Morphological and molecular variation between Australian isolates of Puccinia menthae

Mycol. Res. 103 (12) : 1505–1514 (1999)

1505

Printed in the United Kingdom

Morphological and molecular variation between Australian isolates of Puccinia menthae

J. E D W A R DS, P. K. A D E S, D. G. P A R B E RY, G. M. H A L L O R A N A N D P. W. J. T A Y L O R Institute of Land and Food Resources, The University of Melbourne, Parkville, Victoria 3052, Australia

Puccinia menthae is highly variable, with several varieties and variants within varieties. Two principal groups of races, nominated spearmint rust and peppermint rust, have been recognized according to their host range on commercially-important Mentha species, yet both belong to the same variety, P. menthae var. menthae. Ten collections of P. menthae teliospores from four Mentha species in Victoria, Australia, were examined using light microscopy and SEM. The teliospores from M. spicata, M.icordifolia and M. suaveolens, all hosts to spearmint rust, were verrucose with two equally-sized cells, whereas those from M.ipiperita, host to peppermint rust, were generally smooth-walled, and the apical cell was larger and thicker-walled than the basal cell. Fifteen isolates of P. menthae collected from four Mentha species in Victoria were assessed for genomic variation using RAPD analysis and PCRgenerated length polymorphism of the ITS region of rDNA. Six out of 113 RAPD primers amplified reproducible marker profiles, 58 polymorphic bands were scored and simple matching distances were computed between the isolates. UPGMA cluster analysis was used to produce a dendrogram and non-metric multi-dimensional scaling to produce a two-dimensional map of the isolates. The spearmint rust and peppermint rust isolates clustered into two non-overlapping groups, providing good evidence that there is limited gene flow between them. All isolates had the same sized ITS fragments. The taxonomic implications of these results are discussed and it is suggested that the peppermint and spearmint rusts should be accorded separate varietal or specific status.

The mint rust fungus, Puccinia menthae Pers., is an autoecious, macrocyclic rust occurring on wild and cultivated members of the Lamiaceae throughout the world (Laundon & Waterston, 1964). The host range is extensive, with hosts in 20 genera being listed (species of Blephilia, Bystropogon, Calamintha, Clinopodium, Cunila, Hedeoma, Hyssopus, Lycopus, Melissa, Mentha, Micromeria, Monarda, Monardella, Nepeta, Ocimum, Origanum, Pycnanthemum, Satureja, Thymus and Ziziphora). P. menthae was first found in Australia in 1884 on an indigenous mint, Mentha laxiflora Benth., and then in 1904 on introduced pennyroyal, M. pulegium L., growing in Victoria (McAlpine, 1906), but the rust was not reported again until 1967 when it was recorded on commercially-grown spearmint, M. spicata L., near Sydney, New South Wales (Walker & Conroy, 1969). Morphological and physiological variation within P. menthae has long been recognized. Some forms were first described as separate species, but later incorporated into P. menthae by Arthur (1934). A number of varieties have also been described (Baxter, 1959, 1960), including P. menthae var. menthae which includes the type specimen and is found mainly on hosts within Mentha. Physiologic specialization on the basis of host reactions has often been noted (Vergovsky, 1935, Niederhauser, 1945 ; Baxter & Cummins, 1953 ; Murray, 1961 ; Fletcher, 1963 ; Beresford, 1982 ; Johnson, 1995 ; Edwards et al., 1999), and two principal groups of races infecting Mentha have been recognized (Baxter & Cummins, 1953 ; Murray, 1961 ; Beresford, 1982 ; Ball et al., 1992 ;

Johnson, 1995). One group (spearmint rust) infects M. spicata, spearmint, but not M.ipiperita L., peppermint, and the other group (peppermint rust) infects M.ipiperita but not M. spicata, yet both groups infect M.igracilis Sole (syn. M.icardiaca J. Gerard ex Baker), Scotch spearmint. Both groups are considered the same variety, P. menthae var. menthae, although some teliospore variation between them has been observed (Baxter, 1959). Recent technological advances, such as the use of prism image analysis, have shown that the urediniospores of P. menthae produced on peppermint and spearmint hosts are also morphologically distinct (Ball et al., 1992). Microscopic examination of teliospores from M. pulegium collected by McAlpine in 1904 (Walker & Conroy, 1969) revealed a morphological variant with smooth walls and unequally-sized cells that is outside the published variety descriptions for P. menthae (Grove, 1913, Arthur, 1934, Baxter, 1959). In an initial investigation we found teliospores very similar in appearance to those collected by McAlpine on M.ipiperita growing in Victoria, but never on M. spicata or other spearmint rust hosts. This prompted us to carry out a detailed comparison of teliospore morphology of P. menthae var. menthae on several Mentha species, the results of which are reported here. We also used molecular biology techniques to determine genetic similarity\distance relationships between other isolates from the same hosts. The use of molecular biological techniques has enabled

