp), that of P. lagenophorae 678 bp, and that of P. obscura 670 bp. In P. distincta, the 3k end of 18S rDNA contained 54 bp, the ITS1 region 209 bp, the 5.8S rDNA 156 bp, the ITS2 region 197 bp, and the 5k end of the 26S rDNA 61 bp (see Fig. 3). In addition to the above three species, the sequence alignment used for phylogenetic analysis included P. graminis (sample 20), P. poarum (sample 19) and 18 sequences obtained from GenBank1 (Table 3).
21 Including the gap matrix, the total length of aligned and cut sequences was 700 bp, comprising 18S rDNA (1 bp), ITS1 (264 bp), 5.8S rDNA (156 bp), ITS2 (260 bp) and 26S rDNA (19 bp). The matrix contained 259 constant characters, 105 variable but parsimony-uninformative and 336 variable and parsimony-informative characters. The mean nucleotide composition of all sequences showed a high A–T content (67.7%). Phylogenetic analysis Two most parsimonious trees (Fig. 4) were obtained (length, 1313 steps ; consistency index CI, 0.570 ; retention index RI, 0.645 ; rescaled consistency index RC, 0.368). The grouping of Puccinia distincta with P. lagenophorae, as well as the grouping of these two species with P. obscura, were shown in both most parsimonious trees and were supported by bootstrap values of 99.2 % and 93.8 % (respectively) from 1000 computed parsimony trees (Fig. 6). The distance tree with the smallest minimum evolution score (1.969) is shown in Fig. 5. The consensus tree of 1000 samples (Fig. 6) supports the grouping of P. distincta with P. lagenophorae (99 %), and of these two species with P. obscura (95.8 %). Good bootstrap support from parsimony (90.1 %) and distance (89 %) analyses was obtained also for the grouping of P. carduorum with these three rusts. Among the other rust fungi used in the analysis, strong phylogenetic groupings were observed in both types of analysis for the three non-Pucciniaceae (i.e. Peridermium harknessii, Cronartium ribicola and Pucciniastrum goeppeltianum), and the four rust species associated with Euphorbiaceae/Fabaceae (namely Uromyces fabae, U. pisi, U. scutellatus and U. striatus). Five rusts associated with Brassicaceae (i.e. P. aberrans, P. codyi, P. consimilis, P. monoica and P. thlaspeos) also formed a moderately well-supported cluster, with a bootstrap support of 82.3 % in the parsimony analysis. In contrast, the cereal rusts could not be unequivocally separated from those on Brassicaceae in that P. sorghi and P. coronata f. sp. avenae were grouped weakly with the latter.
DISCUSSION The current study has shown that there were only three differences in the ITS sequences between Puccinia lagenophorae on Senecio vulgaris and P. distincta on Bellis perennis which were, however, consistently present in all British and continental European samples examined. The homogeneity of our data extends to the fact that within either species, all sequences were identical with the exception of the German P. distincta sample 14 which differed at only one position from all other German samples and those from all other countries. This is in agreement with the known epidemiological data for both species which are recent arrivals in Europe, having caused epidemics only within
Puccinia distincta and P. lagenophorae
22 Helicobasidium sp.
82.3
P. thlaspeos P. monoica P. sorghi P. drabae
65.6 69.9 97.0 65.8
Puccinia aberrans P. codyi P. consimilis
87.3 74.0
P. coronata P. graminis P. striiformis
82.3 73.3 65.9 62.2 53.8 84.5
53.6 81.8
81.0 68.2
P. poarum P. carduorum
89.4 90.1
99.2 93.8
99.3 95.6 95.2
87.5
100 100
P. distincta P. lagenophorae P. obscura Uromyces fabae U. scutellatus U. striatus U. pisi Perid. harknessii Cron. ribicola
87.9 89.0
99.0 95.8
89.7 93.8 96.3
88.0
99.6 100
Pucciniastrum goeppeltianum
Fig. 6. Parsimony (left) and distance (right) 50 % majority rule consensus trees of the ITS1-5.8S-ITS2 sequences of rust fungi. Values associated with branch-points indicate the percentage (bootstrap support) from 1000 bootstrapped trees at which the branches clustered together.