Variation within Puccinia menthae substantial advances to be made in measuring genetic variation in fungal populations. Of these, PCR techniques have been particularly useful for analysing genomic variation in biotrophic fungi, such as rusts and powdery mildews, as only minute quantities of DNA are required for analysis (McDonald, 1997). Over the past five years, the RAPD assay developed by Williams et al. (1990) has been used to examine genetic variation in species of several rust genera, including Puccinia (Chen, Roland & Leung 1993 ; Chen, Roland & Leung, 1995 ; Kolmer, Liu & Sies, 1995 ; Park, Jahoor & Felsenstein, 1996 ; Park, Burdon & Jahoor, 1997 ; Engel et al., 1997 ; Jennings, Newton & Buck, 1997), Cronartium (Doudrick, Nelson & Nance, 1993 ; Hamelin, Doudrick & Nance, 1994), Uromyces (Maclean et al., 1995) and Melampsora (Pei et al., 1997). As its name suggests, RAPD analysis does not target any specific region of the chromosomal DNA, and therefore gives a useful estimate of how much total genomic variation exists within and between given populations. A complementary PCR-based technique to RAPD is that which targets a specific region of the DNA. The ribosomal genes of eukaryotes are arranged in highly conserved tandem repeat units (Hillis & Dixon, 1991), and different regions of these rDNA repeat units evolve at different rates. It is possible, therefore, to choose a genomic region that has evolved at a rate suitable for differentiating at the taxonomic level under investigation. Among the spacer regions of this unit, the internal transcribed spacers (ITS) are useful for interspecific comparisons, as variation between organisms in the ITS region is presumed to be negligible within most species yet significant between species (Hillis & Dixon, 1991 ; Goffinet & Bayer, 1997). Length variations of the ITS, generated using fungus-specific oligonucleotide primers, have been used to examine phylogenetic relationships in several fungi (Gardes et al., 1991 ; Nazer et al., 1991 ; Paolocci et al., 1995 ; Liddell & Onsurez-Waugh, 1996 ; Chillali et al., 1997 ; Rohel et al., 1997). Since little is known about the extent of diversity in Puccinia menthae in Australia, the aims of this investigation were to assess the degree of genetic variation present in Australian populations of P. menthae, and to examine the relationship between the spearmint and peppermint rust groups using RAPD analysis, PCR-generated length variation within the ITS region of the rDNA repeat unit, and comparative morphology. MATERIALS AND METHODS Morphological variation Teliospores. Teliospores were collected from four Mentha hosts in 1994, 1996 and 1997. Five collections were from M.ipiperita, host of peppermint rust, and five were from M. spicata, M.icordifolia and M. suaveolens Ehrh. (applemint), all hosts of spearmint rust. The host specificities were confirmed during experiments examining physiologic specialisation (Edwards et al., 1999). Teliospores were lifted from the sori with a fine needle, mounted in lactic acid and examined under bright field i1000 magnification. Photographs were taken using a Leica photomicroscope with Nomarski Interference optics.

1506 From each collection, 50 mature teliospores that had broken off from their points of attachment were measured for : (i) apical cell width and length, measured to the outside of the wall ; (ii) basal cell width and length, measured to the outside of the wall ; (iii) range of wall thickness, measured at the thickest and the thinnest parts ; (iv) septum thickness, measured at the centre of the septum ; (v) pedicel length ; (vi) apical papilla thickness, measured from the centre of the pore in the (vii) spore wall to the top of the papilla ; (viii) apical papilla width at the base. Data were analysed by ANOVA using a hierarchical model : Yijk l µjGijIj(i)jEijk, where µ l mean, Gi l effect of rust groups i, Ij(i) l effect of isolate j within group i, and Eijk l error. Isolates were considered to be random samples, and the group mean square was tested against the isolates within group mean square, rather than overall residual. The relationship between the dimension of the apical and basal cells of the teliospores was examined using a type of covariance analysis, in which the length or width of the basal cell is used as a covariate for the corresponding apical cell dimension. First, the full model is fitted : Model 1 :

Aij l µjGijbi BijjEij,

where Aij l the apical cell measurement and Bij l the corresponding basal cell measurement, µ l mean, Gi l the additive effect of group i, bi l the linear regression coefficient for group i, and Eij l a residual. A reduced model is then fitted : Model 2 :

Aij l µjGijbBijjEij.

This is the same as model 1 except that only one slope (b) is fitted for both groups. A one degree of freedom F test for homogeneity of slopes between the two groups (i.e. H : ! b l b ) is carried out by dividing the difference in residual " # sums of squares between the models by the residual mean square of model 1. Scanning electron microscopy was used to examine teliospore wall ornamentation. Fragments of leaves from M.ipiperita cv. Todd’s Mitcham and M. spicata cv. Native on which teliospores were being produced were air-dried, mounted on an aluminium stub with a carbon sticky tape and then sputter-coated with gold in an Edwards Sputter-coater 5150B. The teliospores were then examined using a Philips XL30 FEG scanning electron microscope over a range of magnifications from i750 to i3600. Urediniospores. Urediniospores collected from M.ipiperita cv. Todd’s Mitcham and M. spicata cv. Native were mounted in lactic acid, examined at i1000 magnification using bright field illumination. The length and width of 20 spores from

J. Edwards and others each were measured. The data were analysed for significant differences between the two groups of rusts by t test. Molecular variation Rust isolates. Infected leaves of P. menthae were collected from 15 commercial fields or home gardens in the state of Victoria, Australia, during 1993–95 from different mint host species (Table 1), and a single-spore isolate from each was established using the technique described by Roelfs, Singh & Saari (1992). The isolates were maintained on their original host genotypes, except for three (95-12, 95-14, 95-18) which were maintained on susceptible hosts (M.igracilis, M.ipiperita cv. Todd’s Mitcham and M. spicata cv. Native respectively), all kept in cages in a glasshouse at 22m\14 mC day\night temperature under natural light. Cages consisted of moulded fibreglass frames, 30 cmi30 cmi67 cm, with solid bases, polythene-covered sides, perspex doors secured with magnetic strips, and the tops vented with 25 µm pore-size dairy filter screen (ACE S272, Australian Catering Equipment Pity, Croydon, Vic.). Five mint hosts were represented : peppermint (M.ipiperita cv. Todd’s Mitcham), eau-decologne mint (M.ipiperita ssp. citrata (Ehrh.) Briq.), spearmint (M. spicata cv. Native), Scotch spearmint (M.igracilis) and garden mint (M.icordifolia Opiz ex Fresen., also considered a form of M. spicata (Wiersema, 1998)). Isolates from two separate geographic regions of Victoria were represented : the river valleys of the north east where peppermint is grown for essential oil production, and metropolitan Melbourne 300 km to the south. Urediniospores from each isolate were collected by tapping infected leaves over aluminium foil, and were stored at 4m until use. DNA extraction. Because P. menthae is not readily cultured axenically, DNA was extracted directly from the urediniospores. For each isolate, 15–50 mg of urediniospores were mixed with sterile sand and liquid nitrogen, and ground into a fine powder with a mortar and pestle (Chen et al., 1993). The DNA was then extracted using the adapted CTAB method of Taylor et al. (1995) and quantified both by measuring the absorbance at 260 nm by spectrophotometer and by uv visualization on an 0n7 % agarose gel stained with ethidium bromide. Individual stock DNA concentrations were adjusted to 10 ng µl−" prior to use in PCR reactions. DNA was extracted three times from isolate 93-09 (in 1995, 1996 and 1997) and twice from isolate 95-21 (in 1996 and 1997), giving a total of 18 DNA samples (Table 1). RAPD-PCR materials and amplification reaction conditions. Of the 113 decamer oligonucleotide primers that were tested for amplification of P. menthae genomic DNA, 108 were arbitrarily chosen primers from Operon Technologies Inc., U.S.A., and five were high-GC primers designed by Kubelik & Szabo (1995) and custom-made by Life Technologies Pty, U.S.A. Reaction volumes of 25 µl were optimized to contain 40 ng of DNA template, 0n8 U of Taq DNA polymerase (Boehringer–Mannheim Biochemica, Germany), 0n24 mmol each of dATP, dCTP, dGTP and dTTP, 0n1 µ of primer, and PCR buffer with a final concentration

1507 of 0n01  Tris-HCl\3 m MgCl \0n05  KCl\0n1 mg ml−" # gelatin, pH 8n3 (Boehringer–Mannheim Biochemica, Germany). PCR was performed with a PTC-100 thermocycler (MJ Research, Inc., U.S.A.) programmed as follows : initial denaturation step at 94m for 1 min followed by 35 cycles of 94m for 10 s, 40m for 30 s and 72m for 1 min, with a final extension at 72m for 5 min. Amplification products were separated by electrophoresis on 1n4 % agarose gel in TAE buffer, stained with ethidium bromide and visualized with uv illumination. The molecular weight marker used was λ DNA digested with EcoRI and HindIII (Promega Inc., U.S.A.). The PCR with each primer was performed at least twice to check consistency of results. RAPD data analysis. PCR products from each DNA sample were scored as either present (1) or absent (0). Only distinct, reproducible bands were scored. Data analysis was conducted using SAS (SAS Institute, 1996). Simple matching distances (Sneath & Sokal, 1973) were calculated using a programme written by the authors. Non-metric multidimensional scaling was carried out using Proc MDS in SAS Release 6.12 to construct a two-dimensional map of the isolates, and UPGMA cluster analysis using Proc Cluster. The dendrogram was produced from the output of Proc Cluster using Proc Tree. Analysis of molecular variance (AMOVA) (Excoffier, Smouse & Quattro, 1992) using WINAMOVA was performed on the similarity matrix to apportion the level of variation within and between the spearmint and peppermint rust groups. (WINAMOVA version 1.04, developed by Laurent Excoffier, University of Geneva, Switzerland, is freely available from http:\\iubio.bio.indiana.edu:71\ROO$ Data\biology\ ibmpc\winamova). AMOVA produces analogues of variance components and F-statistics to analyse the genetic structure of populations using molecular information. While specifically developed for RFLP data of haplotypes (Excoffier et al., 1992), it has been applied to other types of data, including RAPD data of diploid organisms (Huff, Peakall & Smouse, 1993 ; Peakall, Smouse & Huff, 1995 ; Michalakis & Excoffier, 1996 ; Scott et al., 1997). ITS-PCR materials and amplification reaction conditions. Primers used for amplification of the ITS region were ITS4 (dTCCTCCGCTTATTGATATGC) and ITS5 (dGGAAGTAAAAGTCGTAACAAGG) (White et al., 1990). Reaction volumes of 25 µl included 1n0 µ of each primer, 100 ng of DNA template, 0n8 U of Taq DNA polymerase (Boehringer–Mannheim Biochemica, Germany), 0n24 mmol each of dATP, dCTP, dGTP and dTTP, and PCR buffer with a final concentration of 0n01  Tris-HCl\3 m MgCl \0n05  KCl\0n1 mg ml−" gelatin, pH 8n3 # (Boehringer–Mannheim Biochemica, Germany). PCR was performed with a PTC-100 thermocycler (MJ Research, Inc., U.S.A.) programmed as follows : initial denaturation step at 95m for 3 min followed by 35 cycles of annealing at 60m for 1 min, extension at 72m for 1 min and denaturation at 93m for 1 min, with a final extension at 72m for 10 min. Amplification