the past 40 years. Longer-established rust populations might have been expected to show larger variations between different isolates of the same species (Pfunder et al. 2001). The consistent differences between P. distincta and P. lagenophorae indicate that interbreeding does not occur in nature, thus supporting our previous contention that the rust on daisies should be regarded as a species distinct from P. lagenophorae, which makes its naming by McAlpine (1906) particularly apt in retrospect. Differences between these two species can be regularly observed in terms of current (Jurc & Weber 2001) and historical (Preece et al. 2000) field distribution data. Briefly, the European epidemic of P. lagenophorae on Senecio spp. arose in 1960 whereas the new daisy rust became a pan-European epidemic only from 1996 onwards. The comparative measurements of teliospore morphology (Figs 1–2) and dimensions (Table 1) are based on material collected within less than 200 m on two occasions separated by a three-year interval. Their excellent agreement with data reported previously for British and south-east European specimens (Weber et al. 1998b, Mu¨ller 2000, Jurc & Weber 2001) supports the temporal and spatial identity of P. distincta and P. lagenophorae as different species. Koike & Scholler (2001) noted the occurrence of a new rust on B. perennis in North America which they regarded as identical with one on Senecio spp. which is also a recent arrival there. Under controlled conditions, these authors succeeded in infecting B. perennis with rust inoculum obtained from S. vulgaris, as had already
been demonstrated by Wilson et al. (1965) in Britain. At present, it is difficult in the absence of morphological or DNA sequence information to relate the identity of the American daisy rust to the European rust. An examination of rusts on Bellis and Senecio worldwide would be very interesting but may provide only short-term answers as new races or species seem to spread rapidly. Compared with the small ITS sequence differences between P. lagenophorae and P. distincta, the phylogenetic distance between these rusts and P. obscura was considerably greater (37 points of difference). Nonetheless, all three rusts formed a highly coherent cluster in both types of phylogenetic analysis used, and were wellseparated from another rust of Asteraceae, P. poarum (aecial host, Tussilago farfara). The clustering of correlated short- and long-cycled species of grass rusts was observed by Zambino & Szabo (1993) who found that they differed by about 5–10 bp. In contrast, the same authors found that ITS sequences of different special forms of any one species of grass rust varied by only 1–3 bp. In other well-established rusts, a similar distance has been found between different isolates of the same species (Vogler & Bruns 1998, Pfunder et al. 2001), whereas Vogler & Bruns (1993, 1998) found that microand macrocyclic pine rust fungi (e.g. Peridermium harknessii, Cronartium quercuum f. sp. banksiana) had identical ITS sequences but were nonetheless given different names based on differences in the morphology and life-cycles. Numerical base-pair differences, therefore, have to be interpreted within the biological context
R. W. S. Weber, J. Webster and G. Engel of the species concerned. In the case of the recent European arrivals P. lagenophorae and P. distincta, a consistent difference of 3 bp is clearly more significant than it would be between two long-established rust species showing a certain degree of intra-species variation. Rusts with a reduced life-cycle on one host plant are considered to be derived from macrocyclic heteroecious precursor species (Malloch 1995). This relationship is called Tranzschel’s Law if the teliospore state of the derived species is produced on the aecial host of the ancestral species (Shattock & Preece 2000). If we accept a common phylogenetic origin between P. obscura, P. distincta and P. lagenophorae based on Tranzschel’s Law (Weber et al. 1998b), P. distincta is a demicyclic (-opsis type) derivative of P. obscura as indicated by their sharing of a common aecial host (B. perennis) and close morphological similarity and dimensions of their teliospores (Grove 1913). Concerning the relationship of P. lagenophorae to the other two species, our results indicate that P. distincta and P. lagenophorae are more closely related to each other than either is to P. obscura, and this could be taken to imply that P. lagenophorae, like P. distincta, has arisen from P. obscura. There are, however, some obvious difficulties with this hypothesis relating to the place and time where the required evolutionary steps might have occurred. The available evidence suggests that P. lagenophorae is an Australasian species which is known in Europe only since 1960 (Wilson & Walshaw 1963, Wilson & Henderson 1966). In contrast, P. obscura seems to be a long-established European species (Plowright 1889, Grove 1913). P. distincta was first described on cultivated daisies in Australia in 1895 (McAlpine 1906), and we have recently verified that P. obscura was also present in Australia on B. perennis at that time (Weber et al. 1998b). It is possible that P. obscura was introduced into Australia in the mid-nineteenth century, and that P. distincta and P. lagenophorae arose there and were inadvertently reintroduced into Europe on two separate occasions. However, these speculations may never be resolved due to our lack of information on the historical distribution of hosts and pathogens. It would be interesting to compare the sequences of a wider range of rusts on Asteraceae, especially those placed in synonymy with P. lagenophorae by Wilson et al. (1965).
ACKNOWLEDGEMENTS We thank Philip M. Booth, Paolo Davoli, C. Terence Ingold, William B. Jones, Dusˇ an Jurc, Leslie J. Lundy and Thomas F. Preece for collecting rust samples, and we are most grateful to Heidrun Anke (Institute of Biotechnology and Drug Research IBWF, Kaiserslautern) and Timm Anke (University of Kaiserslautern) for their support.
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Corresponding Editors: J. Sugiyama & D. L. Hawksworth