Variation within Puccinia menthae

1508

Table 1. Origin of the single spore isolates of Puccinia menthae used for DNA extraction DNA sample

Isolate no.

Mp-01 Mp-02 Mp-03 Mp-04 Mp-05 Mp-06 Mp-07 Mp-08 Mp-09 Mp-10 Mp-11 Mpc Mg Ms-01 Ms-02 Mc-01 Mc-02 Mc-03

93-09 93-09 93-09 93-08 95-21 95-21 93-01 95-16 93-02 95-02 95-17 95-14 95-09 95-20 93-12 95-12 95-18 95-10

(95) (96) (97) (96) (97)

Host

Origin

M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita L. cv. Todd’s Mitcham M.ipiperita ssp. citrata (Ehrh.) Briq. M.igracilis Sole M. spicata L. cv. Native M. spicata L. cv. Native M.icordifolia Opiz M.icordifolia Opiz M.icordifolia Opiz

Commercially cultivated, Ovens Valley, N.E. Victoria Commercially cultivated, Ovens Valley, N.E. Victoria Commercially cultivated, Ovens Valley, N.E. Victoria Commercially cultivated, Ovens Valley, N.E. Victoria Research plot, Ovens Valley, N.E. Victoria Research plot, Ovens Valley, N.E. Victoria Commercially cultivated, Ovens Valley, N.E. Victoria Commercially cultivated, Ovens Valley, N.E. Victoria Commercially cultivated, Buffalo River Valley, N.E. Victoria Commercially cultivated, Ovens Valley, N.E. Victoria Commercially cultivated, Corryong, N.E. Victoria Garden, Melbourne, Victoria Research plot, Ovens Valley, N.E. Victoria Research plot, Ovens Valley, N.E. Victoria Glasshouse, Ovens Valley, N.E. Victoria Garden, Melbourne, Victoria Garden, Melbourne, Victoria Garden, Melbourne, Victoria

products were resolved and visualized as described for the RAPD analysis, using a 100 bp DNA ladder molecular weight marker (no. 323-1S, New England BioLabs Inc., Australia). RESULTS Morphological variation There was significant variation among the isolates within each rust group for each teliospore morphological character measured, but taking this into account by using isolates as the error term, the two rust groups were also significantly different (P 0n05) in several of these characters (Table 2). Peppermint rust teliospores (Fig. 1) have thicker cell walls and septa, longer pedicels and wider apical papillae than the spearmint rust teliospores (Fig. 2). They also have two unequally-sized cells, the apical cell being larger and thickerwalled than the basal cell, while the spearmint rust teliospores have equal-sized cells with uniform wall thickness.

There was significant differences between isolates within groups, but also between groups, for apical cell length and width. The two groups did not differ significantly in basal cell length and width, although there was significant variation between isolates within groups for these characters. The covariance analysis (Table 3) failed to show any relationship between apical and basal cell length, either within group (model 2) or overall (model 1). The individual measurements overlap, but the group means are clearly different (Fig. 3). There was, however, a highly significant relationship between apical and basal cell widths and the slopes were significantly different between the two groups (Fig. 4). For spearmint rust the fitted regression equation was : apical cell width l 8n23j0n611 basal cell width, while for peppermint rust the equation was : apical cell width l 17n78j0n275 basal cell width. Differences in teliospore wall ornamentation between the

Table 2. Comparison of teliospore morphology of peppermint and spearmint rust isolates of Puccinia menthae

Width of spore Length of spore Wall thickness Thickest part Thinnest part Width of apical cell Length of apical cell Width of basal cell Length of basal cell Pedicel length Septum thickness Apical papilla Width at base Thickness

Peppermint rust (µm)

Spearmint rust (µm)

P-value

Range

Mean

Range

Mean

(Group)

(Isolate)

21–28 24–37

23n8 31n2

19–26 25–34

22n1 29n1

0n40 0n07

0n00 0n00

1n4–3n2 0n5–1n8 20–28 14–23 16–26 6–16 3–64 0n5–2n0

2n4 1n0 23n6 18n0 21n2 12n8 23n1 1n1

0n9–2n8 0n5–1n4 17–25 12–18 18–25 10–18 4–28 0n5–0n9

1n8 0n7 21n3 15n6 21n4 13n7 14n4 0n7

0n00 0n00 0n01 0n00 0n82 0n18 0n05 0n00

0n00 0n00 0n00 0n00 0n00 0n00 0n00 0n00

8–13 2–5

10n4 3n5

8–11 3–5

9n7 3n6

0n05 0n59

0n00 0n00

J. Edwards and others

1509 24

Apical cell length (lm)

20

1

16

12

8 8

12 16 Basal cell length (lm)

20

Fig. 3. Puccinia menthae teliospores : apical cell length versus basal cell length, peppermint rust (j), spearmint rust (#).

Molecular variation

2 Figs 1, 2. Puccinia menthae spores in lactic acid. Bars l 40 µm. Fig. 1. Teliospores and urediniospores from Menthaipiperita (peppermint rust). Note unequally-sized cells. Fig. 2. Teliospores from Mentha spicata (spearmint rust). Note equally-sized cells.

two rust groups were most apparent under SEM (Figs 5, 6). The spearmint rust teliospores are uniformly verrucose, while the peppermint rust teliospores have smooth basal cells and mostly smooth apical cells. Urediniospores collected from M.ipiperita measured 19–28i19–26 µm and those from M. spicata 21-30i1928 µm. They were not significantly different from each other (P l 0n1 and 0n4 for length and width, respectively).

RAPD-PCR analysis. Six out of 113 primers amplified distinct and reproducible marker profiles from the 18 P. menthae DNA samples (Table 4). Each primer amplified between 9 and 18 scorable DNA markers, which ranged in size from 100 to 3000 bp. In total, 83 bands were scored, of which 58 were polymorphic (70 %). None of the five high-GC primers (Kubelik & Szabo, 1995) was successful in amplifying P. menthae DNA. Each of the six primers showed some degree of variation (Table 4). Primer OPX-12 showed the greatest overall degree of polymorphism between the DNA samples (94 %). DNA had been extracted annually from isolate 93-09 to generate three samples, Mp-01, Mp-02 and Mp-03 (1995, 1996 and 1997 respectively), and isolate 95-21 to generate two samples, Mp-05 and Mp-06 (1996 and 1997 respectively). Mp-01 and

Table 3. Analyses of covariance for apical and basal cell length, and apical and basal cell width, of peppermint and spearmint rust teliospores of Puccinia menthae Dependent variable Apical cell length

Apical cell width

Source of variation

..

Sum of squares

Total Group Basal cell length within group (model 1) Homogeneous regressions Residual (model 1) Basal cell length (model 2) Residual (model 2)

452 1 2 1 449 1 450

1813n24 676n56 5n36 0n04 1131n32 5n22 1131n46

Total Group Basal cell width within group (model 1) Homogeneous regressions Residual (model 1) Basal cell width (model 2) Residual (model 2)

451 1 2 1 448 1 442

1696n94 586n79 272n76 37n90 810n38 234n87 848n28

Mean squares

F-ratio

P-value

2n68 0n04 2n52 5n22 2n51

1n06 0n02

0n34 0n89

2n08

0n15

136n38 37n90 1n81 234n87

75n40 20n95

0n00 0n00

124n32

0n00

Variation within Puccinia menthae

1510 Table 4. Decamer oligonucleotide primers selected for RAPD analysis of Puccinia menthae isolates and the level of polymorphism produced

Apical cell width (lm)

30

25

p

20 s

15 15

20 25 Basal cell width (lm)

30

Fig. 4. Puccinia menthae teliospores : apical cell width versus basal cell width, peppermint rust (j), spearmint rust (#). Regression lines fitted by covariate analysis : peppermint rust (p), spearmint rust (s).

5

Primer

Sequence 5h-3h

Scorable bands (no.)

Polymorphic Polymorphism bands (no.) (%)

OPU-01 OPU-05 OPU-13 OPU-15 OPX-12 OPX-13

ACGGACGTCA TTGGCGGCCT GGCTGGTTCC ACGGGCCAGT TCGCCAGCCA ACGGGAGCAA

14 9 16 12 18 14

9 7 12 8 16 6

64 78 75 67 94 43

place. Similarly, Mp-06 differed considerably from Mp-05, also suggesting that contamination of isolate 95-21 had occurred over this time. To ensure that only single-spore isolates, not mixtures of genotypes, were compared, RAPD profiles from Mp-02, Mp-03 and Mp-06 were excluded from subsequent analyses. The distance matrix (calculated as 1ksimilarity coefficient) for the isolates using simple matching distance is presented in Table 5. The two-dimensional map constructed using nonmetric multidimensional scaling (Fig. 7), and the dendrogram constructed using the UPGMA algorithm (Fig. 8), clearly differentiated two non-overlapping clusters. One cluster contained the isolates from M.ipiperita and the other, those from M. spicata, M.igracilis and M.icordifolia. The AMOVA indicated that, of the total variation present, 63 % was between the two rust groups, while 37 % was withingroup variation (Table 6). Length variation within the ITS regions. Length variation within the ITS region was directly assessed by comparison of the amplified DNA, after electrophoresis, with the 100 bp ladder molecular marker. A strong, though diffuse, band at 650–700 bp was common to all isolates, and a smaller band at 600 bp was present in some isolates, varying from strong to faint, and absent from others. The presence of this smaller variable band was not associated with any apparent grouping of the isolates. DISCUSSION

6 Figs 5, 6. Wall ornamentation of P. menthae teliospores. SEM, bars l 10 µm. Fig. 5. From M.ipiperita (peppermint rust). Fig. 6. From M. spicata (spearmint rust).

Mp-02 differed by three bands only, suggesting that some mutations may have occurred over the twelve months. Considerable change was evident between these and Mp-03, however, strongly suggesting that contamination had taken

The RAPD analysis has revealed two distinct groups, one containing the isolates from M.ipiperita, and the other, isolates from M. spicata, M.igracilis and M.icordifolia. The two groups correspond to peppermint and spearmint rusts as mentioned previously. This has been confirmed in experiments examining the extent of physiologic specialisation based on the reactions of Mentha species inoculated in the glasshouse (Edwards et al., 1999). The present study has shown that the two groups are also distinguishable on the basis of teliospore morphology, with many teliospore characters significantly different between the two groups, but they do not differ in the length of their ITS regions. The genetic variation evident within the spearmint rust cluster was not correlated with either location or host species. Within the peppermint rust cluster, however, the isolates showed some separation on the basis of location. Mp-01, Mp04, Mp-05, Mp-07, Mp-08 and Mp-10 were very similar to

J. Edwards and others

1511

Table 5. Genetic distances between 15 Puccinia menthae isolates using simple matching distance (Sneath & Sokal, 1973) DNA sample

Mp-01

Mp-04

Mp-05

Mp-07

Mp-08

Mp-09

Mp-10

Mp-11

Mpc

Mg

Ms-01

Ms-02

Mc-01

Mc-02

Mc-03

Mp-01 Mp-04 Mp-05 Mp-07 Mp-08 Mp-09 Mp-10 Mp-11 Mpc Mg Ms-01 Ms-02 Mc-01 Mc-02 Mc-03

0n00 0n01 0n03 0n03 0n09 0n15 0n07 0n07 0n07 0n39 0n42 0n32 0n33 0n36 0n33

0n00 0n02 0n02 0n08 0n16 0n06 0n08 0n08 0n38 0n40 0n30 0n32 0n35 0n32

0n00 0n02 0n08 0n16 0n08 0n08 0n08 0n40 0n43 0n33 0n34 0n37 0n34

0n00 0n06 0n16 0n06 0n08 0n08 0n38 0n40 0n30 0n32 0n35 0n32

0n00 0n17 0n05 0n14 0n14 0n35 0n42 0n32 0n30 0n34 0n30

0n00 0n17 0n10 0n12 0n40 0n40 0n37 0n36 0n39 0n38

0n00 0n11 0n11 0n33 0n42 0n27 0n28 0n32 0n28

0n00 0n02 0n37 0n44 0n34 0n33 0n38 0n35

0n00 0n39 0n44 0n34 0n35 0n36 0n35

0n00 0n27 0n17 0n16 0n15 0n14

0n00 0n21 0n25 0n19 0n18

0n00 0n12 0n09 0n06

0n00 0n17 0n09

0n00 0n08

0n00

Sample

0·3

Mp_01 Mp_04

0·2

Dimension 2

Ms_01

Mp_07

0·1

0·0

Mp_05

Mp_09

Mp_08

Mp_01 Mp_04 Mp_05 Mp_07 Mpc Mp_10 Mp_11 Mp_08

Mp_10 Ms_02

Mp_11 Mc_02

Mc_03

Mpc

–0·1 Mc_01

–0·2 –0·2

Mg

Mp_09 Mg

– 0·1

0·0 0·1 Dimension 1

0·2

0·3

Fig. 7. Two-dimensional map of 15 Puccinia menthae isolates constructed using non-metric multidimensional scaling from simple matching distances (Sneath & Sokal, 1973). Isolates Mp-01 to Mp-11 from M.ipiperita, Mpc from M.ipiperita ssp. citrata, Mg from M.igracilis, Ms-01 and Ms-02 from M. spicata, and Mc-01 to Mc03 from M.icordifolia.

Ms_02 Mc_03 Mc_02 Mc_01 Ms_01

each other and all come from the Ovens River valley in north east Victoria, while Mp-09 comes from Buffalo River valley. Mp-11 comes from a different north east Victorian region, Corryong. Each of these regions is separated by mountainous country. Mpc, however, is very similar to Mp-11, yet comes from eau-de-cologne mint, M.ipiperita ssp, citrata, in Melbourne. P. menthae is a macrocyclic, autoecious rust which overwinters as teliospores and re-infects new spring growth by means of basidiospores, and can complete its life cycle, including sexual reproduction, on the one host plant. M.igracilis is susceptible to both spearmint and peppermint rust races, and was susceptible to all of the isolates tested in the present study (Edwards et al., 1999), so the potential for

0·00

0·10 0·20 Average distance between clusters

0·30

Fig. 8. Dendrogram of 15 Puccinia menthae isolates constructed using UPGMA cluster analysis from simple matching distances (Sneath & Sokal, 1973). Isolates Mp-01 to Mp-11 from M.ipiperita, Mpc from M.ipiperita ssp. citrata, Mg from M. gracilis, Ms-01 and Ms-02 from M. spicata, and Mc-01 to Mc-03 from M.icordifolia.

gene flow to occur between these groups either by sexual or parasexual means should, on that host, exist. The distinct clustering revealed by the RAPD profiles of the two rust types, with no evidence of intermediate profiles, is a strong indication that the two groups are genetically isolated. For example, DNA samples Mg and Mp-05 (isolates 95-09 and

Variation within Puccinia menthae

1512

Table 6. Analysis of molecular variance for 15 individuals of Puccinia menthae, using 83 RAPD markers. The total data set contains individuals from two rust groups, peppermint rust (9 individuals) and spearmint rust (6 individuals)

Source of variation

..

SSD

MSD

Variance component

% Total

Between rust groups Within rust groups (peppermint rust) (spearmint rust) Total

1 13 (8) (5) 14

0n86 0n84 (0n45) (0n39) 1n70

0n86 0n06

0n11 0n06

63n3 36n7

95-21) were collected from plants of M.igracilis and M.ipiperita, respectively, growing less than a metre apart in plots at the Ovens Research Station, yet they have a simple matching distance of 0n404, i.e. they are 40 % dissimilar. A 3-year investigation of the disease cycle of P. menthae on M.ipiperita cv. Todd’s Mitcham and on M.igracilis has shown that on M.ipiperita the uredinial cycle persists throughout the year and teliospores are produced only sparingly during winter (Edwards et al., 1995). On M.igracilis growing less than 1 m from the M.ipiperita, however, the full life cycle was observed with each of the spore stages appearing in succession. Monitoring the disease cycle has continued from 1993 to 1998, and although some teliospores are produced on M.ipiperita every winter, no spermogonia or aecia have been seen. Sexual reproduction was observed annually on the other spearmint rust hosts, M. spicata and M.icordifolia. For sexual hybridization to occur between rust genotypes, the transfer of spermatia to spermogonia of a sexually different, but compatible, strain must occur. It is possible that hybridization between peppermint and spearmint rusts is not occurring because of the absence of spermogonia on M.ipiperita, but this stage may exist at a very low frequency and not be detected by our sampling technique. The full life cycle occurs on M.ipiperita grown elsewhere (Horner, 1963 ; Laundon & Waterston, 1964 ; Beresford & Mulholland, 1987), including Tasmania, Australia (Mr F. E. Bienvenu, personal communication). It would be of value to examine isolates from regions where spermogonia of both the peppermint and spearmint rusts are produced to see whether the same level of genetic dissimilarity observed here is still apparent. Gene flow between rusts can also occur through parasexual means such as somatic hybridization, which involves ‘ the reassociation of haploid nuclei in uredinial clones in contact with one another ’ (Johnson, 1961), and would be possible on a common host as M.igracilis. There is documented evidence from other rusts that this process occurs within formae speciales, e.g. P. recondita f. sp. tritici (Park et al., 1997), between formae speciales, e.g. P. graminis f. sp. tritici and P. graminis f. sp. secalis (Burdon & Silk, 1997), and even between species, e.g. Melampsora larici-populina and M. medusae (Spiers & Hopcroft, 1994). In addition, the fusion of aeciospore or urediniospore germ tubes with flexuous hyphae of spermagonia has been demonstrated in P. graminis f. sp. tritici by Cotter (1960, cited in Harder, 1984) and Garrett & Silcoxson (1960, cited in Harder, 1984). It would appear, from the evidence presented here, that parasexuality is not occurring between the peppermint and spearmint rusts included in this study. In

P-value 0n010 0n010

Victoria, however, M.igracilis is not grown on a wide scale, so the scarcity of a common host perhaps reduces the chance for hybridization. The two rust groups are also clearly different, based on the more traditional comparison of teliospore morphology. Although the range of values for any one trait may overlap, the means are usually different, and when all the traits are considered together the two groups are quite distinct. As presently defined, P. menthae encompasses many varieties and forms based on teliospore morphology and host range, such as P. menthae var. menthae, P. menthae var. cordillerensis, P. menthae var. rugosa on Bystropogon mollis, P. menthae var. levis on Satureja odora, P. menthae var. pseudomenthae on Mentha cunninghamii and P. menthae var. basiporula on Ocimum sp. (Baxter, 1959, 1960). Savile (1984) argued against the ultrabroad species concept as it fails to distinguish between subtly distinct, but genetically isolated, pathogens. He further stated that Arthur (1934) ‘ seriously oversimplified the taxonomy of many species complexes ’ in his treatment of North American rusts. In this treatment, Arthur reduced P. monardellae Dudley & C. H. Thomps. and P. micromeriae Dudley & C. H. Thomps. to synonymy with P. menthae (Arthur, 1934), making it a species with one of the largest host ranges within the Uredinales (Fletcher, 1958). Prior to Arthur, however, Sydow (1904, cited in Fletcher, 1963) and Grove (1913) considered P. menthae to be a collective species and suggested division into several independent species on the basis of such morphological differences as the presence or absence of warts on the teliospores, variations in the apical papillae, and lengths of the pedicels. The present study shows significant differences between the two groups in each of these teliospore characters.The spearmint rust teliospores described in the present study fit within the description of P. menthae var. menthae (Baxter, 1959), but the peppermint rust teliospores described do not. They do, however, fall within the broad size ranges given for the whole species by Grove (1913) and Arthur (1934), but to our knowledge no variant has been described that has teliospores with unequally sized cells. This cell-size distinction and the lack of ornamentation of this teliospore variant is similar to that noted by Walker & Conroy (1969) in their examination of herbarium specimens of rustinfected M. pulegium collected by McAlpine in 1904, but the teliospores described herein are larger which may merely reflect the limited amount of material available to McAlpine. Many more specimens, including some from M. pulegium and M. laxiflora, would need to be examined before it is possible to ascertain whether this morphological variant represents a rust indigenous to Australia or one that has been introduced.

J. Edwards and others Savile (1976) stated that, in rusts generally, fragile pedicels that break just below the teliospore and well-defined wall ornamentation are indicative of evolution towards aerial dispersal. If so, then the peppermint rust teliospores observed in the present study appear to be of a more primitive type than the spearmint rust teliospores. It is notable that the teliospore variant on M.ipiperita has been recorded only in Australia, yet the separation into spearmint and peppermint rust groups is observed worldwide. Molecular marker technology has proven to be a useful new mycological tool, particularly for re-examination of relationships between morphologically similar fungi including rusts. Molecular marker data provide additional information that can be used to complement more traditionally used traits. Evidence provided by rDNA sequence analysis (Zambino & Szabo, 1993) has confirmed that P. recondita is a species complex, with some isolates more closely related to P. hordei or Uromyces scillarum than to other P. recondita formae speciales. Comparison using restriction fragment length polymorphisms (RFLPs) of the bean rust fungus, Uromyces appendiculatus var. appendiculatus, with a morphologically similar rust, U. appendiculatus var. crassitunicatus, infecting the tropical legume Siratro (Macroptilium atropurpureum) showed conclusively that the two were not closely related genetically, and may be separate species (Braithwaite et al., 1991). Thus there are a number of well documented examples of morphologically similar rusts infecting the same or closely related hosts but being themselves genetically distinct. The length of the ITS region noted in the present study falls within the range reported for other Puccinia species. Primers ITS4 and ITS5 were used by Zambino & Szabo (1993) who found that the ITS regions of different cereal rust species were 690–720 bp long, and by Liddell & Onsurez-Waugh (1996) who reported 650 bp for P. grindeliae. The fragment of 650–700 bp from P. menthae reported herein would not differentiate it from either the cereal rusts or P. grindeliae and therefore, in this case, it would appear that ITS size alone is not a useful indicator of species. Similarly, within Laccaria, Gardes et al. (1991) found that 20 isolates from four species had the same-sized ITS fragments. The advantage of the RAPD technique is that it can show differences at any taxonomic level, provided that enough polymorphic markers are visualized, as this technique allows examination of the whole genome, not just a single region. The results of the RAPD analysis presented here demonstrate a clear genetic distinction between races of spearmint and peppermint rusts while length polymorphism in the ITS region did not. Sequencing of the ITS regions may be necessary to provide more insight into the precise level of taxonomic separation. Differences in host range alone generally characterise formae speciales, whereas morphological differences can indicate varietal distinction. Until now, the morphological differences observed within P. menthae var. menthae have not been deemed sufficient to warrant further separation of the species. We believe that, with the addition of the molecular data, there is a strong case for separation of these two rusts, but the question now posed is at what taxonomic level ? In view of the consistent pattern of difference in the suite of characters, i.e. RAPD profile, teliospore morphology and host specificity, in

1513 the mint rusts of Victoria, we suggest that peppermint rust deserves at least separate varietal or specific rank from spearmint rust. If the biological definition of a species is genetic isolation, then the molecular data points in that direction. Given the insights now possible using molecularbased techniques, the taxonomy of P. menthae as a whole would benefit from re-examination. This work has been generously funded by the Rural Industries Research and Development Corporation of Australia. We would like to thank Mr Fred Bienvenu, peppermint agronomist, Ovens Research Station, Agriculture Victoria, for his collaboration and interest, Ms Jocelyn Carpenter for sharing her expertise with scanning electron microscopy, Dr Vyrna Beilharz and Mr Ian Pascoe for the use of their Leica photomicroscope and their helpful comments regarding fungal taxonomy, and Ms Barbara Engel, Ms Allison Croft and Ms Rebecca Ford for their advice regarding RAPD methods. We are very grateful to Dr John Walker for his thoughtful advice, Dr Robert Park for his suggestions and for providing us with primers ITS4 and ITS5, and to Dr Pauline Gere for her help and advice with the WINAMOVA analysis.

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